Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage

PubMed Central

Dehne, T.; Lindahl, A.; Brittberg, M.; Pruss, A.; Ringe, J.; Sittinger, M.; Karlsson, C.

2012-01-01

Objective: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. Methods: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. Results: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. Conclusions: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage. PMID:26069618

Mining pathway associations for disease-related pathway activity analysis based on gene expression and methylation data.

PubMed

Lee, Hyeonjeong; Shin, Miyoung

2017-01-01

The problem of discovering genetic markers as disease signatures is of great significance for the successful diagnosis, treatment, and prognosis of complex diseases. Even if many earlier studies worked on identifying disease markers from a variety of biological resources, they mostly focused on the markers of genes or gene-sets (i.e., pathways). However, these markers may not be enough to explain biological interactions between genetic variables that are related to diseases. Thus, in this study, our aim is to investigate distinctive associations among active pathways (i.e., pathway-sets) shown each in case and control samples which can be observed from gene expression and/or methylation data. The pathway-sets are obtained by identifying a set of associated pathways that are often active together over a significant number of class samples. For this purpose, gene expression or methylation profiles are first analyzed to identify significant (active) pathways via gene-set enrichment analysis. Then, regarding these active pathways, an association rule mining approach is applied to examine interesting pathway-sets in each class of samples (case or control). By doing so, the sets of associated pathways often working together in activity profiles are finally chosen as our distinctive signature of each class. The identified pathway-sets are aggregated into a pathway activity network (PAN), which facilitates the visualization of differential pathway associations between case and control samples. From our experiments with two publicly available datasets, we could find interesting PAN structures as the distinctive signatures of breast cancer and uterine leiomyoma cancer, respectively. Our pathway-set markers were shown to be superior or very comparable to other genetic markers (such as genes or gene-sets) in disease classification. Furthermore, the PAN structure, which can be constructed from the identified markers of pathway-sets, could provide deeper insights into distinctive associations between pathway activities in case and control samples.

Whole blood gene expression in adolescent chronic fatigue syndrome: an exploratory cross-sectional study suggesting altered B cell differentiation and survival.

PubMed

Nguyen, Chinh Bkrong; Alsøe, Lene; Lindvall, Jessica M; Sulheim, Dag; Fagermoen, Even; Winger, Anette; Kaarbø, Mari; Nilsen, Hilde; Wyller, Vegard Bruun

2017-05-11

Chronic fatigue syndrome (CFS) is a prevalent and disabling condition affecting adolescents. The pathophysiology is poorly understood, but immune alterations might be an important component. This study compared whole blood gene expression in adolescent CFS patients and healthy controls, and explored associations between gene expression and neuroendocrine markers, immune markers and clinical markers within the CFS group. CFS patients (12-18 years old) were recruited nation-wide to a single referral center as part of the NorCAPITAL project. A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls having comparable distribution of gender and age were recruited from local schools. Whole blood samples were subjected to RNA sequencing. Immune markers were blood leukocyte counts, plasma cytokines, serum C-reactive protein and immunoglobulins. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. A total of 29 CFS patients and 18 healthy controls were included. We identified 176 genes as differentially expressed in patients compared to controls, adjusting for age and gender factors. Gene set enrichment analyses suggested impairment of B cell differentiation and survival, as well as enhancement of innate antiviral responses and inflammation in the CFS group. A pattern of co-expression could be identified, and this pattern, as well as single gene transcripts, was significantly associated with indices of autonomic nervous activity, plasma cortisol, and blood monocyte and eosinophil counts. Also, an association with symptoms of post-exertional malaise was demonstrated. Adolescent CFS is characterized by differential gene expression pattern in whole blood suggestive of impaired B cell differentiation and survival, and enhanced innate antiviral responses and inflammation. This expression pattern is associated with neuroendocrine markers of altered HPA axis and autonomic nervous activity, and with symptoms of post-exertional malaise. Trial registration Clinical Trials NCT01040429.

Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

PubMed

Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

2018-04-01

Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice.

PubMed

Quispe-Salcedo, Angela; Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Ohshima, Hayato

2012-04-01

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

HIV turns plasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.

PubMed

Hardy, Andrew W; Graham, David R; Shearer, Gene M; Herbeuval, Jean-Philippe

2007-10-30

Plasmacytoid dendritic cells (pDC) are key players in viral immunity and produce IFN-alpha after HIV-1 exposure, which in turn regulates TNF-related apoptosis-inducing ligand (TRAIL) expression by CD4(+) T cells. We show here that infectious and noninfectious HIV-1 virions induce activation of pDC into TRAIL-expressing IFN-producing killer pDC (IKpDC). IKpDC expressed high levels of activation markers (HLA-DR, CD80, CD83, and CD86) and the migration marker CCR7. Surprisingly, CXCR4 and CCR5 were down-regulated on IKpDC. We also show that HIV-1-induced IKpDC depended on Toll-like receptor 7 (TLR7) activation. HIV-1 or TLR7 agonistexposed IKpDC induced apoptosis of the CD4(+) T cell line SupT1 via the TRAIL pathway. Furthermore, IFN-alpha produced after HIV-induced TLR7 stimulation was responsible for TRAIL expression and the down-regulation of both CXCR4 and CCR5 by IKpDC. In contrast, activation and migration markers were not regulated by IFN-alpha. Finally, IFN-alpha increased the survival of IKpDC. We characterized a subset of pDC with a killer activity that is activated by endosomal-associated viral RNA and not by infection.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

PubMed

Mitachi, Takafumi; Mezaki, Minori; Yamashita, Kunihiko; Itagaki, Hiroshi

2018-01-01

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection

PubMed Central

Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B.; Robinson, Nicola; Brown, Helen; Kinloch, Sabine; Babiker, Abdel; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

2016-01-01

The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. PMID:27415828

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Glial activation and post-synaptic neurotoxicity: the key events in Streptozotocin (ICV) induced memory impairment in rats.

PubMed

Rai, Shivika; Kamat, Pradeep K; Nath, Chandishwar; Shukla, Rakesh

2014-02-01

In the present study the role of glial activation and post synaptic toxicity in ICV Streptozotocin (STZ) induced memory impaired rats was explored. In experiment set up 1: Memory deficit was found in Morris water maze test on 14-16 days after STZ (ICV; 3mg/Kg) administration. STZ causes increased expression of GFAP, CD11b and TNF-α indicating glial activation and neuroinflammation. STZ also significantly increased the level of ROS, nitrite, Ca(2+) and reduced the mitochondrial activity in synaptosomal preparation illustrating free radical generation and excitotoxicity. Increased expression and activity of Caspase-3 was also observed in STZ treated rat which specify apoptotic cell death in hippocampus and cortex. STZ treatment showed decrease expression of post synaptic markers CaMKIIα and PSD-95, while, expression of pre synaptic markers (synaptophysin and SNAP-25) remains unaltered indicating selective post synaptic neurotoxicity. Oral treatment with Memantine (10mg/kg) and Ibuprofen (50 mg/kg) daily for 13 days attenuated STZ induced glial activation, apoptotic cell death and post synaptic neurotoxicity in rat brain. Further, in experiment set up 2: where memory function was not affected i.e. 7-9 days after STZ treatment. The level of GFAP, CD11b, TNF-α, ROS and nitrite levels were increased. On the other hand, apoptotic marker, synaptic markers, mitochondrial activity and Ca(2+) levels remained unaffected. Collective data indicates that neuroinflammatory process and oxidative stress occurs earlier to apoptosis and does not affect memory function. Present study clearly suggests that glial activation and post synaptic neurotoxicity are the key factors in STZ induced memory impairment and neuronal cell death. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparative characterization of stem cell marker expression, metabolic activity and resistance to doxorubicin in adherent and spheroid cells derived from the canine prostate adenocarcinoma cell line CT1258.

PubMed

Liu, Wen; Moulay, Mohammed; Willenbrock, Saskia; Roolf, Catrin; Junghanss, Christian; Ngenazahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

2015-04-01

Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The Tregs' world according to GARP.

PubMed

Battaglia, Manuela; Roncarolo, Maria Grazia

2009-12-01

Naturally occurring CD4+CD25(high) regulatory T cells (nTreg) are essential for maintaining tolerance. FOXP3 has been established as a molecular marker of nTreg; however, FOXP3 cannot be used as a reliable marker for bona fide human nTreg since effector T cells also up-regulate FOXP3 expression upon activation. Despite the important function of nTreg, the underlying molecular mechanisms of nTreg-mediated suppression are far from defined. Previous studies have demonstrated that the TGF-beta latency-associated peptide (LAP) is expressed on the surface of nTreg, and that immunosuppression can be mediated by membrane TGF-beta; however, it remains unknown how LAP is bound to nTreg and what is the functional significance of its selective expression on activated nTreg. The nTreg's world may now change according to GARP, an orphan toll-like receptor composed of leucine-rich repeats. In this issue of the European Journal of Immunology, a study provides further demonstration that GARP is selectively expressed only in activated human nTreg and nTreg cell clones but not in activated effector T cells, confirming GARP as a bona fide nTreg marker. In addition, GARP binds directly to LAP; yet, GARP over-expression is insufficient to induce modification of latent TGF-beta into active TGF-beta further clarifying its role in nTreg-mediated suppression.

Metformin Inhibits Advanced Glycation End Products-Induced Inflammatory Response in Murine Macrophages Partly through AMPK Activation and RAGE/NFκB Pathway Suppression

PubMed Central

Zhou, Zhong'e; Tang, Yong; Chen, Chengjun; Lu, Yi; Liu, Liang

2016-01-01

Advanced glycation end products (AGEs) are major inflammatory mediators in diabetes, affecting atherosclerosis progression via macrophages. Metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated. The present study of murine bone marrow derived macrophages showed that (1) AGEs enhanced proinflammatory cytokines (interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)) mRNA expression, RAGE expression, and NFκB activation; (2) metformin pretreatment inhibited AGEs effects and AGEs-induced cluster designation 86 (CD86) (M1 marker) expression, while promoting CD206 (M2 marker) surface expression and anti-inflammatory cytokine (IL-10) mRNA expression; and (3) the AMPK inhibitor, Compound C, attenuated metformin effects. In conclusion, metformin inhibits AGEs-induced inflammatory response in murine macrophages partly through AMPK activation and RAGE/NFκB pathway suppression. PMID:27761470

Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP.

PubMed

Elkord, Eyad; Abd Al Samid, May; Chaudhary, Belal

2015-08-21

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3- T cells expressing Helios (FoxP3-Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/-Helios+). We show that CD4+GARP+/-LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios- Tregs upon TCR stimulation. Unlike FoxP3+Helios- Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.

Immunosenescence-Related Transcriptomic and Immunologic Changes in Older Individuals Following Influenza Vaccination

PubMed Central

Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Oberg, Ann L.; Zimmermann, Michael T.; Grill, Diane E.; Poland, Gregory A.

2016-01-01

The goal of annual influenza vaccination is to reduce mortality and morbidity associated with this disease through the generation of protective immune responses. The objective of the current study was to examine markers of immunosenescence and identify immunosenescence-related differences in gene expression, gene regulation, cytokine secretion, and immunologic changes in an older study population receiving seasonal influenza A/H1N1 vaccination. Surprisingly, prior studies in this cohort revealed weak correlations between immunosenescence markers and humoral immune response to vaccination. In this report, we further examined the relationship of each immunosenescence marker (age, T cell receptor excision circle frequency, telomerase expression, percentage of CD28− CD4+ T cells, percentage of CD28− CD8+ T cells, and the CD4/CD8 T cell ratio) with additional markers of immune response (serum cytokine and chemokine expression) and measures of gene expression and/or regulation. Many of the immunosenescence markers indeed correlated with distinct sets of individual DNA methylation sites, miRNA expression levels, mRNA expression levels, serum cytokines, and leukocyte subsets. However, when the individual immunosenescence markers were grouped by pathways or functional terms, several shared biological functions were identified: antigen processing and presentation pathways, MAPK, mTOR, TCR, BCR, and calcium signaling pathways, as well as key cellular metabolic, proliferation and survival activities. Furthermore, the percent of CD4+ and/or CD8+ T cells lacking CD28 expression also correlated with miRNAs regulating clusters of genes known to be involved in viral infection. Integrated (DNA methylation, mRNA, miRNA, and protein levels) network biology analysis of immunosenescence-related pathways and genesets identified both known pathways (e.g., chemokine signaling, CTL, and NK cell activity), as well as a gene expression module not previously annotated with a known function. These results may improve our ability to predict immune responses to influenza and aid in new vaccine development, and highlight the need for additional studies to better define and characterize immunosenescence. PMID:27853459

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

PubMed

Kundu, Rakesh; Dasgupta, Suman; Biswas, Anindita; Bhattacharya, Anirban; Pal, Bikas C; Bandyopadhyay, Debashis; Bhattacharya, Shelley; Bhattacharya, Samir

2008-08-13

Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

PubMed

Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

2011-05-26

Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

PubMed

Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

2017-09-01

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

PubMed

Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

2015-10-01

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

PubMed

Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

2015-08-01

The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

Palmitate Attenuates Osteoblast Differentiation of Fetal Rat Calvarial Cells

PubMed Central

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

2014-01-01

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl Co-A carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPAR gamma in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. PMID:24955854

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

PubMed

Yeh, Lee-Chuan C; Ford, Jeffery J; Lee, John C; Adamo, Martin L

2014-07-18

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. Copyright © 2014 Elsevier Inc. All rights reserved.

[DNA hydroxymethylase 10-11 translocation 2 (TET2) inhibits mouse macrophage activation and polarization].

PubMed

Li, Bingyi; Huo, Yi; Lin, Zhifeng; Wang, Tao

2017-09-01

Objective To study the role of DNA hydroxymethylase 10-11 translocation 2 (TET2) in macrophage activation and polarization. Methods RAW264.7 macrophages were cultured in vitro and stimulated with 100 ng/mL LPS for 0, 1, 2, 4, 6 hours. Real-time quantitative PCR was used to detect TET2 mRNA expression. TET2 expression was knocked down with siRNA and the knock-down efficiency was evaluated by real-time quantitative PCR and Western blotting. Following siRNA transfection for 48 hours, RAW264.7 cells were stimulated by LPS for 4 hours, and then real-time quantitative PCR and ELISA were performed to detect the expressions of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and IL-12. The M1 polarizing markers TNF-α, inducible nitric oxide synthase (iNOS) and IL-12, and M2 polarizing markers mannose receptor (MR), arginase 1 (Arg-1) and chitinase 3-like molecule 1 (Ym1) were tested after M1 or M2 induction by LPS/IFN-γ or IL-4. Results TET2 expression increased after LPS treatment in RAW264.7 cells and reached the peak at 2 hours later. The siRNA effectively reduced the expression of TET2. The expressions of IL-6, TNF-α and IL-12 mRNAs increased after TET2 knock-down and LPS stimulation. The expressions of M1 polarization markers and M2 markers were up-regulated by the corresponding stimulations after TET2 knock-down. Conclusion TET2 has the effect of inhibiting LPS-induced macrophage activation and plays an inhibitory role in macrophage M1 and M2 polarization.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

PD-L1 is an activation-independent marker of brown adipocytes.

PubMed

Ingram, Jessica R; Dougan, Michael; Rashidian, Mohammad; Knoll, Marko; Keliher, Edmund J; Garrett, Sarah; Garforth, Scott; Blomberg, Olga S; Espinosa, Camilo; Bhan, Atul; Almo, Steven C; Weissleder, Ralph; Lodish, Harvey; Dougan, Stephanie K; Ploegh, Hidde L

2017-09-21

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or β-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish

PubMed Central

Rovira, Mireia; Arrey, Gerard; Planas, Josep V.

2017-01-01

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a β-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic capacity as a result of swimming-induced exercise. Finally, the induction of myokine expression by swimming-induced exercise support the hypothesis that these myokines may have been produced and secreted by the exercised zebrafish muscle and acted on fast muscle cells to promote metabolic remodeling. These results support the use of zebrafish as a suitable model for studies on muscle remodeling in vertebrates, including humans. PMID:29326600

Immunomodulatory properties of carbon nanotubes are able to compensate immune function dysregulation caused by microgravity conditions

NASA Astrophysics Data System (ADS)

Crescio, Claudia; Orecchioni, Marco; Ménard-Moyon, Cécilia; Sgarrella, Francesco; Pippia, Proto; Manetti, Roberto; Bianco, Alberto; Delogu, Lucia Gemma

2014-07-01

Spaceflights lead to dysregulation of the immune cell functionality affecting the expression of activation markers and cytokine production. Short oxidized multi-walled carbon nanotubes functionalized by 1,3-dipolar cycloaddition have been reported to activate immune cells. In this Communication we have performed surface marker assays and multiplex ELISA on primary monocytes and T cells under microgravity. We have discovered that carbon nanotubes, through their immunostimulatory properties, are able to fight spaceflight immune system dysregulations.Spaceflights lead to dysregulation of the immune cell functionality affecting the expression of activation markers and cytokine production. Short oxidized multi-walled carbon nanotubes functionalized by 1,3-dipolar cycloaddition have been reported to activate immune cells. In this Communication we have performed surface marker assays and multiplex ELISA on primary monocytes and T cells under microgravity. We have discovered that carbon nanotubes, through their immunostimulatory properties, are able to fight spaceflight immune system dysregulations. Electronic supplementary information (ESI) available: Experimental section, structures of f-MWCNTs and uptake by human primary immune cells. See DOI: 10.1039/c4nr02711f

Expression of lactate transporters MCT1, MCT2 and CD147 in the red blood cells of three horse breeds: Finnhorse, Standardbred and Thoroughbred.

PubMed

Mykkänen, A K; Pösö, A R; McGowan, C M; McKane, S A

2010-11-01

In exercising horses, up to 50% of blood lactate is taken up into red blood cells (RBCs). Lactate transporter proteins MCT1, MCT2 and CD147 (an ancillary protein for MCT1) are expressed in the equine RBC membrane. In Standardbreds (SB), lactate transport activity is bimodally distributed and correlates with the amount of MCT1 and CD147. About 75% of SB studied have high lactate transport activity in RBCs. In other breeds, the distribution of lactate transport activity is unknown. To study whether similar bimodal distribution of MCT1 and CD147 is present also in the racing Finnhorse (FH) and Thoroughbred (TB) as in the SB and to study the distribution of MCT2 in all 3 breeds and to determine if there is a connection between MCT expression and performance markers in TB racehorses. Venous blood samples were taken from 118 FHs, 98 TBs and 44 SBs. Red blood cell membranes were purified and MCT1, MCT2 and CD147 measured by western blot. The amount of transporters was compared with TB performance markers. In TBs, the distribution of MCT1 was bimodal and in all breeds distribution of MCT2 unimodal. The amount of CD147 was clearly bimodal in FH and SB, with 85 and 82% expressing high amounts of CD147. In TBs, 88% had high expression of CD147 and 11% low expression, but one horse showed intermediate expression not apparent in FH or SB. Performance markers did not correlate with the amount of MCT1, MCT2 or CD147. High lactate transport activity was present in all 3 racing breeds, with the greatest proportion in the TB, followed by the racing FH, then SB. There was no significant statistical correlation found between lactate transporters in RBC membrane and markers of racing performance in the TB. © 2010 EVJ Ltd.

Combined caveolin-1 and epidermal growth factor receptor expression as a prognostic marker for breast cancer.

PubMed

Liang, Ya-Nan; Liu, Yu; Wang, Letian; Yao, Guodong; Li, Xiaobo; Meng, Xiangning; Wang, Fan; Li, Ming; Tong, Dandan; Geng, Jingshu

2018-06-01

Previous studies have indicated that caveolin-1 (Cav-1) is able to bind the signal transduction factor epidermal growth factor receptor (EGFR) to regulate its tyrosine kinase activity. The aim of the present study was to evaluate the clinical significance of Cav-1 gene expression in association with the expression of EGFR in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Cav-1 and EGFR expression using immunohistochemistry, and clinical significance was assessed using multivariate Cox regression analysis, Kaplan-Meier estimator curves and the log-rank test. Stromal Cav-1 was downregulated in 38.56% (118/306) of tumor tissues, whereas cytoplasmic EGFR and Cav-1 were overexpressed in 53.92% (165/306) and 44.12% (135/306) of breast cancer tissues, respectively. EGFR expression was positively associated with cytoplasmic Cav-1 and not associated with stromal Cav-1 expression in breast cancer samples; however, low expression of stromal Cav-1 was negatively associated with cytoplasmic Cav-1 expression in total tumor tissues, and analogous results were identified in the chemotherapy group. Multivariate Cox's proportional hazards model analysis revealed that, for patients in the estrogen receptor (ER)(+) group, the expression of stromal Cav-1 alone was a significant prognostic marker of breast cancer. However, in the chemotherapy, human epidermal growth factor receptor 2 (HER-2)(-), HER-2(+) and ER(-) groups, the use of combined markers was more effective prognostic marker. Stromal Cav-1 has a tumor suppressor function, and the combined marker stromal Cav-1/EGFR expression was identified as an improved prognostic marker in the diagnosis of breast cancer. Parenchymal expression of Cav-1 is able to promote EGFR signaling in breast cancer, potentially being required for EGFR-mediated initiation of mitosis.

Integrative analysis of multi-omics data for identifying multi-markers for diagnosing pancreatic cancer

PubMed Central

2015-01-01

Background microRNA (miRNA) expression plays an influential role in cancer classification and malignancy, and miRNAs are feasible as alternative diagnostic markers for pancreatic cancer, a highly aggressive neoplasm with silent early symptoms, high metastatic potential, and resistance to conventional therapies. Methods In this study, we evaluated the benefits of multi-omics data analysis by integrating miRNA and mRNA expression data in pancreatic cancer. Using support vector machine (SVM) modelling and leave-one-out cross validation (LOOCV), we evaluated the diagnostic performance of single- or multi-markers based on miRNA and mRNA expression profiles from 104 PDAC tissues and 17 benign pancreatic tissues. For selecting even more reliable and robust markers, we performed validation by independent datasets from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) data depositories. For validation, miRNA activity was estimated by miRNA-target gene interaction and mRNA expression datasets in pancreatic cancer. Results Using a comprehensive identification approach, we successfully identified 705 multi-markers having powerful diagnostic performance for PDAC. In addition, these marker candidates annotated with cancer pathways using gene ontology analysis. Conclusions Our prediction models have strong potential for the diagnosis of pancreatic cancer. PMID:26328610

Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP

PubMed Central

Elkord, Eyad; Abd Al Samid, May; Chaudhary, Belal

2015-01-01

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3− T cells expressing Helios (FoxP3−Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/−Helios+). We show that CD4+GARP+/−LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios− Tregs upon TCR stimulation. Unlike FoxP3+Helios− Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios− Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction. PMID:26343373

Inverse correlation between microtubule-associated protein 1A/1B-light chain 3 and p62/sequestosome-1 expression in the progression of cutaneous squamous cell carcinoma.

PubMed

Yoshihara, Nagisa; Takagi, Atsushi; Ueno, Takashi; Ikeda, Shigaku

2014-04-01

The expression of autophagy-related markers has occasionally been reported to correlate with the clinical stage of disease in patients with solid cancer, indicating autophagy activation. However, there have been no such reports for cutaneous squamous cell carcinoma. In this study, we investigated the expression levels of two autophagy-related markers, microtubule-associated protein IA/IB light chain 3 (LC3) and p62/sequestosome-1 (p62), in cutaneous squamous cell carcinoma specimens and assessed their correlation to clinicopathological factors in patients with this type of cancer. As a marker of the autophagosome, LC3 expression increases with autophagosome formation/accumulation, whereas p62 expression decreases due to selective degradation via autophagy. We performed immunostaining for LC3 and p62 in 50 cutaneous squamous cell carcinoma specimens obtained from patients treated by surgical resection, counted the number of cells that showed positive staining, and calculated the percentage of positive cells per low-power microscopic field. We next investigated the correlations between the expression levels of these markers and various clinicopathological factors. The results indicated that LC3 expression increased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.046). By contrast, the expression of p62 decreased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.001). These results suggest that autophagy becomes activated during disease progression in patients with cutaneous squamous cell carcinoma. © 2014 Japanese Dermatological Association.

Delayed rectifier and A-type potassium channels associated with Kv 2.1 and Kv 4.3 expression in embryonic rat neural progenitor cells.

PubMed

Smith, Dean O; Rosenheimer, Julie L; Kalil, Ronald E

2008-02-13

Because of the importance of voltage-activated K(+) channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and betaIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K(+) currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells.

Markers of nonselective and specific NK cell activation.

PubMed

Fogel, Leslie A; Sun, Michel M; Geurs, Theresa L; Carayannopoulos, Leonidas N; French, Anthony R

2013-06-15

NK cell activation is controlled by the integration of signals from cytokine receptors and germline-encoded activation and inhibitory receptors. NK cells undergo two distinct phases of activation during murine CMV (MCMV) infection: a nonselective phase mediated by proinflammatory cytokines and a specific phase driven by signaling through Ly49H, an NK cell activation receptor that recognizes infected cells. We sought to delineate cell surface markers that could distinguish NK cells that had been activated nonselectively from those that had been specifically activated through NK cell receptors. We demonstrated that stem cell Ag 1 (Sca-1) is highly upregulated during viral infections (to an even greater extent than CD69) and serves as a novel marker of early, nonselective NK cell activation. Indeed, a greater proportion of Sca-1(+) NK cells produced IFN-γ compared with Sca-1(-) NK cells during MCMV infection. In contrast to the universal upregulation of Sca-1 (as well as KLRG1) on NK cells early during MCMV infection, differential expression of Sca-1, as well as CD27 and KLRG1, was observed on Ly49H(+) and Ly49H(-) NK cells late during MCMV infection. Persistently elevated levels of KLRG1 in the context of downregulation of Sca-1 and CD27 were observed on NK cells that expressed Ly49H. Furthermore, the differential expression patterns of these cell surface markers were dependent on Ly49H recognition of its ligand and did not occur solely as a result of cellular proliferation. These findings demonstrate that a combination of Sca-1, CD27, and KLRG1 can distinguish NK cells nonselectively activated by cytokines from those specifically stimulated through activation receptors.

Chronic inflammation is a feature of Achilles tendinopathy and rupture.

PubMed

Dakin, Stephanie Georgina; Newton, Julia; Martinez, Fernando O; Hedley, Robert; Gwilym, Stephen; Jones, Natasha; Reid, Hamish A B; Wood, Simon; Wells, Graham; Appleton, Louise; Wheway, Kim; Watkins, Bridget; Carr, Andrew Jonathan

2018-03-01

Recent investigation of human tissue and cells from positional tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of tendon disease. These mechanisms remain poorly understood in disease of energy-storing tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture. We studied Achilles tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers. Tendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring tendon cells. Tissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles tendon disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Chronic inflammation is a feature of Achilles tendinopathy and rupture

PubMed Central

Newton, Julia; Martinez, Fernando O; Hedley, Robert; Gwilym, Stephen; Jones, Natasha; Reid, Hamish A B; Wood, Simon; Wells, Graham; Appleton, Louise; Wheway, Kim; Watkins, Bridget; Carr, Andrew Jonathan

2018-01-01

Background Recent investigation of human tissue and cells from positional tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of tendon disease. These mechanisms remain poorly understood in disease of energy-storing tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture. Methods We studied Achilles tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers. Results Tendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring tendon cells. Conclusions Tissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles tendon disease. PMID:29118051

IL-32 is a molecular marker of a host defense network in human tuberculosis

PubMed Central

Montoya, Dennis; Inkeles, Megan S.; Liu, Phillip T.; Realegeno, Susan; Teles, Rosane M. B.; Vaidya, Poorva; Munoz, Marcos A.; Schenk, Mirjam; Swindell, William R.; Chun, Rene; Zavala, Kathryn; Hewison, Martin; Adams, John S.; Horvath, Steve; Pellegrini, Matteo; Bloom, Barry R.; Modlin, Robert L.

2014-01-01

Tuberculosis is a leading cause of infectious disease–related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ– and IL-15–induced “defense response” genes. IL-32 induced the vitamin D–dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15–induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15–induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis. PMID:25143364

IL-32 is a molecular marker of a host defense network in human tuberculosis.

PubMed

Montoya, Dennis; Inkeles, Megan S; Liu, Phillip T; Realegeno, Susan; Teles, Rosane M B; Vaidya, Poorva; Munoz, Marcos A; Schenk, Mirjam; Swindell, William R; Chun, Rene; Zavala, Kathryn; Hewison, Martin; Adams, John S; Horvath, Steve; Pellegrini, Matteo; Bloom, Barry R; Modlin, Robert L

2014-08-20

Tuberculosis is a leading cause of infectious disease-related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ- and IL-15-induced "defense response" genes. IL-32 induced the vitamin D-dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15-induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15-induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis. Copyright © 2014, American Association for the Advancement of Science.

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

PubMed

Zogovic, Nevena; Tovilovic-Kovacevic, Gordana; Misirkic-Marjanovic, Maja; Vucicevic, Ljubica; Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Trajkovic, Vladimir

2015-04-01

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process. © 2014 International Society for Neurochemistry.

Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

PubMed

Kaurilind, Eve; Brosché, Mikael

2017-01-01

Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

Impact of traumatic brain injury on sleep structure, electrocorticographic activity and transcriptome in mice.

PubMed

Sabir, Meriem; Gaudreault, Pierre-Olivier; Freyburger, Marlène; Massart, Renaud; Blanchet-Cohen, Alexis; Jaber, Manar; Gosselin, Nadia; Mongrain, Valérie

2015-07-01

Traumatic brain injury (TBI), including mild TBI (mTBI), is importantly associated with vigilance and sleep complaints. Because sleep is required for learning, plasticity and recovery, we here evaluated the bidirectional relationship between mTBI and sleep with two specific objectives: (1) Test that mTBI rapidly impairs sleep-wake architecture and the dynamics of the electrophysiological marker of sleep homeostasis (i.e., non-rapid eye movement sleep delta (1-4Hz) activity); (2) evaluate the impact of sleep loss following mTBI on the expression of plasticity markers that have been linked to sleep homeostasis and on genome-wide gene expression. A closed-head injury model was used to perform a 48h electrocorticographic (ECoG) recording in mice submitted to mTBI or Sham surgery. mTBI was found to immediately decrease the capacity to sustain long bouts of wakefulness as well as the amplitude of the time course of ECoG delta activity during wakefulness. Significant changes in ECoG spectral activity during wakefulness, non-rapid eye movement and rapid eye movement sleep were observed mainly on the second recorded day. A second experiment was performed to measure gene expression in the cerebral cortex and hippocampus after a mTBI followed either by two consecutive days of 6h sleep deprivation (SD) or of undisturbed behavior (quantitative PCR and next-generation sequencing). mTBI modified the expression of genes involved in immunity, inflammation and glial function (e.g., chemokines, glial markers) and SD changed that of genes linked to circadian rhythms, synaptic activity/neuronal plasticity, neuroprotection and cell death and survival. SD appeared to affect gene expression in the cerebral cortex more importantly after mTBI than Sham surgery including that of the astrocytic marker Gfap, which was proposed as a marker of clinical outcome after TBI. Interestingly, SD impacted the hippocampal expression of the plasticity elements Arc and EfnA3 only after mTBI. Overall, our findings reveal alterations in spectral signature across all vigilance states in the first days after mTBI, and show that sleep loss post-mTBI reprograms the transcriptome in a brain area-specific manner and in a way that could be deleterious to brain recovery. Copyright © 2015 Elsevier Inc. All rights reserved.

Acute and subacute IL-1β administrations differentially modulate neuroimmune and neurotrophic systems: possible implications for neuroprotection and neurodegeneration

PubMed Central

2013-01-01

Background In Alzheimer’s disease, stroke and brain injuries, activated microglia can release proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines may change astrocyte and neurotrophin functions, which influences neuronal survival and induces apoptosis. However, the interaction between neuroinflammation and neurotrophin functions in different brain conditions is unknown. The present study hypothesized that acute and subacute elevated IL-1β differentially modulates glial and neurotrophin functions, which are related to their role in neuroprotection and neurodegeneration. Method Rats were i.c.v. injected with saline or IL-1β for 1 or 8 days and tested in a radial maze. mRNA and protein expressions of glial cell markers, neurotrophins, neurotrophin receptors, β-amyloid precursor protein (APP) and the concentrations of pro- and anti-inflammatory cytokines were measured in the hippocampus. Results When compared to controls, memory deficits were found 4 days after IL-1 administrations, however the deficits were attenuated by IL-1 receptor antagonist (RA). Subacute IL-1 administrations increased expressions of APP, microglial active marker CD11b, and p75 neurotrophin receptor, and the concentration of tumor necrosis factor (TNF)-α and IL-1β, but decreased expressions of astrocyte active marker glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF) and TrK B. By contrast, up-regulations of NGF, BDNF and TrK B expressions were found after acute IL-1 administration, which are associated with the increase in both glial marker expressions and IL-10 concentrations. However, TrK A was down-regulated by acute and up-regulated by subacute IL-1 administrations. Subacute IL-1-induced changes in the glial activities, cytokine concentrations and expressions of BDNF and p75 were reversed by IL-1RA treatment. Conclusion These results indicate that acute and subacute IL-1 administrations induce different changes toward neuroprotection after acute IL-1 administrations but neurodegeneration after subacute ones. PMID:23651534

Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

PubMed

Wen, Yan; Wani, Prachi; Zhou, Lu; Baer, Tom; Phadnis, Smruti Madan; Reijo Pera, Renee A; Chen, Bertha

2013-02-01

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated β-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

Reversal of high fat diet-induced obesity through modulating lipid metabolic enzymes and inflammatory markers expressions in rats.

PubMed

A, Kalaivani; Uddandrao, V V Sathibabu; Parim, Brahmanaidu; Ganapathy, Saravanan; P R, Nivedha; Kancharla, Sushma Chandulee; P, Rameshreddy; K, Swapna; Sasikumar, Vadivukkarasi

2018-03-19

In this study, we evaluated the ameliorative potential of Cucurbita maxima seeds oil (CSO (100 mg/kg body weight)) supplementation to high fat diet (HFD)-induced obese rats for 30 days on the changes in body weight, markers of lipid metabolism such as LDL, HDL, triglycerides, total cholesterol, adiponectin, leptin, amylase, and lipase. We also investigated the effects of CSO on the changes of lipid metabolic enzymes such as fatty-acid synthase, acetyl CoA carboxylase, carnitine palmitoyl transferase-1, HMG CoA reductase, and inflammatory markers (TNF-α and IL-6). Administration of CSO revealed significant diminution in body weight gain, altered the activity, expressions of lipid marker enzymes and inflammatory markers. It demonstrated that CSO had considerably altered these parameters when evaluated with HFD control rats. In conclusion, this study suggested that CSO might ameliorate the HFD-induced obesity by altering the enzymes and mRNA expressions important to lipid metabolism.

A novel role of HLA class I in the pathology of medulloblastoma.

PubMed

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-07-12

MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

A novel role of HLA class I in the pathology of medulloblastoma

PubMed Central

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-01-01

Background MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. Methods We investigated expression of four essential components of MHC class I (heavy chain, β2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. Results The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of β2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. Conclusion MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or β2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility. PMID:19594892

Muscle regeneration potential and satellite cell activation profile during recovery following hindlimb immobilization in mice.

PubMed

Guitart, Maria; Lloreta, Josep; Mañas-Garcia, Laura; Barreiro, Esther

2018-05-01

Reduced muscle activity leads to muscle atrophy and function loss in patients and animal models. Satellite cells (SCs) are postnatal muscle stem cells that play a pivotal role in skeletal muscle regeneration following injury. The regenerative potential, satellite cell numbers, and markers during recovery following immobilization of the hindlimb for 7 days were explored. In mice exposed to 7 days of hindlimb immobilization, in those exposed to recovery (7 days, splint removal), and in contralateral control muscles, muscle precursor cells were isolated from all hindlimb muscles (fluorescence-activated cell sorting, FACS) and SCs, and muscle regeneration were identified using immunofluorescence (gastrocnemius and soleus) and electron microscopy (EM, gastrocnemius). Expression of ki67, pax7, myoD, and myogenin was quantified (RT-PCR) from SC FACS yields. Body and grip strength were determined. Following 7 day hindlimb immobilization, a decline in SCs (FACS, immunofluorescence) was observed together with an upregulation of SC activation markers and signs of muscle regeneration including fusion to existing myofibers (EM). Recovery following hindlimb immobilization was characterized by a program of muscle regeneration events. Hindlimb immobilization induced a decline in SCs together with an upregulation of markers of SC activation, suggesting that fusion to existing myofibers takes place during unloading. Muscle recovery induced a significant rise in muscle precursor cells and regeneration events along with reduced SC activation expression markers and a concomitant rise in terminal muscle differentiation expression. These are novel findings of potential applicability for the treatment of disuse muscle atrophy, which is commonly associated with severe chronic and acute conditions. © 2017 Wiley Periodicals, Inc.

ALK Expression Is a Novel Marker for the WNT-activated Type of Pediatric Medulloblastoma and an Indicator of Good Prognosis for Patients.

PubMed

Łastowska, Maria; Trubicka, Joanna; Niemira, Magdalena; Paczkowska-Abdulsalam, Magdalena; Karkucińska-Więckowska, Agnieszka; Kaleta, Magdalena; Drogosiewicz, Monika; Tarasińska, Magdalena; Perek-Polnik, Marta; Krętowski, Adam; Dembowska-Bagińska, Bożenna; Grajkowska, Wiesława; Pronicki, Maciej; Matyja, Ewa

2017-06-01

ALK gene rearrangements were identified in a variety of cancers, including neuroblastoma, where the presence of ALK expression is associated with adverse prognosis. ALK mutations have recently been found in the pediatric brain tumor medulloblastoma, and microarray data indicate that ALK is highly expressed in a subset of these tumors. Therefore, we investigated whether ALK expression correlates with transcriptional profiles and clinical features of medulloblastoma. Tumors from 116 medulloblastoma patients were studied at diagnosis for the detection of ALK expression at the RNA level by an application of NanoString technology and at the protein level by immunohistochemistry using antibody ALK clone D5F3. The results indicate that ALK expression, at both the RNA and the protein levels, is strongly associated with the WNT-activated type of tumors and therefore may serve as a useful marker for the detection of this type of medulloblastoma. Importantly, ALK protein expression alone is also an indicator of good prognosis for medulloblastoma patients.

Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

PubMed Central

Azuma, Sakiko; Yokoyama, Yukihiro; Yamamoto, Akiko; Kyokane, Kazuhiro; Niida, Shumpei; Ishiguro, Hiroshi; Ko, Minoru S. H.

2013-01-01

We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas. PMID:23599043

Advanced Glycation End Products Enhance Macrophages Polarization into M1 Phenotype through Activating RAGE/NF-κB Pathway

PubMed Central

Jin, Xian; Yao, Tongqing; Zhou, Zhong'e; Zhu, Jian; Zhang, Song; Hu, Wei; Shen, Chengxing

2015-01-01

Atherosclerotic lesions are accelerated in patients with diabetes. M1 (classically activated in contrast to M2 alternatively activated) macrophages play key roles in the progression of atherosclerosis. Since advanced glycation end products (AGEs) are major pathogenic factors and active inflammation inducers in diabetes mellitus, this study assessed the effects of AGEs on macrophage polarization. The present study showed that AGEs significantly promoted macrophages to express IL-6 and TNF-α. M1 macrophage markers such as iNOS and surface markers including CD11c and CD86 were significantly upregulated while M2 macrophage markers such as Arg1 and CD206 remained unchanged after AGEs stimulation. AGEs significantly increased RAGE expression in macrophages and activated NF-κB pathway, and the aforementioned effects were partly abolished by administration of anti-RAGE antibody or NF-κB inhibitor PDTC. In conclusion, our results suggest that AGEs enhance macrophage differentiation into proinflammatory M1 phenotype at least partly via RAGE/NF-κB pathway activation. PMID:26114112

Delayed Rectifier and A-Type Potassium Channels Associated with Kv 2.1 and Kv 4.3 Expression in Embryonic Rat Neural Progenitor Cells

PubMed Central

Smith, Dean O.; Rosenheimer, Julie L.; Kalil, Ronald E.

2008-01-01

Background Because of the importance of voltage-activated K+ channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. Methodology/Principal Findings Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and βIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. Conclusions/Significance We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K+ currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells. PMID:18270591

Neutrophil CD64 expression, procalcitonin and presepsin are useful to differentiate infections from flares in SLE patients with SIRS.

PubMed

Echeverri, A; Naranjo-Escobar, J; Posso-Osorio, I; Aguirre-Valencia, D; Zambrano, D; Castaño, G L; Martínez, J D; Cañas, C A; Tobón, G J

2018-06-01

Background/Objective Differentiating systemic lupus erythematosus (SLE) activity from infections in febrile patients is difficult because of similar initial clinical presentation. The aim of this study is to evaluate the usefulness of a number of biomarkers for differentiating infections from activity in SLE patients admitted with systemic inflammatory response (SIRS). Methods Patients with SLE and SIRS admitted to the emergency room were included in this study. Measurements of different markers including procalcitonin, neutrophil CD64 expression and presepsin, were performed. Infection was considered present when positive cultures and/or polymerase chain reaction were obtained. Sensitivity and specificity were calculated for all biomarkers. Results Twenty-seven patients were admitted, 23 women (82.5%), mean age 33.2 years. An infectious disease was confirmed in 12 cases. Markers for SLE activity including anti-DNA titers by IIF ( p = 0.041) and enzyme-linked immunosorbent assay ( p = 0.009) were used for differentiating SLE flares from infection. On the contrary, increased procalcitonin ( p = 0.047), neutrophil CD64 expression by flow cytometry ( p = 0.037) and presepsin ( p = 0.037) levels were observed in infected SLE patients. Conclusions High neutrophil CD64 expression, presepsin and procalcitonin levels are useful to differentiate infections from activity in SLE patients. In most cases, a positive bioscore that includes these three markers demonstrate the presence of an infectious disease.

Comparing chemolithoautotrophic subseafloor communities across geochemical gradients using meta-omics and RNA-SIP

NASA Astrophysics Data System (ADS)

Fortunato, C. S.; Huber, J. A.

2015-12-01

The chemolithoautotrophic microbial community of the rocky subseafloor potentially provides a large amount of organic carbon to the deep ocean, yet our understanding of the activity and metabolic complexity of subseafloor organisms remains poorly described. Past studies have shown that the taxonomic structure of subseafloor communities differs based on the geochemical signatures of individual vents. In this study, we expanded beyond phylogeny and used a combination of metagenomic, metatranscriptomic, and RNA-based stable isotope probing (RNA-SIP) analyses to identify the metabolic potential, expression patterns, and the active autotrophic players and genomic pathways present in venting fluids from Axial Seamount, an active submarine volcano off the coast of Oregon, USA. Low-temperature diffuse vent fluids from three hydrothermal vents, Marker 113, Marker 33, and Anemone, were filtered and preserved on the seafloor for metagenome and metatranscriptome analyses. Fluid for RNA-SIP was also collected and incubated shipboard with 13C-labeled sodium bicarbonate at 30ºC, 55ºC, and 80ºC for each vent. Taxonomically, Epsilonproteobacteria comprised a significant proportion of the community at all three vents, but each vent also had distinct groups that were abundant including SUP05 at Anemone and Methanococcus at Marker 113. Functionally, vents shared many metabolic processes including genes for denitrification, sulfur reduction and sulfur, hydrogen, and ammonium oxidation, which were present and expressed in similar abundance across all three vents. One metabolic difference between vents was the presence and expression of genes for methanogenesis, which were highly abundant and expressed at Marker 113, in lower abundance and expression at Marker 33, and not present at Anemone. RNA-SIP analysis is ongoing but initial results from Marker 113 revealed that at mesophilic, thermophilic, or hyperthemophilic temperatures, different genera and autotrophic metabolisms dominated. In addition, RNA-SIP showed that at 80ºC a different hyperthermophilic group dominated at each vent. Overall, this study demonstrates the metabolic and taxonomic diversity of the subseafloor and better characterizes the subseafloor chemolithoautotrophs and their role in biogeochemical cycling.

Ameliorative potential of gingerol: Promising modulation of inflammatory factors and lipid marker enzymes expressions in HFD induced obesity in rats.

PubMed

Brahma Naidu, Parim; Uddandrao, V V Sathibabu; Ravindar Naik, Ramavat; Suresh, Pothani; Meriga, Balaji; Begum, Mustapha Shabana; Pandiyan, Rajesh; Saravanan, Ganapathy

2016-01-05

Obesity, generally linked to hyperlipidemia, has been occurring of late with distressing alarm and has now become a global phenomenon casting a huge economic burden on the health care system of countries around the world. The present study investigated the effects of gingerol over 30 days on the changes in HFD-induced obese rats in marker enzymes of lipid metabolism such as fatty-acid synthase (FAS), Acetyl CoA Carboxylase (ACC), Carnitine Palmitoyl Transferase-1(CPT-1), HMG co-A Reductase (HMGR), Lecithin Choline Acyl Transferase (LCAT) and Lipoprotein Lipase (LPL) and inflammatory markers (TNF-α and IL-6). The rats were treated orally with gingerol (75 mg kg(-1)) once daily for 30 days with a lorcaserin-treated group (10 mg kg(-1)) included for comparison. Changes in body weight, glucose, insulin resistance and expressions of lipid marker enzymes and inflammatory markers in tissues were observed in experimental rats. The administration of gingerol resulted in a significant reduction in body weight gain, glucose and insulin levels, and insulin resistance, which altered the activity, expressions of lipid marker enzymes and inflammatory markers. It showed that gingerol had significantly altered these parameters when compared with HFD control rats. This study confirms that gingerol prevents HFD-induced hyperlipidemia by modulating the expression of enzymes important to cholesterol metabolism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

MCP-1-Induced Protein Promotes Endothelial-Like and Angiogenic Properties in Human Bone Marrow Monocytic Cells

PubMed Central

Wang, Kangkai; Zhelyabovska, Olga; Saad, Yasser; Kolattukudy, Pappachan E.

2013-01-01

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3′-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases. PMID:24008336

Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription.

PubMed

Gao, Li; Ji, Yue; Lu, Yan; Qiu, Ming; Shen, Yejiao; Wang, Yaqing; Kong, Xiangqing; Shao, Yongfeng; Sheng, Yanhui; Sun, Wei

2018-03-09

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of regulatory proteins and proliferative activity in relation to phenotypic characteristics of upper urothelial carcinoma.

PubMed

Dolićanin, Zana; Velicković, Ljubinka Janković; Djordjević, Biljana; Visnjić, Milan; Pesić, Ivana; Ristić, Ana; Marjanović, Vesna

2011-07-01

Deregulation of the normal cell cycle is common in upper urothelial carcinoma (UUC). The aim of this study was to investigate the expression of regulatory proteins of the cell cycle (p53, p16, cyclin D1, HER-2) and proliferative Ki-67 activity in UUC, and to determine their interaction and influence on the phenotypic characteristics of UUC. In 44 patients with UUC, histopathological and immunohistochemical analyses (p53, p16, cyclin D1, HER-2, and Ki-67) of tumors were done. Overexpression/altered expression of p53, p16, cyclin D1 or HER-2 was detected in 20%, 57%, 64%, and 57% of tumors, respectively. Eleven (25%) UUC had a high proliferative Ki-67 index. Forty patients (91%) had at least one marker altered, while four (9%) tumors had a wild-type status. Analysis of relationship between expressions of molecular markers showed that only high expression of p53 was significantly associated with altered p16 activity (p < 0.05). High Ki-67 index was associated with the high stage (p < 0.005), solid growth (p < 0.01), high grade (p < 0.05), and multifocality p < 0.05) of UUC, while high expression of p53 was associated with the solid growth (p < 0.05). In regression models that included all molecular markers and phenotypic characteristics, only Ki-67 correlated with the growth (p < 0.0001), stage (p < 0.01), grade (p < 0.05) and multifocality (p < 0.05) of UCC; (Ki-67 and HER-2 expression correlated with the lymphovascular invasion (p < 0.05). This investigation showed that only negative regulatory proteins of the cell cycle, p53 and p16, were significantly associated in UUC, while proliferative marker Ki-67 was in relation to the key phenotypic characteristics of UUC in the best way.

Association of Regulatory T Cells with Diabetes Type-1 and Its Renal and Vascular Complications Based on the Expression of Forkhead Box Protein P3 (FoxP3), Helios and Neurophilin-1.

PubMed

Khamechian, Tahereh; Irandoust, Behnaz; Mohammadi, Hanieh; Nikoueinejad, Hassan; Akbari, Hossein

2018-04-01

In recent years, it has been recognized that regulatory T cells (Tregs) play a critical role in maintaining immune tolerance. Moreover, the expression of two markers named Helios and neurophilin-1 (NRP-1) has been highlighted in such cells. Helios, an intracellular transcription marker, largely differentiates twomost operative sub group of Tregs, namely naturally occurring (nTreg) and induced (iTreg) Tregs, and NRP-1 is reckoned as a membranous activity marker of Tregs. We aimed to count peripheral mononuclear cells expressing such markers in a group of type 1 diabetes patients to elucidate the possible role of Tregs in the pathogenesis of such disease and its complications. Blood samples from 61 adult patients with type 1 diabetes and 61 sex and age-matched healthy controls were tested to count two types of Tregs, namely naturally occurring and inducible types, according to the expression of cell surface markers of CD4/CD25/CD47-FITC/PE/APC and intracellular markers of FoxP3/Helios-PE-CY5/eFlour450 by flow cytometry, respectively.We also investigated the relation between expression of such markers with HbA1c, urine albumin/creatinine ratio (UACR), and common carotid intima thickness (CIMT). The circulatory frequency of both Helios+ and Helios- T-cells were significantly decreased in patients compared to those in healthy controls (p<0.001). There was also a significant decrease in circulatory frequency of Helios+ NRP-1+ and Helios- NRP-1+ cells in the patients compared to controls (p=0.029). According to expression of Helios and NRP-1 markers, the number and function of both Tregs were decreased in diabetic patients. Moreover, the neurophilin expression was inversely associated with complications of type 1 diabetes.

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.

Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibitedmore » the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.« less

Sorting Nexin 2 (SNX2): a potential marker of active thyrocytes in normal and hyperfunctioning thyroid disorders.

PubMed

Kanzawa, Maki; Hara, Shigeo; Semba, Shuho; Yokozaki, Hiroshi; Hirokawa, Mitsuyoshi; Itoh, Tomoo

2014-04-01

Sorting nexins (SNXs) are a large, diverse group of cytoplasmic and membrane-associated proteins that function in a variety of cellular processes, including endocytosis, protein trafficking, and the retrieval of transmembrane proteins. In this study, we demonstrated that SNX2 is expressed in columnar and active thyroid follicular cells but not in flattened inactive thyrocytes. Morphometric analysis revealed a significant correlation between SNX2 positivity and columnar cell morphology. Immunohistochemical staining of serial sections of the thyroid tissue indicated that SNX2 localization was similar to sortilin, a protein expressed by active thyrocytes. Expression of SNX2 in thyrocytes is particularly marked and extensive in most hyperstimulated thyroid disorders, including Graves disease (diffusely SNX2 positive in 73.3% patients) and functioning nodules (93.8% patients). SNX2 immunolocalization in hyperstimulated follicular epithelial cells was specific among the SNXs family members examined. These results support the utility of SNX2 as a novel marker of active thyrocytes and reflect the endosomal trafficking activity in these cells.

Distinct Expression of Phenotypic Markers in Placodes- and Neural Crest-Derived Afferent Neurons Innervating the Rat Stomach.

PubMed

Trancikova, Alzbeta; Kovacova, Eva; Ru, Fei; Varga, Kristian; Brozmanova, Mariana; Tatar, Milos; Kollarik, Marian

2018-02-01

Visceral pain is initiated by activation of primary afferent neurons among which the capsaicin-sensitive (TRPV1-positive) neurons play an important role. The stomach is a common source of visceral pain. Similar to other organs, the stomach receives dual spinal and vagal afferent innervation. Developmentally, spinal dorsal root ganglia (DRG) and vagal jugular neurons originate from embryonic neural crest and vagal nodose neurons originate from placodes. In thoracic organs the neural crest- and placodes-derived TRPV1-positive neurons have distinct phenotypes differing in activation profile, neurotrophic regulation and reflex responses. It is unknown to whether such distinction exists in the stomach. We hypothesized that gastric neural crest- and placodes-derived TRPV1-positive neurons express phenotypic markers indicative of placodes and neural crest phenotypes. Gastric DRG and vagal neurons were retrogradely traced by DiI injected into the rat stomach wall. Single-cell RT-PCR was performed on traced gastric neurons. Retrograde tracing demonstrated that vagal gastric neurons locate exclusively into the nodose portion of the rat jugular/petrosal/nodose complex. Gastric DRG TRPV1-positive neurons preferentially expressed markers PPT-A, TrkA and GFRα 3 typical for neural crest-derived TRPV1-positive visceral neurons. In contrast, gastric nodose TRPV1-positive neurons preferentially expressed markers P2X 2 and TrkB typical for placodes-derived TRPV1-positive visceral neurons. Differential expression of neural crest and placodes markers was less pronounced in TRPV1-negative DRG and nodose populations. There are phenotypic distinctions between the neural crest-derived DRG and placodes-derived vagal nodose TRPV1-positive neurons innervating the rat stomach that are similar to those described in thoracic organs.

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

PubMed

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.

FACS-Based Isolation, Propagation and Characterization of Mouse Embryonic Cardiomyocytes Based on VCAM-1 Surface Marker Expression

PubMed Central

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K.; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes. PMID:24386094

Aberrant SSEA-4 upregulation mediates myofibroblast activity to promote pre-cancerous oral submucous fibrosis

NASA Astrophysics Data System (ADS)

Yu, Cheng-Chia; Yu, Chuan-Hang; Chang, Yu-Chao

2016-11-01

Oral submucous fibrosis (OSF), regarded as a precancerous condition, is characterized by juxta-epithelial inflammatory reaction followed by fibro-elastic change in the lamina properia and epithelial atrophy. The pathologic mechanisms of OSF still need to be further clarified. In the study, we investigated the functional expression of SSEA-4, which is a well-known stemness marker, in myofibroblast activity and the clinical significance in OSF tissues. The expression of SSEA-4 in OSF was evaluated by immunohistochemical staining. Functional analysis of SSEA-4 on myofibroblast activity of OSF was achieved by lentiviral silencing ST3GAL2. Immunohisitochemistry demonstrated that SSEA-4 expression was significantly higher expression in areca quid chewing-associated OSF tissues than those of normal oral mucosa tissues. From flow cytometry analysis, arecoline dose-dependently activated SSEA-4 expression in primary human normal buccal mucosal fibroblasts (BMFs). Sorted SSEA-4-positive cells from fibrotic BMFs (fBMFs) have higher colony-forming unit, collagen gel contraction, and α-smooth muscle actin (α-SMA) expression than SSEA-4-negative subset. Knockdown of ST3GAL2 in fBMFs suppressed SSEA-4 expression, collagen contraction, migration, invasiveness, and wound healing capability. Consistently, silencing ST3GAL2 was found to repress arecoline-induced myofibroblast activity in BMFs. The study highlights SSEA-4 as a critical marker for therapeutic intervention to mediate myofibroblast transdifferentiation in areca quid chewing-associated OSF.

Mirror trends of plasticity and stability indicators in primate prefrontal cortex.

PubMed

García-Cabezas, Miguel Á; Joyce, Mary Kate P; John, Yohan J; Zikopoulos, Basilis; Barbas, Helen

2017-10-01

Research on plasticity markers in the cerebral cortex has largely focused on their timing of expression and role in shaping circuits during critical and normal periods. By contrast, little attention has been focused on the spatial dimension of plasticity-stability across cortical areas. The rationale for this analysis is based on the systematic variation in cortical structure that parallels functional specialization and raises the possibility of varying levels of plasticity. Here, we investigated in adult rhesus monkeys the expression of markers related to synaptic plasticity or stability in prefrontal limbic and eulaminate areas that vary in laminar structure. Our findings revealed that limbic areas are impoverished in three markers of stability: intracortical myelin, the lectin Wisteria floribunda agglutinin, which labels perineuronal nets, and parvalbumin, which is expressed in a class of strong inhibitory neurons. By contrast, prefrontal limbic areas were enriched in the enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), known to enhance plasticity. Eulaminate areas have more elaborate laminar architecture than limbic areas and showed the opposite trend: they were enriched in markers of stability and had lower expression of the plasticity-related marker CaMKII. The expression of glial fibrillary acidic protein (GFAP), a marker of activated astrocytes, was also higher in limbic areas, suggesting that cellular stress correlates with the rate of circuit reshaping. Elevated markers of plasticity may endow limbic areas with flexibility necessary for learning and memory within an affective context, but may also render them vulnerable to abnormal structural changes, as seen in neurologic and psychiatric diseases. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Feline Glycoprotein A Repetitions Predominant Anchors Transforming Growth Factor Beta on the Surface of Activated CD4+CD25+ Regulatory T Cells and Mediates AIDS Lentivirus-Induced T Cell Immunodeficiency

PubMed Central

Miller, Michelle M.; Fogle, Jonathan E.; Ross, Peter

2013-01-01

Abstract Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP+TGFb+ Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP+ Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb+ Treg-mediated T cell immune suppression during lentivirus infection. PMID:23373523

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

PubMed

Miller, Michelle M; Fogle, Jonathan E; Ross, Peter; Tompkins, Mary B

2013-04-01

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Impaired granulocyte oxidative burst and decreased expression of leucocyte adhesion molecule-1 (LAM-1) in patients with Wegener's granulomatosis.

PubMed Central

Riecken, B; Gutfleisch, J; Schlesier, M; Peter, H H

1994-01-01

Neutrophils are the target of autoantibodies in Wegener's granulomatosis (WG). In this study, granulocyte function and surface marker expression were investigated in patients with WG. The oxidative burst in response to phorbol myristate acetate (PMA) was tested with granulocytes of 25 patients with histologically proven WG. A significantly diminished percentage of oxygen radical-producing cells was found in patients with active disease. Surface antigen expression of CD11b and LAM-1 was analysed on granulocytes of 20 patients with WG. Whereas the expression of CD11b was normal, surface expression of LAM-1 was decreased in nine cases with WG. The decrease of LAM-1 correlated with disease activity. Phagocytosis of Escherichia coli was tested in 10 patients with WG, and normal values were found in all cases. We conclude that down-regulation of LAM-1 may be a marker of disease activity in WG. The altered response to PMA may indicate functional changes in granulocyte reactivity due to autoantibody-induced damage of the granulocyte membrane. PMID:7512009

The Transcription Factor p53 Influences Microglial Activation Phenotype

PubMed Central

Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

2011-01-01

Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

Normal T-cell activation in elite controllers with preserved CD4+ T-cell counts.

PubMed

Bansal, Anju; Sterrett, Sarah; Erdmann, Nathan; Westfall, Andrew O; Dionne-Odom, Jodie; Overton, Edgar T; Goepfert, Paul A

2015-11-01

HIV elite controllers suppress HIV viremia without antiretroviral therapy (ART), yet previous studies demonstrated that elite controllers maintain an activated T-cell phenotype. Chronic immune activation has detrimental consequences and thus ART has been advocated for all elite controllers. However, elite controllers are not a clinically homogenous group. Since CD4% is among the best predictors of AIDS-related events, in the current study, we assessed whether this marker can be used to stratify elite controllers needing ART. Sixteen elite controllers were divided into two groups based on CD4% (EC > 40% and EC ≤40%), and T-cell subsets were analyzed for markers of memory/differentiation (CD45RA, CCR7, CD28), activation (CD38/HLA-DR), immunosenescence (CD57), costimulation (CD73, CD28) and exhaustion (PD-1, CD160, Tim-3). Monocyte subsets (CD14, CD16) were also analyzed and sCD14 levels were quantified using ELISA. In the EC group, expression of activation, exhaustion, and immunosensescence markers on T cells were significantly reduced compared with the EC group and similar to the seronegative controls. The EC group expressed higher levels of costimulatory molecules CD28 and CD73 and had lower levels of monocyte activation (HLA-DR expression) with a reduced frequency of inflammatory monocyte (CD14 CD16) subset. Furthermore, the EC group maintained a stable CD4% during a median follow-up of 6 years. Elite controllers with preserved CD4T cells (EC) have normal T-cell and monocyte phenotypes and therefore may have limited benefit from ART. CD4% can be an important marker for evaluating future studies aimed at determining the need for ART in this group of individuals.

ILK Induces Cardiomyogenesis in the Human Heart

PubMed Central

Traister, Alexandra; Aafaqi, Shabana; Masse, Stephane; Dai, Xiaojing; Li, Mark; Hinek, Aleksander; Nanthakumar, Kumaraswamy; Hannigan, Gregory; Coles, John G.

2012-01-01

Background Integrin-linked kinase (ILK) is a widely conserved serine/threonine kinase that regulates diverse signal transduction pathways implicated in cardiac hypertrophy and contractility. In this study we explored whether experimental overexpression of ILK would up-regulate morphogenesis in the human fetal heart. Methodology/Principal Findings Primary cultures of human fetal myocardial cells (19–22 weeks gestation) yielded scattered aggregates of cardioblasts positive for the early cardiac lineage marker nk×2.5 and containing nascent sarcomeres. Cardiac cells in colonies uniformly expressed the gap junction protein connexin 43 (C×43) and displayed a spectrum of differentiation with only a subset of cells exhibiting the late cardiomyogenic marker troponin T (cTnT) and evidence of electrical excitability. Adenovirus-mediated overexpression of ILK potently increased the number of new aggregates of primitive cardioblasts (p<0.001). The number of cardioblast colonies was significantly decreased (p<0.05) when ILK expression was knocked down with ILK targeted siRNA. Interestingly, overexpression of the activation resistant ILK mutant (ILKR211A) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILKWT). The cardiomyogenic effects of ILKR211A and ILKWT were accompanied by concurrent activation of β-catenin (p<0.001) and increase expression of progenitor cell marker islet-1, which was also observed in lysates of transgenic mice with cardiac-specific over-expression of ILKR211A and ILKWT. Finally, endogenous ILK expression was shown to increase in concert with those of cardiomyogenic markers during directed cardiomyogenic differentiation in human embryonic stem cells (hESCs). Conclusions/Significance In the human fetal heart ILK activation is instructive to the specification of mesodermal precursor cells towards a cardiomyogenic lineage. Induction of cardiomyogenesis by ILK overexpression bypasses the requirement of proximal PI3K activation for transduction of growth factor- and β1-integrin-mediated differentiation signals. Altogether, our data indicate that ILK represents a novel regulatory checkpoint during human cardiomyogenesis. PMID:22666394

Gas1 expression in parietal cells of Bowman's capsule in experimental diabetic nephropathy.

PubMed

Luna-Antonio, Brenda I; Rodriguez-Muñoz, Rafael; Namorado-Tonix, Carmen; Vergara, Paula; Segovia, Jose; Reyes, Jose L

2017-07-01

Gas1 (Growth Arrest-Specific 1) is a pleiotropic protein with novel functions including anti-proliferative and proapoptotic activities. In the kidney, the expression of Gas1 has been described in mesangial cells. In this study, we described that renal parietal cells of Bowman's capsule (BC) and the distal nephron cells also express Gas1. The role of Gas1 in the kidney is not yet known. There is a subpopulation of progenitor cells in Bowman's capsule with self-renewal properties which can eventually differentiate into podocytes as a possible mechanism of regeneration in the early stages of diabetic nephropathy. We analyzed the expression of Gas1 in the parietal cells of Bowman's capsule in murine experimental diabetes. We found that diabetes reduced the expression of Gas1 and increased the expression of progenitor markers like NCAM, CD24, and SIX1/2, and mesenchymal markers like PAX2 in the Bowman's capsule. We also analyzed the expression of WT1 (a podocyte-specific marker) on BC and observed an increase in the number of WT1 positive cells in diabetes. In contrast, nephrin, another podocyte-specific protein, decreases its expression in the first week of diabetes in the glomerular tuft, which is gradually restored during the second and third weeks of diabetes. These results suggest that in diabetes the decrease of Gas1 promotes the activation of parietal progenitor cells of Bowman's capsule that might differentiate into podocytes and compensate their loss observed in this pathology.

Dermal Stem Cells Can Differentiate Down an Endothelial Lineage

PubMed Central

Bell, Emma; Richardson, Gavin D.; Jahoda, Colin A.; Gledhill, Karl; Phillips, Helen M.; Henderson, Deborah; Owens, W. Andrew

2012-01-01

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle. PMID:22571645

The role of proteinase 3 (PR3) and the protease-activated receptor-2 (PAR-2) pathway in dendritic cell (DC) maturation of human-DC-like monocytes and murine DC.

PubMed

Jiang, Bo; Grage-Griebenow, Evelin; Csernok, Elena; Butherus, Kristine; Ehlers, Stefan; Gross, Wolfgang L; Holle, Julia U

2010-01-01

The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC. Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h. There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC. PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

PubMed

Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation. These events are manifest in inflammatory diseases, such as atherosclerosis. Therefore, our results suggest that DBMSCs could be usefully employed as a therapeutic strategy for atherosclerosis.

A Novel Strategy for Enrichment and Isolation of Osteoprogenitor Cells from Induced Pluripotent Stem Cells Based on Surface Marker Combination

PubMed Central

Ochiai-Shino, Hiromi; Kato, Hiroshi; Sawada, Takashi; Onodera, Shoko; Saito, Akiko; Takato, Tsuyoshi; Shibahara, Takahiko; Muramatsu, Takashi; Azuma, Toshifumi

2014-01-01

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development. PMID:24911063

YB-1 overexpression promotes a TGF-β1-induced epithelial–mesenchymal transition via Akt activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ha, Bin; Lee, Eun Byul; Cui, Jun

2015-03-06

The Y-box binding protein-1 (YB-1) is a transcription/translation regulatory protein, and the expression thereof is associated with cancer aggressiveness. In the present study, we explored the regulatory effects of YB-1 during the transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells. Downregulation of YB-1 increased E-cadherin promoter activity, and upregulation of YB-1 decreased promoter activity, suggesting that the YB-1 level may be correlated with the EMT. TGF-β1 induced YB-1 expression, and TGF-β1 translocated cytosolic YB-1 into the nucleus. YB-1 overexpression promoted TGF-β1-induced downregulation of epithelial markers, upregulation of mesenchymal markers, and cell migration. Moreover, YB-1 overexpression enhanced themore » expression of E-cadherin transcriptional repressors via TGF-β1-induced Akt activation. Our findings afford new insights into the role played by YB-1 in the TGF-β1 signaling pathway. - Highlights: • YB-1 regulates E-cadherin expression in A549 cells. • TGF-β1 induces upregulating and nuclear localization of YB-1. • YB-1 overexpression accelerates TGF-β1-induced EMT and cell migration. • YB-1 regulates Snail and Slug expression via Akt activation.« less

Prognostic Impact of Activated Leucocyte Cell Adhesion Molecule (ALCAM/CD166) in Infantile Neuroblastoma.

PubMed

Wachowiak, Robin; Mayer, Steffi; Kaifi, Jussuf; Gebauer, Florian; Izbicki, Jakob R; Lacher, Martin; Bockhorn, Maximilian; Tachezy, Michael

2016-08-01

Activated leukocyte cell adhesion molecule (ALCAM/CD166) as a member of the 'immunoglobulin superfamily' is known to be involved in cancer cell proliferation and migration. The aim of this study was to investigate the expression of ALCAM in neuroblastoma tissues. ALCAM expression was analyzed in primary neuroblastoma specimens by immunohistochemistry on microarray sections. Histopathological and clinical data were correlated with ALCAM expression and survival analysis was performed. Sixty-six children were included in the study. Strong expression of ALCAM was detected in 52 (79%) of the samples. Weak expression was significantly correlated with the International Neuroblastoma Staging System (INSS) stage (p=0.024) and positive n-MYC amplification (p=0.019). Recurrence-free survival (RFS) and overall survival (OS) were significantly shorter if ALCAM was expressed weakly (p=0.032 and p=0.001). Weak ALCAM expression was significantly correlated with established markers for poor prognosis, as well as shorter RFS and OS. ALCAM might be considered as a prognostic marker for infantile neuroblastoma. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

PubMed

Carlson, Signe; Helterline, Deri; Asbe, Laura; Dupras, Sarah; Minami, Elina; Farris, Stephen; Stempien-Otero, April

2017-07-01

Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, P<0.007). There was significant up-regulation of cardiac mac Arg1 and YM1 with MI in both WT and uPA null mice (n=4-9 per genotype and condition). Treatment with plasmin increased expression of Arg1 and YM1 in cultured cardiac macs. Histologic analysis revealed increased density of activated fibroblasts and M2 macs in SR-uPA hearts post-infarction with associated increases in fibrosis. Cardiac macs isolated from human hearts with ischemic heart disease expressed increased levels of the M2 marker CD206 in comparison to blood-derived macs (4.9±1.3). Cardiac macs in mouse and human hearts adopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combining FoxP3 and Helios with GARP/LAP markers can identify expanded Treg subsets in cancer patients.

PubMed

Abd Al Samid, May; Chaudhary, Belal; Khaled, Yazan S; Ammori, Basil J; Elkord, Eyad

2016-03-22

Regulatory T cells (Tregs) comprise numerous heterogeneous subsets with distinct phenotypic and functional features. Identifying Treg markers is critical to investigate the role and clinical impact of various Treg subsets in pathological settings, and also for developing more effective immunotherapies. We have recently shown that non-activated FoxP3-Helios+ and activated FoxP3+/-Helios+ CD4+ T cells express GARP/LAP immunosuppressive markers in healthy donors. In this study we report similar observations in the peripheral blood of patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC). Comparing levels of different Treg subpopulations in cancer patients and controls, we report that in PC patients, and unlike LICRC patients, there was no increase in Treg levels as defined by FoxP3 and Helios. However, defining Tregs based on GARP/LAP expression showed that FoxP3-LAP+ Tregs in non-activated and activated settings, and FoxP3+Helios+GARP+LAP+ activated Tregs were significantly increased in both groups of patients, compared with controls. This work implies that a combination of Treg-specific markers could be used to more accurately determine expanded Treg subsets and to understand their contribution in cancer settings. Additionally, GARP-/+LAP+ CD4+ T cells made IL-10, and not IFN-γ, and levels of IL-10-secreting CD4+ T cells were elevated in LICRC patients, especially with higher tumor staging. Taken together, our results indicate that investigations of Treg levels in different cancers should consider diverse Treg-related markers such as GARP, LAP, Helios, and others and not only FoxP3 as a sole Treg-specific marker.

Src is activated by the nuclear receptor peroxisome proliferator-activated receptor β/δ in ultraviolet radiation-induced skin cancer.

PubMed

Montagner, Alexandra; Delgado, Maria B; Tallichet-Blanc, Corinne; Chan, Jeremy S K; Sng, Ming K; Mottaz, Hélén; Degueurce, Gwendoline; Lippi, Yannick; Moret, Catherine; Baruchet, Michael; Antsiferova, Maria; Werner, Sabine; Hohl, Daniel; Saati, Talal Al; Farmer, Pierre J; Tan, Nguan S; Michalik, Liliane; Wahli, Walter

2014-01-01

Although non-melanoma skin cancer (NMSC) is the most common human cancer and its incidence continues to rise worldwide, the mechanisms underlying its development remain incompletely understood. Here, we unveil a cascade of events involving peroxisome proliferator-activated receptor (PPAR) β/δ and the oncogene Src, which promotes the development of ultraviolet (UV)-induced skin cancer in mice. UV-induced PPARβ/δ activity, which directly stimulated Src expression, increased Src kinase activity and enhanced the EGFR/Erk1/2 signalling pathway, resulting in increased epithelial-to-mesenchymal transition (EMT) marker expression. Consistent with these observations, PPARβ/δ-null mice developed fewer and smaller skin tumours, and a PPARβ/δ antagonist prevented UV-dependent Src stimulation. Furthermore, the expression of PPARβ/δ positively correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma (SCC), and critically, linear models applied to several human epithelial cancers revealed an interaction between PPARβ/δ and SRC and TGFβ1 transcriptional levels. Taken together, these observations motivate the future evaluation of PPARβ/δ modulators to attenuate the development of several epithelial cancers.

Src is activated by the nuclear receptor peroxisome proliferator-activated receptor β/δ in ultraviolet radiation-induced skin cancer

PubMed Central

Montagner, Alexandra; Delgado, Maria B; Tallichet-Blanc, Corinne; Chan, Jeremy S K; Sng, Ming K; Mottaz, Hélène; Degueurce, Gwendoline; Lippi, Yannick; Moret, Catherine; Baruchet, Michael; Antsiferova, Maria; Werner, Sabine; Hohl, Daniel; Al Saati, Talal; Farmer, Pierre J; Tan, Nguan S; Michalik, Liliane; Wahli, Walter

2014-01-01

Although non-melanoma skin cancer (NMSC) is the most common human cancer and its incidence continues to rise worldwide, the mechanisms underlying its development remain incompletely understood. Here, we unveil a cascade of events involving peroxisome proliferator-activated receptor (PPAR) β/δ and the oncogene Src, which promotes the development of ultraviolet (UV)-induced skin cancer in mice. UV-induced PPARβ/δ activity, which directly stimulated Src expression, increased Src kinase activity and enhanced the EGFR/Erk1/2 signalling pathway, resulting in increased epithelial-to-mesenchymal transition (EMT) marker expression. Consistent with these observations, PPARβ/δ-null mice developed fewer and smaller skin tumours, and a PPARβ/δ antagonist prevented UV-dependent Src stimulation. Furthermore, the expression of PPARβ/δ positively correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma (SCC), and critically, linear models applied to several human epithelial cancers revealed an interaction between PPARβ/δ and SRC and TGFβ1 transcriptional levels. Taken together, these observations motivate the future evaluation of PPARβ/δ modulators to attenuate the development of several epithelial cancers. PMID:24203162

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.

PubMed

Kawakami, Kyojiro; Fujita, Yasunori; Matsuda, Yoko; Arai, Tomio; Horie, Kengo; Kameyama, Koji; Kato, Taku; Masunaga, Koichi; Kasuya, Yutaka; Tanaka, Masashi; Mizutani, Kosuke; Deguchi, Takashi; Ito, Masafumi

2017-05-05

Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.

Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells

PubMed Central

2017-01-01

Purpose Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated. Conclusions SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease. PMID:29093986

Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms

PubMed Central

Rőszer, Tamás

2015-01-01

The alternatively activated or M2 macrophages are immune cells with high phenotypic heterogeneity and are governing functions at the interface of immunity, tissue homeostasis, metabolism, and endocrine signaling. Today the M2 macrophages are identified based on the expression pattern of a set of M2 markers. These markers are transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. This review discusses whether these M2 markers can be reliably used to identify M2 macrophages and define their functional subdivisions. Also, it provides an update on the novel signals of the tissue environment and the neuroendocrine system which shape the M2 activation. The possible evolutionary roots of the M2 macrophage functions are also discussed. PMID:26089604

The bile acid receptor GPBAR1 (TGR5) is expressed in human gastric cancers and promotes epithelial-mesenchymal transition in gastric cancer cell lines

PubMed Central

Cipriani, Sabrina; Marchianò, Silvia; Marino, Elisabetta; Zampella, Angela; Rende, Mario; Mosci, Paolo; Distrutti, Eleonora; Donini, Annibale; Fiorucci, Stefano

2016-01-01

GPBAR1 (also known as TGR5) is a bile acid activated receptor expressed in several adenocarcinomas and its activation by secondary bile acids increases intestinal cell proliferation. Here, we have examined the expression of GPBAR1 in human gastric adenocarcinomas and investigated whether its activation promotes the acquisition of a pro-metastatic phenotype. By immunohistochemistry and RT-PCR analysis we found that expression of GPBAR1 associates with advanced gastric cancers (Stage III-IV). GPBAR1 expression in tumors correlates with the expression of N-cadherin, a markers of epithelial-mesenchymal transition (EMT) (r=0.52; P<0.01). Expression of GPBAR1, mRNA and protein, was detected in cancer cell lines, with MKN 45 having the higher expression. Exposure of MKN45 cells to GPBAR1 ligands, TLCA, oleanolic acid or 6-ECDCA (a dual FXR and GPBAR1 ligand) increased the expression of genes associated with EMT including KDKN2A, HRAS, IGB3, MMP10 and MMP13 and downregulated the expression of CD44 and FAT1 (P<0.01 versus control cells). GPBAR1 activation in MKN45 cells associated with EGF-R and ERK1 phosphorylation. These effects were inhibited by DFN406, a GPBAR1 antagonist, and cetuximab. GPBAR1 ligands increase MKN45 migration, adhesion to peritoneum and wound healing. Pretreating MKN45 cells with TLCA increased propensity toward peritoneal dissemination in vivo. These effects were abrogated by cetuximab. In summary, we report that GPBAR1 is expressed in advanced gastric cancers and its expression correlates with markers of EMT. GPBAR1 activation in MKN45 cells promotes EMT. These data suggest that GPBAR1 antagonist might have utility in the treatment of gastric cancers. PMID:27409173

Bone marrow-derived macrophages from aged rats are more responsive to inflammatory stimuli.

PubMed

Barrett, James P; Costello, Derek A; O'Sullivan, Joan; Cowley, Thelma R; Lynch, Marina A

2015-04-09

Lipopolysaccharide (LPS) and interferon-γ (IFNγ) increase expression of tumour necrosis factor-α (TNFα) that characterizes the M1 activation state of macrophages. Whereas it is accepted that the immune system undergoes changes with age, there is inconsistency in the literature with respect to the impact of age on the response of macrophages to inflammatory stimuli. Here, we investigate the effect of age on the responsiveness of bone marrow-derived macrophages (BMDMs) to LPS and IFNγ. The context for addressing this question is that macrophages, which infiltrate the brain of aged animals, will encounter the neuroinflammatory environment that has been described with age. Brain tissue, prepared from young and aged rats, was assessed for expression of inflammatory markers by PCR and for evidence of infiltration of macrophages by flow cytometry. BMDMs were prepared from the long bones of young and aged rats, maintained in culture for 8 days and incubated in the presence or absence of LPS (100 ng/ml) or IFNγ (50 ng/ml). Cells were harvested and assessed for mRNA expression of markers of M1 activation including TNFα and NOS2, or for expression of IFNγR1 and TLR4 by western immunoblotting. To assess whether BMDMs induced glial activation, mixed glial cultures were incubated in the presence of conditioned media obtained from unstimulated BMDMs of young and aged rats and evaluated for expression of inflammatory markers. Markers associated with M1 activation were expressed to a greater extent in BMDMs from aged rats in response to LPS and IFNγ, compared with cells from young rats. The increased responsiveness was associated with increases in IFNγ receptor (IFNγR) and Toll-like receptor 4 (TLR4). The data show that conditioned media from BMDMs of aged rats increased the expression of pro-inflammatory mediators in glial cells. Significantly, there was an age-related increase in macrophage infiltration into the brain, and this was combined with increased expression of IFNγ and the Toll-like receptor 4 agonist, high-mobility group protein B1 (HMGB1). Exposure of infiltrating macrophages to the inflammatory microenvironment that develops in the brain with age is likely to contribute to a damaging cascade that negatively impacts neuronal function.

Inflammatory markers in relation to body composition, physical activity and assessment of nutritional status of the adolescents.

PubMed

Neves Miranda, Valter Paulo; Gouveia Peluzio, Maria do Carmo; Rodrigues de Faria, Eliana; Castro Franceschini, Sylvia do Carmo; Eloiza Priore, Silvia

2015-05-01

The evaluation of inflammatory markers during adolescence can monitor different stages and manifestation of chronic diseases in adulthood. The control of the subclinical inflammation process through changes in lifestyle, especially in the practice of physical activity and dietary education can mitigate the effects of risk factors that trigger the process of atherosclerosis. To do a critical review regarding inflammatory markers as a risk factor of cardiovascular disease in relation to body composition, physical activity and assessment of nutritional status of adolescents. A literature review was performed in the following electronic databases: PUBMED, SCIELO and CONCHRANE COLLECTION. The following associated terms were used "inflammation AND cardiovascular diseases AND nutritional status OR body composition OR physical activity". There were topics created for the discussion of subjects: obesity and risk factors for cardiovascular disease during adolescence; expression of inflammatory markers in adolescence; development of cardiovascular disease with inflammatory markers, and finally, inflammatory markers, physical activity and nutritional evaluation. It was observed that the inflammatory markers may manifest in adolescence and be related to risk factors for cardiovascular diseases. Physical activity and nutritional evaluation featured as non-pharmacological measures to control the incidence of inflammatory markers and cardiovascular risk factor. Intervention studies may clarify how the adoption of a more proper lifestyle can influence the inflammatory process. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Glial Fibrillary Acidic Protein (GFAP) as a Mesenchymal marker of Early Hepatic Stellate Cells Activation in Liver Fibrosis in Chronic Hepatitis C Infection

PubMed Central

Hassan, Sobia; Syed, Serajuddaula; Kehar, Shahnaz Imdad

2014-01-01

Objective: This study aims to determine expression of Glial Fibrillary Acidic Protein and of Alpha Smooth Muscle Actin (α-SMA) in hepatic stellate cells of CHC cases and their association with stage of fibrosis. Methods: The study was conducted at Ziauddin University, Clifton Campus during the year 2010-2012. Sixty Chronic Hepatitis C cases were immmunostained using anti α-SMA antibody and anti-GFAP antibody. Semi quantitative scoring in pericentral, periportal and perisinusoidal area of each case was done to assess immunoexpression of each marker. Results : Immunoexpression of GFAP showed significant association with α-SMA. GFAP expression was inversely correlated with progression of fibrosis. Conclusion : GFAP could represent a useful marker for early hepatic stellate cells activation. Follow up biopsies showing decline in GFAP levels may help identify the target group requiring aggressive therapy. PMID:25225520

C-fos expression in the pons and medulla of the cat during carbachol-induced active sleep.

PubMed

Yamuy, J; Mancillas, J R; Morales, F R; Chase, M H

1993-06-01

Microinjection of carbachol into the rostral pontine tegmentum of the cat induces a state that is comparable to naturally occurring active (REM, rapid eye movement) sleep. We sought to determine, during this pharmacologically induced behavioral state, which we refer to as active sleep-carbachol, the distribution of activated neuron within the pons and medulla using c-fos immunocytochemistry as a functional marker. Compared with control cats, which were injected with saline, active sleep-carbachol cats exhibited higher numbers of c-fos-expressing neurons in (1) the medial and portions of the lateral reticular formation of the pons and medulla, (2) nuclei in the dorsolateral rostral pons, (3) various raphe nuclei, including the dorsal, central superior, magnus, pallidus, and obscurus, (4) the medial and lateral vestibular, prepositus hypoglossi, and intercalatus nuclei, and (5) the abducens nuclei. On the other hand, the mean number of c-fos-expressing neurons found in the masseter, facial, and hypoglossal nuclei was lower in carbachol-injected than in control cats. The data indicate that c-fos expression can be employed as a marker of state-dependent neuronal activity. The specific sites in which there were greater numbers of c-fos-expressing neurons during active sleep-carbachol are discussed in relation to the state of active sleep, as well as the functional role that these sites play in generating the various physiological patterns of activity that occur during this state.

Elevated amygdala activity to sad facial expressions: a state marker of bipolar but not unipolar depression.

PubMed

Almeida, Jorge R C; Versace, Amelia; Hassel, Stefanie; Kupfer, David J; Phillips, Mary L

2010-03-01

Difficulties in emotion processing and poor social function are common to bipolar disorder (BD) and major depressive disorder (MDD) depression, resulting in many BD depressed individuals being misdiagnosed with MDD. The amygdala is a key region implicated in processing emotionally salient stimuli, including emotional facial expressions. It is unclear, however, whether abnormal amygdala activity during positive and negative emotion processing represents a persistent marker of BD regardless of illness phase or a state marker of depression common or specific to BD and MDD depression. Sixty adults were recruited: 15 depressed with BD type 1 (BDd), 15 depressed with recurrent MDD, 15 with BD in remission (BDr), diagnosed with DSM-IV and Structured Clinical Interview for DSM-IV Research Version criteria; and 15 healthy control subjects (HC). Groups were age- and gender ratio-matched; patient groups were matched for age of illness onset and illness duration; depressed groups were matched for depression severity. The BDd were taking more psychotropic medication than other patient groups. All individuals participated in three separate 3T neuroimaging event-related experiments, where they viewed mild and intense emotional and neutral faces of fear, happiness, or sadness from a standardized series. The BDd-relative to HC, BDr, and MDD-showed elevated left amygdala activity to mild and neutral facial expressions in the sad (p < .009) but not other emotion experiments that was not associated with medication. There were no other significant between-group differences in amygdala activity. Abnormally elevated left amygdala activity to mild sad and neutral faces might be a depression-specific marker in BD but not MDD, suggesting different pathophysiologic processes for BD versus MDD depression. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates.

PubMed

Singh, Sukhi; Shams Hakimi, Caroline; Jeppsson, Anders; Hesse, Camilla

2017-12-01

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Contribution of Fetal, but Not Adult, Pulmonary Mesothelium to Mesenchymal Lineages in Lung Homeostasis and Fibrosis.

PubMed

von Gise, Alexander; Stevens, Sean M; Honor, Leah B; Oh, Jin Hee; Gao, Chi; Zhou, Bin; Pu, William T

2016-02-01

The lung is enveloped by a layer of specialized epithelium, the pulmonary mesothelium. In other organs, mesothelial cells undergo epithelial-mesenchymal transition and contribute to organ stromal cells. The contribution of pulmonary mesothelial cells (PMCs) to the developing lung has been evaluated with differing conclusions. PMCs have also been indirectly implicated in lung fibrosis in the progressive, fatal lung disease idiopathic pulmonary fibrosis. We used fetal or postnatal genetic pulse labeling of PMCs to assess their fate in murine development, normal lung homeostasis, and models of pulmonary fibrosis. We found that most fetal PMC-derived mesenchymal cells (PMCDCs) expressed markers of pericytes and fibroblasts, only a small minority expressed smooth muscle markers, and none expressed endothelial cell markers. Postnatal PMCs did not contribute to lung mesenchyme during normal lung homeostasis or in models of lung fibrosis. However, fetal PMCDCs were abundant and actively proliferating within fibrotic regions in lung fibrosis models, suggesting that they actively participate in the fibrotic process. These data clarify the role of fetal and postnatal PMCDCs in lung development and disease.

Time-course of changes in neuronal activity markers following iTBS-TMS of the rat neocortex.

PubMed

Hoppenrath, Kathrin; Funke, Klaus

2013-03-01

In a rat model of transcranial magnetic stimulation we could recently show that intermittent theta-burst stimulation (iTBS) affects the neocortical expression of the immediate early gene products c-Fos and zif268 as well as that of the two glutamic acid decarboxylase isoforms GAD65 and GAD67 and that of the calcium-binding proteins calbindin (CB) and parvalbumin (PV), known as markers of excitatory and inhibitory activity. We now analyzed in more detail the time course of changes in the expression of these proteins at 10, 20, 40, 80 and 160min following a single block of iTBS consisting of 600 stimuli. Initial increase in c-Fos, zif268 and GAD65 (20min) signals transient activation of excitatory and inhibitory neurons, thereafter first followed by a decrease in markers of activity of inhibitory neurons (GAD67, PV, CB: 20-80min) and then by a late decrease in c-Fos and GAD65 expression (160min). The results demonstrate that one iTBS block may have an after-effect of at least two different phases, an early phase with increased neuronal activity (c-Fos, zif268) but also the likelihood of increased GABA-release (GAD65), followed by a late phase (>40min) of reduced neuronal activity in excitatory and inhibitory systems which may indicate a state of reduced excitability. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas.

PubMed

Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-Sound; Ailles, Laurie; Moghal, Nadeem

2014-12-31

The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers like KRT5 and TP63. While some evidence suggests that basal cells are not all functionally equivalent, few heterogeneously expressed markers have been identified to purify and study subpopulations. In addition, few signaling pathways have been identified that regulate their cell behavior. The goals of this work were to investigate tracheal basal cell diversity and to identify new signaling pathways that regulate basal cell behavior. We used flow cytometry (FACS) to profile cell surface marker expression at a single cell level in primary human tracheal basal cell cultures that maintain stem cell/progenitor activity. FACS results were validated with tissue staining, in silico comparisons with normal basal cell and lung cancer datasets, and an in vitro proliferation assay. We identified 105 surface markers, with 47 markers identifying potential subpopulations. These subpopulations generally fell into more (~ > 13%) or less abundant (~ < 6%) groups. Microarray gene expression profiling supported the heterogeneous expression of these markers in the total population, and immunostaining of large airway tissue suggested that some of these markers are relevant in vivo. 24 markers were enriched in lung SQCCs relative to adenocarcinomas, with four markers having prognostic significance in SQCCs. We also identified 33 signaling receptors, including the MST1R/RON growth factor receptor, whose ligand MST1/MSP was mitogenic for basal cells. This work provides the largest description to date of molecular diversity among human large airway basal cells. Furthermore, these markers can be used to further study basal cell function in repair and disease, and may aid in the classification and study of SQCCs.

In vitro Production of IL-6 and IFN-γ is Influenced by Dietary Variables and Predicts Upper Respiratory Tract Infection Incidence and Severity Respectively in Young Adults

PubMed Central

Meng, Huicui; Lee, Yujin; Ba, Zhaoyong; Fleming, Jennifer A.; Furumoto, Emily J.; Roberts, Robert F.; Kris-Etherton, Penny M.; Rogers, Connie J.

2015-01-01

Assessment of immune responses in healthy adults following dietary or lifestyle interventions is challenging due to significant inter-individual variability. Thus, gaining a better understanding of host factors that contribute to the heterogeneity in immunity is necessary. To address this question, healthy adults [n = 36, 18–40 years old, body mass index (BMI) 20–35 kg/m2] were recruited. Dietary intake was obtained via 3-day dietary recall records, physical activity level was evaluated using the International Physical Activity Questionnaire, and peripheral blood mononuclear cells were isolated from peripheral blood. Expression of activation markers on unstimulated immune subsets was assessed by flow cytometry. T-cell proliferation and cytokine secretion was assessed following in vitro stimulation with anti-CD3 or lipopolysaccharide. Furthermore, the incidence and severity of cold or flu symptoms were obtained from self-reported upper respiratory tract infection (URTI) questionnaires. The relationship between activation marker expression on T cells and T-cell effector functions; and in vitro cytokine secretion and URTI was determined by linear or logistic regression. CD69 and CD25 expression on unstimulated T cells was significantly associated with T-cell proliferation and interleukin-2 secretion. Incidence and severity of cold or flu symptoms was significantly associated with in vitro interleukin-6 and interferon-gamma secretion, respectively. Furthermore, host factors (e.g., age, BMI, physical activity, and diet) contributed significantly to the relationship between activation marker expression and T-cell effector function, and cytokine secretion and cold and flu status. In conclusion, these results suggest that lifestyle and dietary factors are important variables that contribute to immune responses and should be included in human clinical trials that assess immune endpoints. PMID:25788896

Hypothalamic AMPK-induced autophagy ameliorates hypercatabolism in septic rats by regulating POMC expression.

PubMed

Cao, Chun; Gao, Tao; Cheng, Yan; Cheng, Minhua; Su, Ting; Xi, Fengchan; Wu, Cuili; Yu, Wenkui

2018-03-18

Hypercatabolism plays a critical role in the pathogenesis of post-critical care debility in critical patients. Central nervous system may exerte a critical role in the regulation of hypercatabolism. However, little is known about the exact mechanisms of the central role. Here, we reported that actived hypothalamic AMP-activated protein kinase (AMPK)-induced autophagy modulated the expression of POMC to ameliorate hypercatabolism in septic rats. Firstly, rats were i.c.v. injected with the lentiviral vector containing shRNA against POMC. Two weeks after injections, rats were intraperitoneally injected with LPS or saline. Twenty-four hours later, blood, skeletal muscle and hypothalamus tissues were obtained. Hypercatabolism markers and neuropeptides expression were detected. Then, rats were injected with AICAR or saline into third ventricle and promptly intraperitoneally injected with LPS or saline. Twenty-four hours after infection, blood, skeletal muscle and hypothalamus tissues were obtained. Hypercatabolism, hypothalamic AMPK-induced autophagy markers and neuropeptides expression were also detected. Results showed that sepsis would decrease the level of hypothalamic autophagy accompany with the alterations of POMC expression and hypercatabolism. Knocking out hypothalamus POMC expression could significantly ameliorate hypercatabolism. Moreover, Central activation of AMPK-induced autophagy pathway via third ventricle injection of AICAR, an AMPK activator, could efficiently ameliorate hypercatabolism as well as attenuate the elevated POMC expression rather than other neuropeptides. Taken together, these results suggested that hypothalamic AMPK-autophagy pathway as a regulatory pathway for POMC expression was essential for hypercatabolism during sepsis. And hypothalamic AMPK-autophagy activation could attenuate the POMC expression to ameliorate hypercatabolism. Pharmaceuticals with the ability of activating hypothalamic AMPK-autophagy pathway may be a therapeutic potential for hypercatabolism in septic patients. Copyright © 2018 Elsevier Inc. All rights reserved.

Keratoacanthoma of the Lip

PubMed Central

Dillenburg, Caroline Siviero; Martins, Manoela Domingues; Meurer, Luise; Castilho, Rogerio Moraes; Squarize, Cristiane Helena

2015-01-01

Abstract The PI3K-PTEN-mTOR is one of the most important pathways involved in cancer development and progression; however, its role in keratoacanthoma (KA) is poorly understood. In this study, we investigated the activation of key proteins in the PI3K-mTOR pathway in lip KA. We analyzed the activation of the PI3K-PTEN-mTOR pathway using human tumor samples stained for well-established protein markers in this pathway, including pS6 and pAKT phosphoproteins. We assessed proliferation using Ki-67 and performed additional morphological and immunohistochemical analysis using anti-PTEN and anti-p16 antibodies. We found that the majority of KA labeled to pS6 and not pAKT. PTEN expression was inversely correlated with Ki-67 expression. In addition to PTEN expression, KA cells were positive for p16Ink4 senescence marker. PI3K-PTEN-mTOR pathway is activated in lip KA, leading to downstream activation of mTORC1, but not mTORC2. This pathway plays an important role in KA progression by promoting proliferation and activation of oncogenic-induced senescence. PMID:26402814

Calcium-activated potassium channels as potential early markers of human cervical cancer

PubMed Central

Ramírez, Ana; Vera, Eunice; Gamboa-Domínguez, Armando; Lambert, Paul; Gariglio, Patricio; Camacho, Javier

2018-01-01

Cervical cancer is a major cause of cancer-associated mortality in women in developing countries. Thus, novel early markers are required. Ion channels have gained great interest as tumor markers, including cervical cancer. The calcium-activated potassium channel KCNMA1 (subunit α-1 from subfamily M) has been associated with different malignancies, including tumors such as breast and ovarian cancer that are influenced by hormones. The KCNMA1 channel blocker iberiotoxin decreases the proliferation of HeLa cervical cancer cells. Nevertheless, KCNMA1 channel expression during cervical carcinogenesis remains elusive. Therefore, KCNMA1 expression was studied in cervical cancer development. FVB transgenic mice expressing the E7-oncogene of high-risk human papilloma virus, and non-transgenic mice were treated with estradiol-releasing pellets during 3 or 6 months to induce cervical lesions. Twenty-four human cervical biopsies from non-cancerous, low- or high-grade intraepithelial lesions, or cervical cancer were also studied. mRNA and protein expression was assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. Cervical dysplasia and carcinoma were observed only in the transgenic mice treated with estradiol for 3 and 6 months, respectively. Estradiol treatment increased KCNMA1 mRNA and protein expression in all groups; however, the highest levels were observed in the transgenic mice with carcinoma. KCNMA1 protein expression in the squamous cells of the transformation zone was observed only in the transgenic mice with cervical dysplasia or cancer. Human biopsies from non-cancerous cervix did not display KCNMA1 protein expression; in contrast, the majority of the tissues with cervical lesions (16/18) displayed KCNMA1 protein expression. The lowest channel immunostaining intensity was observed in biopsies from low-grade dysplasia and the strongest in the carcinoma tissues. These results suggest KCNMA1 channels as potential early cervical cancer markers. PMID:29725443

Correlation of mRNA Expression and Signal Variability in Chronic Intracortical Electrodes.

PubMed

Falcone, Jessica D; Carroll, Sheridan L; Saxena, Tarun; Mandavia, Dev; Clark, Alexus; Yarabarla, Varun; Bellamkonda, Ravi V

2018-01-01

The goal for this research was to identify molecular mechanisms that explain animal-to-animal variability in chronic intracortical recordings. Microwire electrodes were implanted into Sprague Dawley rats at an acute (1 week) and a chronic (14 weeks) time point. Weekly recordings were conducted, and action potentials were evoked in the barrel cortex by deflecting the rat's whiskers. At 1 and 14 weeks, tissue was collected, and mRNA was extracted. mRNA expression was compared between 1 and 14 weeks using a high throughput multiplexed qRT-PCR. Pearson correlation coefficients were calculated between mRNA expression and signal-to-noise ratios at 14 weeks. At 14 weeks, a positive correlation between signal-to-noise ratio (SNR) and NeuN and GFAP mRNA expression was observed, indicating a relationship between recording strength and neuronal population, as well as reactive astrocyte activity. The inflammatory state around the electrode interface was evaluated using M1-like and M2-like markers. Expression for both M1-like and M2-like mRNA markers remained steady from 1 to 14 weeks. Anti-inflammatory markers, CD206 and CD163, however, demonstrated a significant positive correlation with SNR quality at 14 weeks. VE-cadherin, a marker for adherens junctions, and PDGFR-β, a marker for pericytes, both partial representatives of blood-brain barrier health, had a positive correlation with SNR at 14 weeks. Endothelial adhesion markers revealed a significant increase in expression at 14 weeks, while CD45, a pan-leukocyte marker, significantly decreased at 14 weeks. No significant correlation was found for either the endothelial adhesion or pan-leukocyte markers. A positive correlation between anti-inflammatory and blood-brain barrier health mRNA markers with electrophysiological efficacy of implanted intracortical electrodes has been demonstrated. These data reveal potential mechanisms for further evaluation to determine potential target mechanisms to improve consistency of intracortical electrodes recordings and reduce animal-to-animal/implant-to-implant variability.

Chronic crude garlic-feeding modified adult male rat testicular markers: mechanisms of action

PubMed Central

Hammami, Imen; Amara, Souheila; Benahmed, Mohamed; El May, Michèle V; Mauduit, Claire

2009-01-01

Background Garlic or Allium sativum (As) shows therapeutic effects such as reduction of blood pressure or hypercholesterolemia but side-effects on reproductive functions remain poorly investigated. Because of garlic's chemical complexity, the processing methods and yield in preparations differ in efficacy and safety. In this context, we clarify the mechanisms of action of crushed crude garlic on testicular markers. Methods During one month of treatment, 24 male rats were fed 5%, 10% and 15% crude garlic. Results We showed that crude garlic-feeding induced apoptosis in testicular germ cells (spermatocytes and spermatids). This cell death process was characterized by increased levels of active CASP3 but not CASP6. Expression of the caspase inhibitors BIRC3 and BIRC2 was increased at all doses of As while expression of XIAP and BIRC5 was unchanged. Moreover, expression of the IAP inhibitor DIABLO was increased at doses 10% and 15% of As. The germ cell death process induced by As might be related to a decrease in testosterone production because of the reduced expression of steroidogenic enzymes (Star, Cyp11a, Hsd3b5 and Hsd17b). Evaluation of Sertoli markers showed that TUBB3 and GSTA2 expression was unchanged. In contrast, AMH, RHOX5 and CDKN1B expression was decreased while GATA4 expression was increased. Conclusion In summary, we showed that feeding with crude garlic inhibited Leydig steroidogenic enzyme expression and Sertoli cell markers. These alterations might induce apoptosis in testicular germ cells. PMID:19552815

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis*

PubMed Central

Nelson, Shakira M.; Shay, Ashley E.; James, Jamaal L.; Carlson, Bradley A.; Urban, Joseph F.; Prabhu, K. Sandeep

2016-01-01

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trspfl/flCreWT mice that express selenoproteins driven by tRNASec (Trsp), whereas N. brasiliensis-infected Trspfl/flCreLysM selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ12-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses. PMID:26644468

Occludin as a functional marker of vascular endothelial cells on tube-forming activity.

PubMed

Kanayasu-Toyoda, Toshie; Ishii-Watabe, Akiko; Kikuchi, Yutaka; Kitagawa, Hiroko; Suzuki, Hiroko; Tamura, Hiroomi; Tada, Minoru; Suzuki, Takuo; Mizuguchi, Hiroyuki; Yamaguchi, Teruhide

2018-02-01

Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G 2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis. © 2017 Wiley Periodicals, Inc.

Aged keratinocyte phenotyping: morphology, biochemical markers and effects of Dead Sea minerals.

PubMed

Soroka, Yoram; Ma'or, Zeev; Leshem, Yael; Verochovsky, Lilian; Neuman, Rami; Brégégère, François Menahem; Milner, Yoram

2008-10-01

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging. We showed that the expression of 16 biochemical markers was altered in aged cultured cells and in tissues, including caspases 1 and 3 and beta-galactosidase activities, immunoreactivities of p16, Ki67, 20S proteasome and effectors of the Fas-dependent apoptotic pathway. Aged cells diversity, and individual variability of aging markers, call for a multifunctional assessment of the aging phenomenon, and of its modulation by drugs. As a test case, we have measured the effects of Dead Sea minerals on keratinocyte cultures and human skin, and found that they stimulate proliferation and mitochondrial activity, decrease the expression of some aging markers, and limit apoptotic damage after UVB irradiation.

The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells.

PubMed

Fan, J Z; Yang, X; Bi, Z G

2015-07-01

We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

PubMed Central

Fan, J.Z.; Yang, X.; Bi, Z.G.

2015-01-01

We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation. PMID:25923459

Normalized levels of red blood cells expressing phosphatidylserine, their microparticles, and activated platelets in young patients with β-thalassemia following bone marrow transplantation.

PubMed

Klaihmon, Phatchanat; Vimonpatranon, Sinmanus; Noulsri, Egarit; Lertthammakiat, Surapong; Anurathapan, Usanarat; Sirachainan, Nongnuch; Hongeng, Suradej; Pattanapanyasat, Kovit

2017-10-01

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with β-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young β-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in β-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.

BMP signaling and podocyte markers are decreased in human diabetic nephropathy in association with CTGF overexpression.

PubMed

Turk, Tamara; Leeuwis, Jan Willem; Gray, Julia; Torti, Suzy V; Lyons, Karen M; Nguyen, Tri Q; Goldschmeding, Roel

2009-07-01

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF(+/+) and CTGF(+/-) mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF(+/-) mice, pSmad1/5/8 was preserved, compared with diabetic CTGF(+/+) mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

PubMed

Alexander, Matthew R; Murgai, Meera; Moehle, Christopher W; Owens, Gary K

2012-04-02

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

Mitochondrial impairment, apoptosis and autophagy in a rat brain as immediate and long-term effects of perinatal phencyclidine treatment - influence of restraint stress.

PubMed

Jevtić, Gordana; Nikolić, Tatjana; Mirčić, Aleksandar; Stojković, Tihomir; Velimirović, Milica; Trajković, Vladimir; Marković, Ivanka; Trbovich, Alexander M; Radonjić, Nevena V; Petronijević, Nataša D

2016-04-03

Phencyclidine (PCP) acts as a non-competitive antagonist of glutamatergic N-methyl-d-aspartate receptor. Its perinatal administration to rats causes pathophysiological changes that mimick some pathological features of schizophrenia (SCH). Numerous data indicate that abnormalities in mitochondrial structure and function could be associated with the development of SCH. Mitochondrial dysfunction could result in the activation of apoptosis and/or autophagy. The aim of this study was to assess immediate and long-term effects of perinatal PCP administration and acute restraint stress on the activity of respiratory chain enzymes, expression of apoptosis and autophagy markers and ultrastructural changes in the cortex and hippocampus of the rat brain. Six groups of rats were subcutaneously treated on 2nd, 6th, 9th and 12th postnatal days (P), with either PCP (10mg/kg) or saline (0.9% NaCl). One NaCl and one PCP group were sacrificed on P13, while other two NaCl and PCP groups were sacrificed on P70. The remaining two NaCl and PCP groups were subjected to 1h restraint stress prior sacrifice on P70. Activities of respiratory chain enzymes were assessed spectrophotometrically. Expression of caspase 3 and AIF as markers of apoptosis and Beclin 1, p62 and LC3, as autophagy markers, was assessed by Western blot. Morphological changes of cortical and hippocampal ultrastructure were determined by transmission electron microscopy. Immediate effects of perinatal PCP administration at P13 were increased activities of complex I in the hippocampus and cytochrome c oxidase (COX) in the cortex and hippocampus implying mitochondrial dysfunction. These changes were followed by increased expression of apoptotic markers. However the measurement of autophagy markers at this time point has revealed decrease of this process in cortex and the absence of changes in hippocampus. At P70 the activity of complex I was unchanged while COX activity was significantly decreased in cortex and increased in the hippocampus. Expressions of apoptotic markers were still significantly higher in PCP perinatally treated rats in all investigated structures, but the changes of autophagy markers have indicated increased level of autophagy also in both structures. Restraint stress on P70 has caused increase of COX activity both in NaCl and PCP perinatally treated rats, but this increase was lower in PCP group. Also, restraint stress resulted in decrease of apoptotic and increase of autophagy processes especially in the hippocampus of PCP perinatally treated group. The presence of apoptosis and autophagy in the brain was confirmed by transmission electron microscopy. In this study we have demonstrated for the first time the presence of autophagy in PCP model of SCH. Also, we have shown increased sensitivity of PCP perinatally treated rats to restraint stress, manifested in alterations of apoptotic and autophagy markers. The future studies are necessary to elucidate the role of mitochondria in the pathophysiology of SCH and putative significance for development of novel therapeutic strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression

PubMed Central

Moyo, Nathifa A; Marchi, Emanuele; Steinbach, Falko

2013-01-01

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte–macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system. PMID:23461413

Aryl hydrocarbon receptor suppresses the osteogenesis of mesenchymal stem cells in collagen-induced arthritic mice through the inhibition of β-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Yulong; Niu, Menglin; Department of Blood Transfusion, Peking University Cancer Hospital & Institute, No. 52 Fucheng Rd., Beijing 100142

The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis (RA), particularly bone loss, have not been clearly explored. The imbalance between osteoblasts and osteoclasts is a major reason for bone loss. The dysfunction of osteoblasts, which are derived from mesenchymal stem cells (MSCs), induced bone erosion occurs earlier and is characterized as more insidious. Here, we showed that the nuclear expression and translocation of Ahr were both significantly increased in MSCs from collagen-induced arthritis (CIA) mice. The enhanced Ahr suppressed the mRNA levels of osteoblastic markers including Alkaline phosphatase (Alp) and Runt-related transcription factor 2 (Runx2)more » in the differentiation of MSCs to osteoblasts in CIA. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated activation of Ahr dose-dependently suppressed the expression of osteoblastic markers. In addition, the expression of β-catenin was reduced in CIA MSCs compared with control, and the TCDD-mediated activation of the Ahr significantly inhibited β-catenin expression. The Wnt3a-induced the activation of Wnt/β-catenin pathway partly rescued the osteogenesis decline induced by TCDD. Taken together, these results indicate that activated Ahr plays a negative role in CIA MSCs osteogenesis, possibly by suppressing the expression of β-catenin. - Highlights: • The Ahr pathway displays an activated profile in CIA MSCs. • The activation of Ahr suppresses osteogenesis in CIA MSCs. • TCDD suppresses osteogenesis in a dose-dependent manner. • The activation of Ahr inhibits β-catenin expression to exacerbate bone erosion.« less

Conditions in blood sampling procedures that extend the ex vivo stability of eosinophil activity markers in peripheral blood from allergic patients and healthy controls.

PubMed

Halldén, G; Nopp, A; Ihre, E; Peterson, C; Lundahl, J

1999-11-01

Serum-ECP, EG2-epitope on intracellular ECP and surface expression of CD9 and CD11b in peripheral blood eosinophils (PBE) are considered to be markers that mirror clinical parameters in allergic inflammation. The aim was to investigate the impact of the blood sampling procedure on PBE markers and to identify optimal conditions for extended pre-analysis storage. Blood, from healthy individuals and patients with allergic rhinitis/asthma, was collected in tubes with EDTA, citrate, or without anti-coagulant. The expression of EG2-epitope, CD9, and CD11b were analyzed in eosinophils and neutrophils after 1, 5, and 24 hours of storage at +4 degrees C, according to the FOG-method and flow cytometry. In vitro stimulation with fMLP/PMA was used for metabolic activity analysis and CD11b mobilization. Following a 1-hour clotting period at +20 to 22 degrees C, samples were stored at +4 degrees C and serum-ECP levels were measured. The EG2-epitope, serum-ECP, and CD9 were stable in samples from both healthy controls and allergic patients at all storage conditions. The EG2-epitope, serum-ECP and PBE count were significantly increased in the patient group, whereas no differences were observed in the expression of CD9 or CD11b. Both granulocytes and monocytes retained their metabolic activity for 24 hours. Neutrophils in citrate-blood increased their ability to respond to fMLP, as compared with EDTA-blood. In vitro analysis of selected activity markers and functional tests could be performed on granulocytes from both healthy individuals and allergic patients after 24 hours storage at +4 degrees C. The anticoagulant citrate seems to be preferable to EDTA when monocytes or CD11b expression are analyzed.

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

PubMed

Al-Adsani, Amani; Burke, Zoë D; Eberhard, Daniel; Lawrence, Katherine L; Shen, Chia-Ning; Rustgi, Anil K; Sakaue, Hiroshi; Farrant, J Mark; Tosh, David

2010-10-27

The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

The epithelial-mesenchymal transition generates cells with properties of stem cells.

PubMed

Mani, Sendurai A; Guo, Wenjun; Liao, Mai-Jing; Eaton, Elinor Ng; Ayyanan, Ayyakkannu; Zhou, Alicia Y; Brooks, Mary; Reinhard, Ferenc; Zhang, Cheng Cheng; Shipitsin, Michail; Campbell, Lauren L; Polyak, Kornelia; Brisken, Cathrin; Yang, Jing; Weinberg, Robert A

2008-05-16

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer

PubMed Central

Alvarez-Cubero, Maria J.; Nadal, Rosa; Sanchez-Rovira, Pedro; Salido, Marta; Rodríguez, María; García-Puche, Jose L.; Delgado-Rodriguez, Miguel; Solé, Francisco; García, Maria A.; Perán, Macarena; Rosell, Rafael; Marchal, Juan A.; Lorente, Jose A.

2014-01-01

Circulating tumor cells (CTCs) are frequently associated with epithelialmesenchymal transition (EMT). The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer. PMID:25277187

Altered Innate and Lymphocytic Immune Responses in Mouse Splenocytes Post-Flight

NASA Technical Reports Server (NTRS)

Hwang, ShenAn; Crucian, Brian E.; Sams, Clarence F.; Actor, Jeffrey K.

2011-01-01

Space flight is known to affect immune responses of astronauts and animals, decreasing lymphocytic responses to mitogenic stimuli, delayed typed hypersensitivity reactions, and T-cell activation. Despite changes in immune suppression, there are no reports of consistent adverse clinical events post flight. To further investigate the spectrum of affected immune responses, murine splenocytes were stimulated immediately post-shuttle flight (14 days on STS-135) with T-cell stimulators or toll-like receptor agonists. Comparisons were made to ground control splenocytes from age-matched mice. Cell phenotypes were assessed, as well as activation markers and associated cytokine production. The CD4+ population decreased with no concurrent decrease in CD8+ cells from shuttle mice post flight compared to ground controls. Regarding antigen presenting cell populations, the number of CD11c+ cells were slightly elevated post flight, compared to ground controls, with increased MHC Class I expression (I-A(sup b)) and no change in Class II expression (H-2K(sup b)). CD86+ populations were also significantly diminished. However, the decreased markers did not correlate with activity. Stimulation of splenocytes post flight showed significant increase in bead uptake, increased Class I expression, increased TNF-alpha and IL-6 production in response to TLR-2 (zymosan) and TLR-4 (LPS) agonists. While most activated (ConA or anti-CD3/anti-CD28) CD4+ cells showed markedly diminished responses (reduced IL-2 production), non-specific T cell responses to superantigen (SEA/SEB) increased post flight as determined by expression of early activation markers. Production of additional cytokines was also dysregulated postflight. Overall, persistent immune changes during space flight could represent unique clinical risks for exploration class missions. The consequences of pathogenic encounter remain an important concern that should be addressed.

NP001 regulation of macrophage activation markers in ALS: A phase I clinical and biomarker study

PubMed Central

MILLER, ROBERT G.; ZHANG, RONGZHEN; BLOCK, GILBERT; KATZ, JONATHAN; BAROHN, RICHARD; KASARSKIS, EDWARD; FORSHEW, DALLAS; GOPALAKRISHNAN, VIDHYA; MCGRATH, MICHAEL S.

2017-01-01

This is a phase I, placebo-controlled, single ascending dose safety and tolerability study of NP001 in patients with ALS. NP001 is a novel regulator of inflammatory macrophages and monocytes. As ALS progression is thought to be related to neuroinflammation, an additional objective of the study was to assess the effects of NP001 administration on monocyte activation markers. Thirty-two ALS patients were enrolled and received either placebo (eight) or one of four (six at each dose) ascending single i.v. doses (0.2, 0.8, 1.6 and 3.2 mg/kg NP001). Patients were monitored for safety, and blood monocyte immune activation markers CD16 and HLA-DR were assessed pre- and 24 h post-dosing. Changes from baseline were calculated. Results showed that NP001 was generally safe and well tolerated. Importantly, a single dose of NP001 caused a dose-dependent reduction in expression of monocyte CD16, a marker of monocyte activation/inflammation. Additionally, monocyte HLA-DR expression was also decreased in those patients with elevated values at baseline. In conclusion, these data indicate that NP001 has an acute effect on inflammatory monocytes in ALS patient blood. The potential for modulation of inflammation in the context of ALS disease progression will require further study with long-term follow-up. PMID:25192333

MMP‐2 and MMP‐14 Silencing Inhibits VEGFR2 Cleavage and Induces the Differentiation of Porcine Adipose‐Derived Mesenchymal Stem Cells to Endothelial Cells

PubMed Central

Almalki, Sami G.; Llamas Valle, Yovani

2017-01-01

Abstract The molecular mechanisms that control the ability of adipose‐derived mesenchymal stem cells (AMSCs) to remodel three‐dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme‐linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP‐2 and MMP‐14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP‐14 and MMP‐2 expression, and MMP2 enzyme activity. Silencing of MMP‐2 and MMP‐14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated‐low‐density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP‐2 and MMP‐14 in the re‐endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385–1398 PMID:28213979

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

PubMed

Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

2016-04-01

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Biomarkers for evaluation of mast cell and basophil activation.

PubMed

Kabashima, Kenji; Nakashima, Chisa; Nonomura, Yumi; Otsuka, Atsushi; Cardamone, Chiara; Parente, Roberta; De Feo, Giulia; Triggiani, Massimo

2018-03-01

Mast cells and basophils play a pathogenetic role in allergic, inflammatory, and autoimmune disorders. These cells have different development, anatomical location and life span but share many similarities in mechanisms of activation and type of mediators. Mediators secreted by mast cells and basophils correlate with clinical severity in asthma, chronic urticaria, anaphylaxis, and other diseases. Therefore, effective biomarkers to measure mast cell and basophil activation in vivo could potentially have high diagnostic and prognostic values. An ideal biomarker should be specific for mast cells or basophils, easily and reproducibly detectable in blood or biological fluids and should be metabolically stable. Markers of mast cell and basophil include molecules secreted by stimulated cells and surface molecules expressed upon activation. Some markers, such as histamine and lipid mediators are common to mast cells and basophils whereas others, such as tryptase and other proteases, are relatively specific for mast cells. The best surface markers of activation expressed on mast cells and basophils are CD63 and CD203. While these mediators and surface molecules have been associated to a variety of diseases, none of them fulfills requirements for an optimal biomarker and search for better indicators of mast cell/basophil activation in vivo is ongoing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Anti-tumorigenic action of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran: evidence for involvement of GPR30/EGFR signaling pathway.

PubMed

Chandra, V; Fatima, I; Saxena, R; Hussain, M K; Hajela, K; Sankhwar, P; Roy, B G; Chandna, S; Dwivedi, A

2013-05-01

The aim of the present study was to investigate the effect of non-steroidal, pure antiestrogenic benzopyran derivative i.e., 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) on the growth of human endometrial cancer cells in vivo and in vitro and to elucidate its mechanism of action. Cell proliferation was assayed by measuring the incorporation of 5'-bromo-2'-deoxyuridine in Ishikawa and primary endometrial cancer cells. The expression of proliferation and apoptotic markers was analyzed by immunoblotting. The effect of K-1 on GPR30-regulated proteins was analyzed by ELISA and by immunoblotting. Nude mice bearing subcutaneous implanted-Ishikawa tumors, were treated for 14days with K-1 (200μg/kg body weight/day/orally). The proliferation markers, GPR30-regulated proteins and apoptotic markers were analyzed by immunoblotting in tumor xenograft. The apoptotic effect of compound K-1 was determined by TUNEL assay. Compound K-1 inhibited proliferation of endometrial adenocarcinoma cells and decreased the expression of proliferation markers. It caused apoptosis by increasing the expression of apoptotic markers (NOXA, PUMAα) and reducing the expression of p-CREB and BclxL. Compound interfered with GPR30-regulated-EGFR activation, decreased p-ERK, p-c-jun, c-fos, cyclinD1 and c-myc expression. Treatment of tumor-bearing mice with K-1 resulted in a significant decrease in tumor volume and weight. Decreased expression of p-ERK and its downstream molecules and increased expression of apoptotic markers were observed in tumor in K-1 treated animals. Findings suggest the potent inhibitory effect of compound K-1 on endometrial cancer cellular growth (in-vitro) and on tumor size (in-vivo) which is mediated at least, in part, by interference with GPR30-signaling. Copyright © 2013 Elsevier Inc. All rights reserved.

Forskolin, a hedgehog signalling inhibitor, attenuates carbon tetrachloride-induced liver fibrosis in rats.

PubMed

El-Agroudy, Nermeen N; El-Naga, Reem N; El-Razeq, Rania Abd; El-Demerdash, Ebtehal

2016-11-01

Liver fibrosis is one of the leading causes of morbidity and mortality worldwide with very limited therapeutic options. Given the pivotal role of activated hepatic stellate cells in liver fibrosis, attention has been directed towards the signalling pathways underlying their activation and fibrogenic functions. Recently, the hedgehog (Hh) signalling pathway has been identified as a potentially important therapeutic target in liver fibrosis. The present study was designed to explore the antifibrotic effects of the potent Hh signalling inhibitor, forskolin, and the possible molecular mechanisms underlying these effects. Male Sprague-Dawley rats were treated with either CCl 4 and/or forskolin for 6 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed. Hepatic fibrosis was assessed by measuring α-SMA expression and collagen deposition by Masson's trichrome staining and hydroxyproline content. The effects of forskolin on oxidative stress markers (GSH, GPx, lipid peroxides), inflammatory markers (NF-κB, TNF-α, COX-2, IL-1β), TGF-β1 and Hh signalling markers (Ptch-1, Smo, Gli-2) were also assessed. Hepatic fibrosis induced by CCl 4 was significantly reduced by forskolin, as indicated by decreased α-SMA expression and collagen deposition. Forskolin co-treatment significantly attenuated oxidative stress and inflammation, reduced TGF-β1 levels and down-regulated mRNA expression of Ptch-1, Smo and Gli-2 through cAMP-dependent PKA activation. In our model, forskolin exerted promising antifibrotic effects which could be partly attributed to its antioxidant and anti-inflammatory effects, as well as to its inhibition of Hh signalling, mediated by cAMP-dependent activation of PKA. © 2016 The British Pharmacological Society.

Forskolin, a hedgehog signalling inhibitor, attenuates carbon tetrachloride‐induced liver fibrosis in rats

PubMed Central

El‐Agroudy, Nermeen N; El‐Naga, Reem N; El‐Razeq, Rania Abd

2016-01-01

Background and Purpose Liver fibrosis is one of the leading causes of morbidity and mortality worldwide with very limited therapeutic options. Given the pivotal role of activated hepatic stellate cells in liver fibrosis, attention has been directed towards the signalling pathways underlying their activation and fibrogenic functions. Recently, the hedgehog (Hh) signalling pathway has been identified as a potentially important therapeutic target in liver fibrosis. The present study was designed to explore the antifibrotic effects of the potent Hh signalling inhibitor, forskolin, and the possible molecular mechanisms underlying these effects. Experimental Approach Male Sprague‐Dawley rats were treated with either CCl4 and/or forskolin for 6 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed. Hepatic fibrosis was assessed by measuring α‐SMA expression and collagen deposition by Masson's trichrome staining and hydroxyproline content. The effects of forskolin on oxidative stress markers (GSH, GPx, lipid peroxides), inflammatory markers (NF‐κB, TNF‐α, COX‐2, IL‐1β), TGF‐β1 and Hh signalling markers (Ptch‐1, Smo, Gli‐2) were also assessed. Key Results Hepatic fibrosis induced by CCl4 was significantly reduced by forskolin, as indicated by decreased α‐SMA expression and collagen deposition. Forskolin co‐treatment significantly attenuated oxidative stress and inflammation, reduced TGF‐β1 levels and down‐regulated mRNA expression of Ptch‐1, Smo and Gli‐2 through cAMP‐dependent PKA activation. Conclusion and Implications In our model, forskolin exerted promising antifibrotic effects which could be partly attributed to its antioxidant and anti‐inflammatory effects, as well as to its inhibition of Hh signalling, mediated by cAMP–dependent activation of PKA. PMID:27590029

Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity.

PubMed

Saurer, Leslie; Rihs, Silvia; Birrer, Michèle; Saxer-Seculic, Nikolina; Radsak, Markus; Mueller, Christoph

2012-10-01

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory responses. We have previously demonstrated a substantial increase in TREM-1-expressing macrophages in the inflamed intestinal mucosa of patients with inflammatory bowel diseases (IBD). TREM-1 is also produced as a soluble receptor (sTREM-1). Here, we aimed to determine whether serum sTREM-1 could be used as a surrogate marker of disease activity in patients with IBD. Intestinal biopsies and concurrently collected sera from patients with Crohn's disease (CD) and Ulcerative colitis (UC) enrolled in the Swiss IBD cohort study were analyzed for intestinal TREM-1 mRNA and serum sTREM-1 expression. TREM-1 mRNA and sTREM-1 were correlated with the endoscopically determined disease activity. Serum sTREM-1 and TREM-1 mRNA expression levels were further determined in sera and colonic tissues collected at various time-points post disease induction in an experimental mouse model of colitis and correlated with disease activity. Expression of TREM-1 mRNA was upregulated in intestinal biopsies from patients with active disease but not in patients with quiescent disease. Serum sTREM-1 was elevated in IBD patients compared to normal controls. No substantial differences in sTREM-1 expression levels were found in patients with active versus quiescent disease. In colitic mice, colonic TREM-1 mRNA and serum sTREM-1 were also upregulated. While colonic TREM-1 mRNA expression levels correlated with disease activity, augmented serum sTREM-1 in fact associated with a milder course of disease. Analysis of sTREM-1 as a surrogate marker of disease activity in patients with IBD warrants caution. Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yanling; Xu, Sanpeng; Xiao, Fei

2010-05-28

Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of 'immune coagulation' and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as wellmore » as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.« less

Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production

PubMed Central

Fang, Wei-Yu; Chen, Yi-Wen; Hsiao, Jenn-Ren; Liu, Chiang-Shin; Kuo, Yi-Zih; Wang, Yi-Ching; Chang, Kung-Chao; Tsai, Sen-Tien; Chang, Mei-Zhu; Lin, Siao-Han; Wu, Li-Wha

2015-01-01

S100A9 is a calcium-binding protein with two EF-hands and frequently deregulated in several cancer types, however, with no clear role in oral cancer. In this report, the expression of S100A9 in cancer and adjacent tissues from 79 early-stage oral cancer patients was detected by immunohistochemical staining. Although S100A9 protein was present in both tumor and stromal cells, only the early-stage oral cancer patients with high stromal expression had reduced recurrence-free survival. High stromal S100A9 expression was also significantly associated with non-well differentiation and recurrence. In addition to increasing cell migration and invasion, ectopic S100A9 expression in tumor cells promoted xenograft tumorigenesis as well as the dominant expression of myeloid cell markers and pro-inflammatory IL-6. The expression of S100A9 in one stromal component, monocytes, stimulated the aggressiveness of co-cultured oral cancer cells. We also detected the elevation of serum S100A9 levels in early-stage oral cancer patients of a separate cohort of 73 oral cancer patients. The release of S100A9 protein into extracellular milieu enhanced tumor cell invasion, transendothelial monocyte migration and angiogenic activity. S100A9-mediated release of IL-6 requires the crosstalk of tumor cells with monocytes through the activation of NF-κB and STAT-3. Early-stage oral cancer patients with both high S100A9 expression and high CD68+ immune infiltrates in stroma had shortest recurrence-free survival, suggesting the use of both S100A9 and CD68 as poor prognostic markers for oral cancer. Together, both intracellular and extracellular S100A9 exerts a tumor-promoting action through the activation of oral cancer cells and their associated stroma in oral carcinogenesis. PMID:26315114

Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production.

PubMed

Fang, Wei-Yu; Chen, Yi-Wen; Hsiao, Jenn-Ren; Liu, Chiang-Shin; Kuo, Yi-Zih; Wang, Yi-Ching; Chang, Kung-Chao; Tsai, Sen-Tien; Chang, Mei-Zhu; Lin, Siao-Han; Wu, Li-Wha

2015-09-29

S100A9 is a calcium-binding protein with two EF-hands and frequently deregulated in several cancer types, however, with no clear role in oral cancer. In this report, the expression of S100A9 in cancer and adjacent tissues from 79 early-stage oral cancer patients was detected by immunohistochemical staining. Although S100A9 protein was present in both tumor and stromal cells, only the early-stage oral cancer patients with high stromal expression had reduced recurrence-free survival. High stromal S100A9 expression was also significantly associated with non-well differentiation and recurrence. In addition to increasing cell migration and invasion, ectopic S100A9 expression in tumor cells promoted xenograft tumorigenesis as well as the dominant expression of myeloid cell markers and pro-inflammatory IL-6. The expression of S100A9 in one stromal component, monocytes, stimulated the aggressiveness of co-cultured oral cancer cells. We also detected the elevation of serum S100A9 levels in early-stage oral cancer patients of a separate cohort of 73 oral cancer patients. The release of S100A9 protein into extracellular milieu enhanced tumor cell invasion, transendothelial monocyte migration and angiogenic activity. S100A9-mediated release of IL-6 requires the crosstalk of tumor cells with monocytes through the activation of NF-κB and STAT-3. Early-stage oral cancer patients with both high S100A9 expression and high CD68+ immune infiltrates in stroma had shortest recurrence-free survival, suggesting the use of both S100A9 and CD68 as poor prognostic markers for oral cancer. Together, both intracellular and extracellular S100A9 exerts a tumor-promoting action through the activation of oral cancer cells and their associated stroma in oral carcinogenesis.

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B.

PubMed

Hu, Wei; Lagarias, J Clark

2017-01-01

Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis (Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection, we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly sensitive reporters of off-target transgene expression. © 2017 American Society of Plant Biologists. All Rights Reserved.

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B1[OPEN

PubMed Central

2017-01-01

Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis (Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection, we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly sensitive reporters of off-target transgene expression. PMID:27881727

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

PubMed

Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

2013-12-01

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice

PubMed Central

Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E; Alkana, Ronald L; Tsukamoto, Hidekatsu; Davies, Daryl L.

2015-01-01

The present investigation tested the role of ATP-activated P2X7 receptors (P2X7Rs) in alcohol-induced brain damage using a model that combines intragastric (iG) ethanol feeding and high fat diet in C57BL/6J mice (Hybrid). The Hybrid paradigm caused increased levels of pro-inflammatory markers, changes in microglia and astrocytes, reduced levels of neuronal marker NeuN and increased P2X7R expression in ethanol-sensitive brain regions. Observed changes in P2X7R and NeuN expression were more pronounced in Hybrid paradigm with inclusion of additional weekly binges. In addition, high fat diet during Hybrid exposure aggravated the increase in P2X7R expression and activation of glial cells. PMID:26198936

PPARgamma is not a critical mediator of primary monocyte differentiation or foam cell formation.

PubMed

Patel, Lisa; Charlton, Steven J; Marshall, Ian C; Moore, Gary B T; Coxon, Phil; Moores, Kitty; Clapham, John C; Newman, Suzanna J; Smith, Stephen A; Macphee, Colin H

2002-01-18

In the present report we clarify the role of PPARgamma in differentiation and function of human-derived monocyte/macrophages in vitro. Rosiglitazone, a selective PPARgamma activator, had no effect on the kinetics of appearance of monocyte/macrophage differentiation markers or on cell size or granularity. Depletion of PPARgamma by more than 90% using antisense oligonucleotides did not influence accumulation of oxidized LDL or prevent the upregulation of CD36 that normally accompanies oxLDL treatment. In contrast, PPARgamma depletion reduced the expression of ABCA1 and LXRalpha mRNAs. Metalloproteinase-9 expression, a marker of atherosclerotic plaque vulnerability, was suppressed by rosiglitazone. We conclude that activation of PPARgamma does not affect monocyte/macrophage differentiation. In addition, PPARgamma is not absolutely required for oxLDL-driven lipid accumulation, but is required for full expression of ABCA1 and LXRalpha. Our data support a role for rosiglitazone as a potential directly acting antiatherosclerotic agent.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

PubMed

Kero, Darko; Kalibovic Govorko, Danijela; Medvedec Mikic, Ivana; Vukojevic, Katarina; Cigic, Livia; Saraga-Babic, Mirna

2015-10-01

To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of IGF-I and Protein Degradation Markers During Hindlimb Unloading and Growth Hormone Administration in Rats

NASA Astrophysics Data System (ADS)

Leinsoo, T. A.; Turtikova, O. V.; Shenkman, B. S.

2013-02-01

It is known that hindlimb unloading or spaceflight produce atrophy and a number of phenotypic alterations in skeletal muscles. Many of these processes are triggered by the axis growth hormone/insulin-like growth factor I. However growth hormone (GH) and insulin-like growth factor I (IGF-I) expression relationship in rodent models of gravitational unloading is weakly investigated. We supposed the IGF-I is involved in regulation of protein turnover. In this study we examined the IGF-I expression by RT-PCR assay in the rat soleus, tibialis anterior and liver after 3 day of hindlimb suspension with growth hormone administration. Simultaneously were studied expression levels of MuRF-1 and MAFbx/atrogin as a key markers of intracellular proteolysis. We demonstrated that GH administration did not prevent IGF-I expression decreasing under the conditions of simulated weightlessness. It was concluded there are separate mechanisms of action of GH and IGF-I on protein metabolism in skeletal muscles. Gravitational unloading activate proteolysis independently of growth hormone activity.

Antipsychotics increase vesicular glutamate transporter 2 (VGLUT2) expression in thalamolimbic pathways.

PubMed

Moutsimilli, Larissa; Farley, Severine; El Khoury, Marie-Anne; Chamot, Christophe; Sibarita, Jean-Baptiste; Racine, Victor; El Mestikawy, Salah; Mathieu, Flavie; Dumas, Sylvie; Giros, Bruno; Tzavara, Eleni T

2008-03-01

Recently the two vesicular-glutamate-transporters VGLUT1 and VGLUT2 have been cloned and characterized. VGLUT1 and VGLUT2 together label all glutamatergic neurons, but because of their distinct expression patterns in the brain they facilitate our ability to define between a VGLUT1-positive cortical and a VGLUT2-positive subcortical glutamatergic systems. We have previously demonstrated an increased cortical VGLUT1 expression as marker of antidepressant activity. Here, we assessed the effects of different psychotropic drugs on brain VGLUT2 mRNA and protein expression. The typical antipsychotic haloperidol, and the atypicals clozapine and risperidone increased VGLUT2 mRNA selectively in the central medial/medial parafascicular, paraventricular and intermediodorsal thalamic nuclei; VGLUT2 protein was accordingly amplified in paraventricular and ventral striatum and in prefrontal cortex. The antidepressants fluoxetine and desipramine and the sedative anxiolytic diazepam had no effect. These results highlight the implication of thalamo-limbic glutamatergic pathways in the action of antipsychotics. Increased VGLUT2 expression in these neurons might constitute a marker for antipsychotic activity and subcortical glutamate neurotransmission might be a possible novel target for future generation antipsychotics.

Evaluation of Accessory Lacrimal Gland in Muller's Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease.

PubMed

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H; Jassim Jaboori, Assraa; Setabutr, Pete; Aakalu, Vinay K

2017-04-01

The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Characterization of a Unique Cell Population Marked by Transgene Expression in the Adult Cochlea of Nestin-CreERT2/tdTomato-Reporter Mice

PubMed Central

Chow, Cynthia L.; Guo, Weixiang; Trivedi, Parul; Zhao, Xinyu; Gubbels, Samuel P.

2015-01-01

Hair cells in the adult mammalian cochlea cannot spontaneously regenerate after damage resulting in the permanency of hearing loss. Stem cells have been found to be present in the cochlea of young rodents; however, there has been little evidence for their existence into adulthood. We used nestin-CreERT2/tdTomato-reporter mice to trace the lineage of putative nestin-expressing cells and their progeny in the cochleae of adult mice. Nestin, an intermediate filament found in neural progenitor cells during early development and adulthood, is regarded as a multi-potent and neural stem cell marker. Other investigators have reported its presence in postnatal and young adult rodents; however, there are discrepancies amongst these reports. Using lineage tracing, we documented a robust population of tdTomato-expressing cells and evaluated these cells at a series of adult time points. Upon activation of the nestin promoter, tdTomato was observed just below and medial to the inner hair cell layer. All cells co-localized with the stem cell and cochlear-supporting-cell marker Sox2 as well as the supporting cell and Schwann cell marker Sox10; however, they did not co-localize with the Schwann cell marker Krox20, spiral ganglion marker NF200, or GFAP-expressing supporting cell marker. The cellular identity of this unique population of tdTomato-expressing cells in the adult cochlea of nestin-CreERT2/tdTomato mice remains unclear however these cells may represent a type of supporting cell on the neural aspect of the inner hair cell layer. PMID:25611038

Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells

PubMed Central

Hamatani, Hiroko; Sakairi, Toru; Takahashi, Satoshi; Watanabe, Mitsuharu; Maeshima, Akito; Ohse, Takamoto; Pippin, Jeffery W.; Shankland, Stuart J.; Nojima, Yoshihisa

2014-01-01

Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury. PMID:25056347

Immunohistochemical and Image Analysis-Based Study Shows That Several Immune Checkpoints are Co-expressed in Non-Small Cell Lung Carcinoma Tumors.

PubMed

Parra, Edwin Roger; Villalobos, Pamela; Zhang, Jiexin; Behrens, Carmen; Mino, Barbara; Swisher, Stephen; Sepesi, Boris; Weissferdt, Annika; Kalhor, Neda; Heymach, John Victor; Moran, Cesar; Zhang, Jianjun; Lee, Jack; Rodriguez-Canales, Jaime; Gibbons, Don; Wistuba, Ignacio I

2018-06-01

The understanding of immune checkpoint molecules' co-expression in non-small cell lung carcinoma (NCLC) is important to potentially design combinatorial immunotherapy approaches. We studied 225 formalin-fixed, paraffin-embedded tumor tissues from stage I-III NCLCs - 142 adenocarcinomas (ADCs) and 83 squamous cell carcinomas (SCCs) - placed in tissue microarrays. Nine immune checkpoint markers were evaluated; four (programmed death ligand 1 [PD-L1], B7-H3, B7-H4, and indoleamine 2,3-dioxygenase 1 [IDO-1]) expressed predominantly in malignant cells (MCs) and five (inducible T cell costimulator, V-set immunoregulatory receptor, T-cell immunoglobulin mucin family member 3, lymphocyte activating 3, and OX40) expressed mostly in stromal tumor-associated inflammatory cells (TAICs). All markers were examined using a quantitative image analysis and correlated with clinicopathologic features, TAICs, and molecular characteristics. Using above the median value as positive expression in MCs and high density of TAICs expressing those markers, we identified higher expression of immune checkpoints in SCC than ADC. Common simultaneous expression by MCs was PD-L1 + B7-H3 + IDO-1 in ADC and PD-L1 + B7-H3, or B7-H3 + B7-H4, in SCC. TAICs expressing checkpoint were significantly higher in current smokers than in never smokers. Almost all the immune checkpoint markers showed positive correlation with TAICs expressing inflammatory cell markers. KRAS-mutant ADC specimens showed higher expression of PD-L1 in MCs and of B7-H3, T-cell immunoglobulin mucin family member 3, and IDO-1 in TAICs than wild type. Kaplan-Meier survival curves showed worse prognosis in ADC patients with higher B7-H4 expression by MCs. We found frequent immunohistochemical co-expression of immune checkpoints in surgically resected NCLC tumors and correlated with tumor histology, smoking history, tumor size, and the density of inflammatory cells and tumor mutational status. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Effect of lifelong football training on the expression of muscle molecular markers involved in healthy longevity.

PubMed

Mancini, A; Vitucci, D; Labruna, G; Imperlini, E; Randers, M B; Schmidt, J F; Hagman, M; Andersen, T R; Russo, R; Orrù, S; Krustrup, P; Salvatore, F; Buono, P

2017-04-01

We investigated whether lifelong football training affects the expression of healthy longevity-related muscle molecular markers. Biopsies were collected from the vastus lateralis muscle of 10 lifelong football-trained men (68.2 ± 3.0 years) and of 10 active untrained healthy men (66.7 ± 1.3 years). Gene and protein expression was measured by RTqPCR on RNA and by western blotting on protein extracts from muscle biopsies, respectively. The expression of AMPKα1/α2, NAMPT, TFAM and PGC1α, which are markers of oxidative metabolism, and MyHC β isoform expression was higher in the muscle of football-trained men vs untrained men. Also citrate synthase activity was higher in trained than in untrained men (109.3 ± 9.2 vs 75.1 ± 9.2 mU/mg). These findings were associated with a healthier body composition in trained than in untrained men [body weight: 78.2 ± 6.5 vs 91.2 ± 11.2 kg; body mass index BMI: 24.4 ± 1.6 vs 28.8 ± 4.0 kg m -2 ; fat%: 22.6 ± 8.0 vs 31.4 ± 5.0%)] and with a higher maximal oxygen uptake (VO 2 max: 34.7 ± 3.8 vs 27.3 ± 4.0 ml/min/kg). Also the expression of proteins involved in DNA repair and in senescence suppression (Erk1/2, Akt and FoxM1) was higher in trained than in untrained men. At BMI- and age-adjusted multiple linear regression analysis, fat percentage was independently associated with Akt protein expression, and VO 2 max was independently associated with TFAM mRNA and with Erk1/2 protein expression. Lifelong football training increases the expression of key markers involved in muscle oxidative metabolism, and in the DNA repair and senescence suppression pathways, thus providing the molecular basis for healthy longevity.

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

PubMed

Jin, Hye Jin; Kwon, Ji Hye; Kim, Miyeon; Bae, Yun Kyung; Choi, Soo Jin; Oh, Wonil; Yang, Yoon Sun; Jeon, Hong Bae

2016-04-01

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy. ©AlphaMed Press.

Ligand-activated PPARδ upregulates α-smooth muscle actin expression in human dermal fibroblasts: A potential role for PPARδ in wound healing.

PubMed

Ham, Sun Ah; Hwang, Jung Seok; Yoo, Taesik; Lee, Won Jin; Paek, Kyung Shin; Oh, Jae-Wook; Park, Chan-Kyu; Kim, Jin-Hoi; Do, Jung Tae; Kim, Jae-Hwan; Seo, Han Geuk

2015-12-01

The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. PPARδ plays pivotal roles in wound healing by promoting fibroblast-to-myofibroblast differentiation via TGF-β/Smad3 signaling. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

LGR5/GPR49 is implicated in motor neuron specification in nervous system.

PubMed

Song, Shao-jun; Mao, Xing-gang; Wang, Chao; Han, An-guo; Yan, Ming; Xue, Xiao-yan

2015-01-01

The biological roles of stem cell marker LGR5, the receptor for the Wnt-agonistic R-spondins, for nervous system are poorly known. Bioinformatics analysis in normal human brain tissues revealed that LGR5 is closely related with neuron development and functions. Interestingly, LGR5 and its ligands R-spondins (RSPO2 and RSPO3) are specifically highly expressed in projection motor neurons in the spinal cord, brain stem and cerebral. Inhibition of Notch activity in neural stem cells (NSCs) increased the percentage of neuronal cells and promoted LGR5 expression, while activation of Notch signal decreased neuronal cells and inhibited the LGR5 expression. Furthermore, knockdown of LGR5 inhibited the expression of neuronal markers MAP2, NeuN, GAP43, SYP and CHRM3, and also reduced the expression of genes that program the identity of motor neurons, including Isl1, Lhx3, PHOX2A, TBX20 and NEUROG2. Our data demonstrated that LGR5 is highly expressed in motor neurons in nervous system and is involved in their development by regulating transcription factors that program motor neuron identity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells.

PubMed

Mihaylova, Ivana; DeRuyter, Marcel; Rummens, Jean-Luc; Bosmans, Eugene; Maes, Michael

2007-08-01

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.

Telomerase expression in the mammalian heart

PubMed Central

Richardson, Gavin D.; Breault, David; Horrocks, Grace; Cormack, Suzanne; Hole, Nicholas; Owens, W. Andrew

2012-01-01

While the mammalian heart has low, but functionally significant, levels of telomerase expression, the cellular population responsible remains incompletely characterized. This study aimed to identify the cell types responsible for cardiac telomerase activity in neonatal, adult, and cryoinjured adult hearts using transgenic mice expressing green fluorescent protein (GFP), driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate-limiting component of telomerase. A rare population of mTert-GFP-expressing cells was identified that possessed all detectable cardiac telomerase RNA and telomerase activity. It was heterogeneous and included cells coexpressing markers of cardiomyocytic, endothelial, and mesenchymal lineages, putative cardiac stem cell markers, and, interestingly, cardiomyocytes with a differentiated phenotype. Quantification using both flow cytometry and immunofluorescence identified a significant decline in mTert-GFP cells in adult animals compared to neonates (∼9- and ∼20-fold, respectively). Cardiac injury resulted in a ∼6.45-fold expansion of this population (P<0.005) compared with sham-operated controls. This study identifies the cells responsible for cardiac telomerase activity, demonstrates a significant diminution with age but a marked response to injury, and, given the relationship between telomerase activity and stem cell populations, suggests that they represent a potential target for further investigation of cardiac regenerative potential.—Richardson, G. D., Breault, D., Horrocks, G., Cormack, S., Hole, N., Owens, W. A. Telomerase expression in the mammalian heart. PMID:22919071

Brain Region–Specific Alterations in the Gene Expression of Cytokines, Immune Cell Markers and Cholinergic System Components during Peripheral Endotoxin–Induced Inflammation

PubMed Central

Silverman, Harold A; Dancho, Meghan; Regnier-Golanov, Angelique; Nasim, Mansoor; Ochani, Mahendar; Olofsson, Peder S; Ahmed, Mohamed; Miller, Edmund J; Chavan, Sangeeta S; Golanov, Eugene; Metz, Christine N; Tracey, Kevin J; Pavlov, Valentin A

2014-01-01

Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune–brain communication, including the impact of peripheral inflammation on brain region–specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1β, IL-6 and other cytokines and brain region–specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches. PMID:25299421

BMP Signaling and Podocyte Markers Are Decreased in Human Diabetic Nephropathy in Association With CTGF Overexpression

PubMed Central

Turk, Tamara; Leeuwis, Jan Willem; Gray, Julia; Torti, Suzy V.; Lyons, Karen M.; Nguyen, Tri Q.; Goldschmeding, Roel

2009-01-01

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF+/+ and CTGF+/− mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF+/− mice, pSmad1/5/8 was preserved, compared with diabetic CTGF+/+ mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1. (J Histochem Cytochem 57:623–631, 2009) PMID:19255250

The antidepressant-like effects of pioglitazone in a chronic mild stress mouse model are associated with PPARγ-mediated alteration of microglial activation phenotypes.

PubMed

Zhao, Qiuying; Wu, Xiaohui; Yan, Shuo; Xie, Xiaofang; Fan, Yonghua; Zhang, Jinqiang; Peng, Cheng; You, Zili

2016-10-04

Discoveries that microglia-mediated neuroinflammation is involved in the pathological process of depression provided a new strategy for novel antidepressant therapy. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor regulating inflammation and microglial polarization and, therefore, a potential target for resolving depressive disorders. Our hypothesis was that antidepressant effects could be achieved through anti-inflammatory and neuroprotective activities by PPARγ-dependent microglia-modulating agents. Chronic mild stress (CMS) treatment was performed on C57BL/6 mice for 6 weeks. After 3 weeks with the CMS procedure, depressive-like behaviors were evaluated by sucrose preference (SP), tail suspension test (TST), forced swimming test (FST), and locomotor activity. Pioglitazone was administered intragastrically once per day for 3 weeks at different doses. Neuroinflammatory cytokines were determined by real time-PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot. The activated microglial state was confirmed by immunohistochemistry. N9 microglial cells were subjected to lipopolysaccharide, pioglitazone, and GW9662 to discuss the phenotype of activated microglia by RT-PCR, ELISA, and western blot. It was demonstrated that the PPARγ agonist pioglitazone (2.5 mg/kg) ameliorated depression-like behaviors in CMS-treated mice, as indicated by body weight (BW), the SP test, the FST, and the TST. The amelioration of the depression was blocked by the PPARγ antagonist GW9662. The expression of M1 markers (IL-1β, IL-6, TNFα, iNOS, and CCL2) increased, and the gene expression of M2 markers (Ym1, Arg1, IL-4, IL-10, and TGFβ) decreased in the hippocampus of the stress-treated mice. Pioglitazone significantly inhibited the increased numbers and morphological alterations of microglia in the hippocampus, reduced the elevated expression of microglial M1 markers, and increased the downgraded expression of microglial M2 markers in C57BL/6 mice exposed to CMS. In an in vitro experiment, pioglitazone reversed the imbalance of M1 and M2 inflammatory cytokines, which is correlated with the inhibition of nuclear factor kB activation and is expressed in LPS-stimulated N9 microglial cells. We showed that pioglitazone administration induce the neuroprotective phenotype of microglia and ameliorate depression-like behaviors in CMS-treated C57BL/6 mice. These data suggested that the microglia-modulating agent pioglitazone present a beneficial choice for depression.

Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure.

PubMed

Ghafourian, Mehri; Karami, Najmeh; Khodadadi, Ali; Nikbakht, Roshan

2014-06-01

Recurrent spontaneous abortion (RSA) and in vitro fertilization (IVF) failure with unknown causes are the controversial issues that are probably related to the immune system. To compare circulating NK cells expressing activation and inhibition surface markers between patients with RSA and IVF failure with those of healthy multiparous and successful IVF control women, respectively. In this case-control study peripheral blood samples were collected from 43 patients who included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising of 36 normal multiparous women and seven women with successful IVF. The expression of CD69, CD94 and CD161 surface markers on CD56+NK cells were assessed using specific monoclonal antibodies by flowcytometry. The percentage of NK cells increased significantly in patients with RSA and in women with IVF failure in comparison to healthy multiparous and successful IVF control groups (p<0.001). The overall expression of CD69, CD94, CD161 were also increased significantly on NK cells in both patient groups compared to control groups (p<0.001). Elevated expression of CD69 and CD161 on NK cells can be considered as immunological risk markers in RSA and IVF failure. However, it is not clear if high expression of CD94 on peripheral blood NK cells is related to abnormal activity of endometrial NK cells.

Prognostic relevance of epithelial-mesenchymal transition and proliferation in surgically treated primary parotid gland cancer.

PubMed

Busch, Alina; Bauer, Larissa; Wardelmann, Eva; Rudack, Claudia; Grünewald, Inga; Stenner, Markus

2017-05-01

Cancer of the major salivary glands comprises a morphologically diverse group of rare tumours of largely unknown cause. Epithelial-mesenchymal transition (EMT) has been shown to play a significant prognostic role in various human cancers. The aim was to assess the expression of EMT markers in different histological subtypes of parotid gland cancer (PGC) and analyse their prognostic value. We examined 94 PGC samples (13 histological subtypes) for the expression of MIB-1, epithelial cadherin (E-cadherin), β-catenin, vimentin and cytokeratin 8/18 (CK8/18) by means of immunohistochemistry. The experimental findings were correlated with clinicopathological and survival parameters. We detected all analysed EMT and proliferation markers in specifically different constellations within the examined histological subtypes of PGC. We found high epithelial marker expressions (CK8/18, E-cadherin, membranous β-catenin) only in a distinct variety of carcinomas. A high proliferation rate (high MIB-1 expression) as well as a combination of high CK8/18 and low vimentin expression was associated with a significantly worse survival. Our findings indicate that activation of the EMT pathway is a relevant explanation for tumour progression in individual histological subtypes of malignant parotid gland lesions, but by far not in all. Evidence of EMT activation in PGC cannot be seen as an isolated prognostic factor. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation*

PubMed Central

Fortier, Anne-Marie; Asselin, Eric; Cadrin, Monique

2013-01-01

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression. PMID:23449973

Enhanced endothelial cell senescence by lithium-induced matrix metalloproteinase-1 expression.

PubMed

Struewing, Ian T; Durham, Samuel N; Barnett, Corey D; Mao, Catherine D

2009-06-26

Endothelial cell (EC) senescence and dysfunction occurring after chronic injury and inflammation are highly associated with the development and progression of cardiovascular diseases. However, the factors involved in the establishment of EC senescence remain poorly understood. We have previously shown that lithium, an inhibitor of glycogen synthase kinase (GSK)-3beta and activator of the Wnt/beta-catenin signaling pathway, induces an EC senescent-like phenotype. Herein, we show that lithium induces a rapid and pronounced up-regulation of the matrix metalloproteinase (MMP)-1, an inflammation and senescent cell marker, at the mRNA and protein levels, whereas the induction of two other senescent cell markers is either weak (interleukin-8) or delayed (plasminogen activator inhibitor-1). Lithium effect on MMP-1 expression is also specific among other MMPs and not mediated by GSK3beta inhibition. Lithium affects MMP-1 expression mainly at the transcriptional level but neither the AP1/Ets regulatory sites nor the redox sensitive (-1607/2G) site in MMP-1 promoter are involved in lithium-dependent MMP-1 regulation. However, down-regulation of p53, a target of lithium in EC, dampens both basal and lithium-induced MMP-1 expression, which further links MMP-1 up-regulation with the establishment of cell senescence. Although increased MMP-1 levels are usually associated with angiogenesis in enabled proliferative EC, the exogenous addition of activated MMP-1 on lithium- arrested EC increases the number of EC positive for the senescent-associated-beta-galactosidase marker. Conversely, down-regulation of MMP-1 expression by small interfering RNAs blunts the lithium-dependent increase in senescent-associated-beta-galactosidase positive cells. Altogether our data indicate that lithium-induced MMP-1 may participate in the reinforcement of EC senescence and reveal a novel mechanism for lithium-induced tissue remodeling.

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

PubMed Central

Rosiak, Kamila; Smolarz, Maciej; Stec, Wojciech J.; Peciak, Joanna; Grzela, Dawid; Winiecka-Klimek, Marta; Stoczynska-Fidelus, Ewelina; Krynska, Barbara; Piaskowski, Sylwester; Rieske, Piotr

2016-01-01

Background The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. Methods Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. Results Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. Conclusions Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells. PMID:27145078

Upregulation of CD147 Promotes Metastasis of Cholangiocarcinoma by Modulating the Epithelial-to-Mesenchymal Transitional Process.

PubMed

Dana, Paweena; Kariya, Ryusho; Vaeteewoottacharn, Kulthida; Sawanyawisuth, Kanlayanee; Seubwai, Wunchana; Matsuda, Kouki; Okada, Seiji; Wongkham, Sopit

2017-08-07

CD147 is a transmembrane protein that can induce the expression and activity of matrix metalloproteinases (MMPs). Expression of CD147 has been shown to potentiate cell migration, invasion, and metastasis of cancer. In this study, the critical role of CD147 in metastasis was elucidated using CD147-overexpressing cholangiocarcinoma (CCA) cells in vitro and in vivo. The molecular mechanism, demonstrated herein, supported the hypothesis that metastasis increased in CD147-overexpressing cells. Five CD147-overexpressing clones (Ex-CD147) were established from a low CD147-expressing CCA cell line, KKU-055, using lentivirus containing pReceiver-Lenti-CD147. The metastatic capability was determined using the tail vein injection mouse model and an in vitro 3D invasion assay. Liver colonization was assessed using anti-HLA class I immunohistochemistry. Adhesion abilities, cytoskeletal arrangements, MMP activities, the expressions of adhesion molecules, and epithelial-mesenchymal transitional markers were analyzed. All Ex-CD147 clones exhibited a high CD147 expression and high liver colonization in the tail vein-injected mouse model, whereas parental cells lacked this ability. Ex-CD147 clones exhibited metastatic phenotypes (i.e., an increase in F-actin rearrangement) and cell invasion and a decrease in cell adhesion. The molecular mechanisms were shown to be via the induction of MMP-2 activity and enhancement of epithelial-mesenchymal transitions. An increase in mesenchymal markers Slug, vimentin, and N-cadherin, and a decrease in epithelial markers E-cadherin and claudin-1, together with suppression of the adhesion molecule ICAM-1, were observed in the Ex-CD147 clones. Moreover, suppression of CD147 expression using siCD147 in two CCA cell lines with high CD147 expression significantly decreased cell migration and invasion of these CCA cells. These findings emphasize the essential role of CD147 in CCA metastasis and suggest CD147 as a promising target for the effective treatment of CCA.

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

PubMed

Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

2017-08-31

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Glucagon-like peptide-1 receptor expression on human eosinophils and its regulation of eosinophil activation.

PubMed

Mitchell, P D; Salter, B M; Oliveria, J P; El-Gammal, A; Tworek, D; Smith, S G; Sehmi, R; Gauvreau, G M; Butler, M; O'Byrne, P M

2017-03-01

Glucagon-like peptide-1 (GLP-1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP-1 also has immune modulatory roles. To measure the expression of the GLP-1 receptor (GLP-1R) on eosinophils and neutrophils in normal and asthmatic subjects and evaluate effects of a GLP-1 analog on eosinophil function. Peripheral blood samples were taken from 10 normal and 10 allergic asthmatic subjects. GLP-1R expression was measured on eosinophils and neutrophils. Subsequently, the asthmatic subjects underwent allergen and diluent inhalation challenges, and GLP-1R expression was measured. Purified eosinophils, collected from mild asthmatic subjects, were stimulated with lipopolysaccharide (LPS) and a GLP-1 analog to evaluate eosinophil cell activation markers CD11b and CD69 and cytokine (IL-4, IL-5, IL-8 and IL-13) production. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. Eosinophil, but not neutrophil, expression of GLP-1R is significantly higher in normal controls compared to allergic asthmatics. The expression of GLP-1R did not change on either eosinophils or neutrophils following allergen challenge. A GLP-1 analog significantly decreased the expression of eosinophil-surface activation markers following LPS stimulation and decreased eosinophil production of IL-4, IL-8 and IL-13, but not IL-5. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. A GLP-1 analog attenuates LPS-stimulated eosinophil activation. GLP-1 agonists may have additional adjunctive indications in treating persons with concomitant type 2 diabetes mellitus and asthma. © 2016 John Wiley & Sons Ltd.

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

PubMed

Mirza, Sameer; Rakha, Emad A; Alshareeda, Alaa; Mohibi, Shakur; Zhao, Xiangshan; Katafiasz, Bryan J; Wang, Jun; Gurumurthy, Channabasavaiah Basavaraju; Bele, Aditya; Ellis, Ian O; Green, Andrew R; Band, Hamid; Band, Vimla

2013-02-01

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients' outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.

Trichloroethylene perturbs HNF4a expression and activity in the developing chick heart.

PubMed

Harris, Alondra P; Ismail, Kareem A; Nunez, Martha; Martopullo, Ira; Lencinas, Alejandro; Selmin, Ornella I; Runyan, Raymond B

2018-03-15

Exposure to trichloroethylene (TCE) is linked to formation of congenital heart defects in humans and animals. Prior interactome analysis identified the transcription factor, Hepatocyte Nuclear Factor 4 alpha (HNF4a), as a potential target of TCE exposure. As a role for HNF4a is unknown in the heart, we examined developing avian hearts for HNF4a expression and for sensitivity to TCE and the HNF4a agonist, Benfluorex. In vitro analysis using a HNF4a reporter construct showed both TCE and HFN4a to be antagonists of HNF4a-mediated transcription at the concentrations tested. HNF4a mRNA is expressed transiently in the embryonic heart during valve formation and cardiac development. Embryos were examined for altered gene expression in the presence of TCE or Benfluorex. TCE altered expression of selected mRNAs including HNF4a, TRAF6 and CYP2C45. There was a transition between inhibition and induction of marker gene expression in embryos as TCE concentration increased. Benfluorex was largely inhibitory to selected markers. Echocardiography of exposed embryos showed reduced cardiac function with both TCE and Benfluorex. Cardiac contraction was reduced by 29% and 23%, respectively at 10 ppb. The effects of TCE and Benfluorex on autocrine regulation of HNF4a, selected markers and cardiac function argue for a functional interaction of TCE and HNF4a. Further, the dose-sensitive shift between inhibition and induction of marker expression may explain the nonmonotonic-like dose response observed with TCE exposure in the heart. Copyright © 2018 Elsevier B.V. All rights reserved.

Lifestyle impacts on the aging associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

PubMed Central

Song, Zhangfa; von Figura, Guido; Liu, Yan; Kraus, Johann M.; Torrice, Chad; Dillon, Patric; Rudolph-Watabe, Masami; Ju, Zhenyu; Kestler, Hans A.; Sanoff, Hanna; Rudolph, K. Lenhard

2010-01-01

Summary Cellular aging is characterised by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and that activation of DNA damage responses. There is some evidence the DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging associated expression of serum markers of DNA damage (CRAMP, EF-1α, Stathmin, n-acetyl-glucosaminidase, and chitinase) in comparison to other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging. PMID:20560902

Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

PubMed Central

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H.; Jaboori, Assraa Jassim; Setabutr, Pete; Aakalu, Vinay K.

2017-01-01

Purpose The accessory lacrimal glands (ALG) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods ALG tissues obtained from Muller’s Muscle Conjunctival Resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2 and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions Thus, we report for the first time that human ALG tissues contain precursor marker positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES and isolate candidate precursor cells from ALG tissue. PMID:27612554

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation.

PubMed

Gladka, Monika M; Molenaar, Bas; de Ruiter, Hesther; van der Elst, Stefan; Tsui, Hoyee; Versteeg, Danielle; Lacraz, Grègory P A; Huibers, Manon M H; van Oudenaarden, Alexander; van Rooij, Eva

2018-01-31

Background -Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods -Here, we present a method to obtain high quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results -After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression we were also able to identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and the injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton associated protein 4 ( Ckap4 ) as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Ckap4 inhibition in activated fibroblasts treated with TGFβ triggered a greater increase in the expression of genes related to activated fibroblasts compared to control, suggesting a role of Ckap4 in modulating fibroblast activation in the injured heart. Conclusions -Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

Vitamin D increases programmed death receptor-1 expression in Crohn’s disease

PubMed Central

Bendix, Mia; Greisen, Stinne; Dige, Anders; Hvas, Christian L.; Bak, Nina; Jørgensen, Søren P.; Dahlerup, Jens F.; Deleuran, Bent; Agnholt, Jørgen

2017-01-01

Background: Vitamin D modulates inflammation in Crohns disease (CD). Programmed death (PD)-1 receptor contributes to the maintenance of immune tolerance. Vitamin D might modulate PD-1 signalling in CD. Aim: To investigate PD-1 expression on T cell subsets in CD patients treated with vitamin D or placebo. Methods: We included 40 CD patients who received 1200 IU vitamin D3 for 26 weeks or placebo and eight healthy controls. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated at baseline and week 26. The expressions of PD-1, PD-L1, and surface activation markers were analysed by flow cytometry. Soluble PD-1 plasma levels were measured by ELISA. Results: PD-1 expression upon T cell stimulation was increased in CD4+CD25+int T cells in vitamin D treated CD patients from 19% (range 10 39%) to 29% (11 79%)(p = 0.03) compared with placebo-treated patients. Vitamin D treatment, but not placebo, decreased the expression of the T cell activation marker CD69 from 42% (31 62%) to 33% (19 - 54%)(p = 0.01). Soluble PD-1 levels were not influenced by vitamin D treatment. Conclusions: Vitamin D treatment increases CD4+CD25+int T cells ability to up-regulate PD-1 in response to activation and reduces the CD69 expression in CD patients. PMID:28412753

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marengo, Barbara; Bottini, Consuelo; La Porta, C.A.M.

Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme that converts phosphatidylethanolamine (PE) into phosphatidylcholine. We have previously shown that PEMT suppressed hepatoma growth by triggering apoptosis. We investigate whether PEMT controlled cell death and cell proliferation triggered by fasting/refeeding and whether it is a marker of early preneoplastic lesions. The induction of programmed cell death and suppression of cell proliferation by fasting were associated with enhanced PEMT expression and activity, and with a decrease in CTP:phosphocholine cytidylyltransferase expression. Refeeding returned the liver growth and expression of CTP:phosphocholine cytidylyltransferase to control levels, while the expression of PEMT decreased to below control values. Aftermore » DENA administration, PEMT protein, evaluated by Western blotting, slightly increased, but it remained below control levels. The treatment with 20 mg/kg DENA to refed rats induced the appearance of initiated hepatocytes that were negative for PEMT expression. Present findings indicate that PEMT is a novel tumour marker for early liver preneoplastic lesions.« less

Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

PubMed Central

Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

2011-01-01

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis

PubMed Central

Wormley, Floyd L.; Chaiban, Joseph; Fidel, Paul L.

2001-01-01

Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection. PMID:11447188

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

New therapeutic aspect for carvedilol: Antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamdy, Nadia; El-Demerdash, Ebtehal, E-mail: ebtehal_dm@yahoo.com

2012-06-15

Portal hypertension is a common complication of chronic liver diseases associated with liver fibrosis and cirrhosis. At present, beta-blockers such as carvedilol remain the medical treatment of choice for protection against variceal bleeding and other complications. Since carvedilol has powerful antioxidant properties we assessed the potential antifibrotic effects of carvedilol and the underlying mechanisms that may add further benefits for its clinical usefulness using a chronic model of carbon tetrachloride (CCl4)-induced hepatotoxicity. Two weeks after CCl4 induction of chronic hepatotoxicity, rats were co-treated with carvedilol (10 mg/kg, orally) daily for 6 weeks. It was found that treatment of animals withmore » carvedilol significantly counteracted the changes in liver function and histopathological lesions induced by CCl4. Also, carvedilol significantly counteracted lipid peroxidation, GSH depletion, and reduction in antioxidant enzyme activities; glutathione-S-transferase and catalase that was induced by CCl4. In addition, carvedilol ameliorated the inflammation induced by CCl4 as indicated by reducing the serum level of acute phase protein marker; alpha-2-macroglobulin and the liver expression of nuclear factor-kappa B (NF-κB). Finally, carvedilol significantly reduced liver fibrosis markers including hydroxyproline, collagen accumulation, and the expression of the hepatic stellate cell (HSC) activation marker; alpha smooth muscle actin. In conclusion, the present study provides evidences for the promising antifibrotic effects of carvedilol that can be explained by amelioration of oxidative stress through mainly, replenishment of GSH, restoration of antioxidant enzyme activities and reduction of lipid peroxides as well as amelioration of inflammation and fibrosis by decreasing collagen accumulation, acute phase protein level, NF-κB expression and finally HSC activation. -- Highlights: ► Carvedilol is a beta blocker with antioxidant and antifibrotic properties. ► It restores GSH and antioxidant enzyme activities and reduces lipid peroxidation. ► It ameliorates inflammation and nuclear factor kappa-B expression. ► It ameliorates fibrosis by decreasing collagen accumulation and HSC activation.« less

Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts.

PubMed

Zhang, Yu; Yang, Jian-Hong

2013-11-01

Diabetes mellitus is associated with increased risk of osteopenia and bone fracture that may be related to hyperglycemia. However, the mechanisms accounting for diabetic bone disorder are unclear. Here, we showed that high glucose significantly promoted the production of reactive oxygen species (ROS) in rat primary osteoblasts. Most importantly, we reported for the first time that ROS induced by high glucose increased alkaline phosphatase activity, inhibited type I collagen (collagen I) protein level and cell mineralization, as well as gene expression of osteogenic markers including runt-related transcription factor 2 (Runx2), collagen I, and osteocalcin, but promoted lipid droplet formation and gene expression of adipogenic markers including peroxisome proliferator-activated receptor gamma, adipocyte fatty acid binding protein (aP2), and adipsin, which were restored by pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, high glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation but stimulated adipogenic differentiation. In contrast, NAC and a PI3K inhibitor, LY-294002, reversed the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of Akt under high glucose. These results indicated that oxidative stress played a key role in high glucose-induced increase of adipogenic differentiation, which contributed to the inhibition of osteogenic differentiation. This process was mediated by PI3K/Akt pathway in rat primary osteoblasts. Hence, suppression of oxidative stress could be a potential therapeutic approach for diabetic osteopenia. © 2013 Wiley Periodicals, Inc.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

PubMed

Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

2017-05-01

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289. © 2017 AlphaMed Press.

Interferon-γ regulates the function of mesenchymal stem cells from oral lichen planus via indoleamine 2,3-dioxygenase activity.

PubMed

Zhang, Zhihui; Han, Ying; Song, Jiangyuan; Luo, Ruxi; Jin, Xin; Mu, Dongdong; Su, Sha; Ji, Xiaoli; Ren, Yan-Fang; Liu, Hongwei

2015-01-01

Little is known about mesenchymal stem cells (MSCs) in normal or inflammatory oral mucosal tissues, such as in oral lichen planus (OLP). Our objectives were to identify, isolate, and characterize MSCs from normal human oral mucosa and OLP lesions, and to evaluate indoleamine 2,3 dioxygenase (IDO) activity in mediating immunomodulation of MSCs from these tissues. Expressions of MSCs-related markers were examined in isolated cells by flow cytometry. Self-renewal and multilineage differentiations were studied to characterize these MSCs. Interferon-γ (IFN-γ), IDO, and STRO-1 were assessed by immunofluorescence. MSCs from oral mucosa and OLP or IFN-γ-pretreated MSCs were co-cultured with allogeneic mixed lymphocyte reaction assays (MLR). Proliferation and apoptosis of MLR or MSCs were detected by CCK8 and the annexin V-FITC apoptosis detection kit, respectively. IDO expression and activity were measured by real-time PCR, Western blotting, and high-performance liquid chromatography. Isolated cells from oral mucosa and OLP expressed MSC-related markers STRO-1, CD105, and CD90 but were absent for hematopoietic stem cell markers CD34. Besides, they all showed self-renewal and multilineage differentiation capacities. MSCs in OLP presented STRO-1/IDO+ phenotype by immunofluorescence. MSCs and IFN-γ-pretreated MSCs could inhibit lymphocyte proliferation via IDO activity, but not via cell apoptosis. Long-term IFN-γ could also inhibit MSC proliferation via IDO activity. Mesenchymal stem cells can be isolated from human oral mucosa and OLP tissues. Besides self-renewal and multilineage differentiation properties, these cells may participate in immunomodulation mediated by IFN-γ via IDO activity in human OLP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule.

PubMed

Witek, Małgorzata A; Aufforth, Rachel D; Wang, Hong; Kamande, Joyce W; Jackson, Joshua M; Pullagurla, Swathi R; Hupert, Mateusz L; Usary, Jerry; Wysham, Weiya Z; Hilliard, Dawud; Montgomery, Stephanie; Bae-Jump, Victoria; Carey, Lisa A; Gehrig, Paola A; Milowsky, Matthew I; Perou, Charles M; Soper, John T; Whang, Young E; Yeh, Jen Jen; Martin, George; Soper, Steven A

2017-01-01

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.

FoxP3 Expression in Macrophages, Cancer, and B Cells-Is It Real?

PubMed

Vadasz, Zahava; Toubi, Elias

2017-06-01

During the last decade, B regulatory cells are appreciated to have a central role in preventing autoimmunity and maintaining self-tolerance. They are characterized by expressing different phenotypic markers and the production of either IL-10 or TGF-β or both. The recent recognition of Fas ligand expressing B regulatory cells as "killer" cells established their role in maintaining viral persistence by preventing effective antiviral immune responses. The forkhead lineage-transcription factor (FoxP3) was considered for many years to be a highly specific intracellular regulatory marker of CD4+CD25+ T regulatory cells. The possibility of FoxP3 being expressed in B regulatory cells was suggested in many studies. Though controversial, FoxP3 expression was also reported in macrophages and cancer cells. Aiming to avoid artifact staining, many researchers required the usage of FoxP3 messenger RNA (mRNA) and PCR in order to prove a true expression of FoxP3 in these different cells. In addition, most studies' report on that FoxP3 expression in all abovementioned cells is related to their status of activation since naïve (non-activated cells) were found poorly FoxP3 expressing. In this review, we present the existing data on FoxP3 expression in non-T-regulatory cells, but we suggest that further studies are needed to better establish this concept.

LPL is the strongest prognostic factor in a comparative analysis of RNA-based markers in early chronic lymphocytic leukemia.

PubMed

Kaderi, Mohd Arifin; Kanduri, Meena; Buhl, Anne Mette; Sevov, Marie; Cahill, Nicola; Gunnarsson, Rebeqa; Jansson, Mattias; Smedby, Karin Ekström; Hjalgrim, Henrik; Jurlander, Jesper; Juliusson, Gunnar; Mansouri, Larry; Rosenquist, Richard

2011-08-01

The expression levels of LPL, ZAP70, TCL1A, CLLU1 and MCL1 have recently been proposed as prognostic factors in chronic lymphocytic leukemia. However, few studies have systematically compared these different RNA-based markers. Using real-time quantitative PCR, we measured the mRNA expression levels of these genes in unsorted samples from 252 newly diagnosed chronic lymphocytic leukemia patients and correlated our data with established prognostic markers (for example Binet stage, CD38, IGHV gene mutational status and genomic aberrations) and clinical outcome. High expression levels of all RNA-based markers, except MCL1, predicted shorter overall survival and time to treatment, with LPL being the most significant. In multivariate analysis including the RNA-based markers, LPL expression was the only independent prognostic marker for overall survival and time to treatment. When studying LPL expression and the established markers, LPL expression retained its independent prognostic strength for overall survival. All of the RNA-based markers, albeit with varying ability, added prognostic information to established markers, with LPL expression giving the most significant results. Notably, high LPL expression predicted a worse outcome in good-prognosis subgroups, such as patients with mutated IGHV genes, Binet stage A, CD38 negativity or favorable cytogenetics. In particular, the combination of LPL expression and CD38 could further stratify Binet stage A patients. LPL expression is the strongest RNA-based prognostic marker in chronic lymphocytic leukemia that could potentially be applied to predict outcome in the clinical setting, particularly in the large group of patients with favorable prognosis.

Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*

PubMed Central

G. Lavoie, Elise; Dranoff, Jonathan A.

2017-01-01

Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression. PMID:28898276

Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

PubMed Central

Krönig, Malte; Walter, Max; Drendel, Vanessa; Werner, Martin; Jilg, Cordula A.; Richter, Andreas S.; Backofen, Rolf; McGarry, David; Follo, Marie; Schultze-Seemann, Wolfgang; Schüle, Roland

2015-01-01

A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. PMID:25514598

Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter

PubMed Central

Peshdary, Vian

2017-01-01

Firemaster® 550 (FM550) is a chemical mixture currently used as an additive flame retardant in commercial products, and is comprised of 2-ethylhexyl-2,3,4,5-tertrabromobenzoate (TBB), bis(2-ethylhexyl) tetrabromophthalate (TBPH), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (IPTP). Animal and in vitro studies suggest that FM550, TPP and IPTP may have adipogenic effects and may exert these effects through PPARγ activation. Using murine 3T3-L1 preadipocytes, we investigated the detailed expression of transcription factors and adipogenic markers in response to FM550 and its components. Further we investigated the mechanism of action of the peroxisome proliferator-activated receptor gamma (PPARγ) on downstream targets of the receptor by focussing on the mature adipocyte marker, adipocyte protein 2 (aP2). In addition, we set to elucidate the components responsible for the adipogenic effects seen in the FM550 mixture. We show that FM550 and its components TPP, IPTP, and TBPH, but not TBB induced lipid accumulation in a dose-dependent manner. Interestingly, despite displaying enhanced lipid accumulation, TBPH did not alter the mRNA or protein expression of terminal differentiation markers. In contrast, FM550, TPP, and IPTP treatment enhanced lipid accumulation, and mRNA and protein expression of terminal differentiation markers. To further delineate the mechanisms of action of FM550 and its components we focussed on aP2 promoter activity. For this purpose we used the enhancer region of the mouse aP2 promoter using a 584-bp reporter construct containing an active PPRE located 5.4 kb away from the transcription start site of aP2. Exposure to FM550, IPTP, and TPP significantly increased PPARγ mediated aP2 enhancer activity. Furthermore, we show that TPP- and IPTP-dependent upregulation of aP2 was significantly inhibited by the selective PPARγ antagonist GW9662. In addition, chromatin immunoprecipitation experiments showed that IPTP and TPP treatment led to the recruitment of PPARγ to the regulatory region of aP2. PMID:28437481

Activation status of mucosal-associated invariant T cells reflects disease activity and pathology of systemic lupus erythematosus.

PubMed

Chiba, Asako; Tamura, Naoto; Yoshikiyo, Kazunori; Murayama, Goh; Kitagaichi, Mie; Yamaji, Ken; Takasaki, Yoshinari; Miyake, Sachiko

2017-03-14

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes constituting a large proportion of peripheral blood T cells expressing αβ T-cell receptor in humans. In this study, we aimed to investigate their involvement in systemic lupus erythematosus (SLE). Peripheral blood MAIT cells from patients with SLE were assessed for their frequency, activation markers, and cell death by flow cytometry. The correlation between plasma cytokine levels and CD69 expression on MAIT cells was analyzed. The major histocompatibility complex class I-related protein MR1-restricted antigen-presenting capacity of antigen-presenting cells was investigated. Cytokine-mediated activation of MAIT cells in the absence of exogenous antigens was also examined. The frequency of MAIT cells was markedly reduced in SLE. The reduced number of MAIT cells was not attributable to the downregulation of surface markers, but it was partially due to the enhanced cell death of MAIT cells, possibly by activation-induced cell death. The CD69 expression levels on MAIT cells in SLE correlated with disease activity. Moreover, monocytes from patients with SLE exhibited increased ability to induce MAIT cell activation. The plasma concentration of interleukin (IL)-6, IL-18, and interferon (IFN)-α positively correlated with the expression levels of CD69 on MAIT cells in SLE. MAIT cells were activated by cytokines, including IFN-α, IL-15, and IL-12 plus IL-18, in the absence of exogenous antigens. These results suggest that MAIT cells reflect the pathological condition of SLE and that their activated status correlates with presence of disease.

Selective cortical VGLUT1 increase as a marker for antidepressant activity.

PubMed

Moutsimilli, Larissa; Farley, Severine; Dumas, Sylvie; El Mestikawy, Salah; Giros, Bruno; Tzavara, Eleni T

2005-11-01

The two recently characterized vesicular glutamate transporters (VGLUT) presynaptically mark and differentiate two distinct excitatory neuronal populations and thus define a cortical and a subcortical glutamatergic system (VGLUT1 and VGLUT2 positive, respectively). These two systems might be differentially implicated in brain neuropathology. Still, little is known on the modalities of VGLUT1 and VGLUT2 regulations in response to pharmacological or physiological stimuli. Given the importance of cortical neuronal activity in psychosis we investigated VGLUT1 mRNA and protein expression in response to chronic treatment with commonly prescribed psychotropic medications. We show that agents with antidepressant activity, namely the antidepressants fluoxetine and desipramine, the atypical antipsychotic clozapine, and the mood stabilizer lithium increased VGLUT1 mRNA expression in neurons of the cerebral cortex and the hippocampus and in concert enhanced VGLUT1 protein expression in their projection fields. In contrast the typical antipsychotic haloperidol, the cognitive enhancers memantine and tacrine, and the anxiolytic diazepam were without effect. We suggest that VGLUT1 could be a useful marker for antidepressant activity. Furthermore, adaptive changes in VGLUT1 positive neurons could constitute a common functional endpoint for structurally unrelated antidepressants, representing promising antidepressant targets in tracking specificity, mechanism, and onset at action.

A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells

PubMed Central

Guo, Xuan; Wang, Shu; Dou, Ya-ling; Guo, Xiang-fei; Chen, Zhao-li; Wang, Xin-wei; Shen, Zhi-qiang; Qiu, Zhi-gang

2015-01-01

Abstract Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies. PMID:25556695

A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.

PubMed

Guo, Xuan; Wang, Shu; Dou, Ya-ling; Guo, Xiang-fei; Chen, Zhao-li; Wang, Xin-wei; Shen, Zhi-qiang; Qiu, Zhi-gang; Jin, Min; Li, Jun-wen

2015-06-01

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

PubMed

Martinez, Fernando O; Helming, Laura; Milde, Ronny; Varin, Audrey; Melgert, Barbro N; Draijer, Christina; Thomas, Benjamin; Fabbri, Marco; Crawshaw, Anjali; Ho, Ling Pei; Ten Hacken, Nick H; Cobos Jiménez, Viviana; Kootstra, Neeltje A; Hamann, Jörg; Greaves, David R; Locati, Massimo; Mantovani, Alberto; Gordon, Siamon

2013-02-28

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis

PubMed Central

Goettsch, Claudia; Babelova, Andrea; Trummer, Olivia; Erben, Reinhold G.; Rauner, Martina; Rammelt, Stefan; Weissmann, Norbert; Weinberger, Valeska; Benkhoff, Sebastian; Kampschulte, Marian; Obermayer-Pietsch, Barbara; Hofbauer, Lorenz C.; Brandes, Ralf P.; Schröder, Katrin

2013-01-01

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4–/– mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis. PMID:24216508

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

PubMed

Sessa, F; Bonato, M; Frigerio, B; Capella, C; Solcia, E; Prat, M; Bara, J; Samloff, I M

1990-06-01

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dongjing; Wu, Jilin, E-mail: 6296082@qq.com; Liu, Meizhou

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulatedmore » miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the carcinogenesis and progression of HCC. • Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.« less

Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta

PubMed Central

Boisset, Jean-Charles; Clapes, Thomas; Van Der Linden, Reinier; Dzierzak, Elaine; Robin, Catherine

2013-01-01

Summary Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The αIIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. αIIb pairs exclusively with β3 to form the αIIbβ3 integrin. β3 (CD61) also pairs with αv (CD51) to form the αvβ3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express αIIbβ3 and αvβ3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express αvβ3, and most fetal liver HSCs do not express either integrin. By using αIIb deficient embryos, we show that αIIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta. PMID:23789102

Development of paratransgenic Artemia as a platform for control of infectious diseases in shrimp mariculture.

PubMed

Subhadra, B; Hurwitz, I; Fieck, A; Rao, D V S; Rao, G Subba; Durvasula, R

2010-03-01

To study the accumulation and retention of recombinant proteins in Artemia gut for optimizing paratransgenic disease control in shrimp aquaculture. Transgenic Escherichia coli expressing fluorescent marker proteins and the transgenic cyanobacterium Synechococcus bacillarus expressing a functional murine single chain antibody, DB3, were fed to Artemia franciscana. Stable expression and retention of several marker molecules (e.g. GFP, DS Red and DB3) up to 10 h after of feeding with E. coli were evident within the gut of Artemia. Engineered strains of S. bacillarus expressing DB3 accumulated within the gut of Artemia with detectable antibody activity for 8-10 h of feeding via ELISA, coincident with the time period of the highest density of transgenic S. bacillarus in the Artemia gut. Artemia fed transgenic bacteria or algae accumulated recombinant proteins for up to 10 h that retained biological activity. Co-delivery of multiple recombinant proteins simultaneously in the gut of Artemia was also demonstrated. Expression of molecules that target infectious agents of mariculture in shrimp via commonly deployed feed organisms such as Artemia could potentially offer powerful new tools in the ongoing global effort to increase food supply.

Targeting melanoma stem cells with the Vitamin E derivative δ-tocotrienol.

PubMed

Marzagalli, Monica; Moretti, Roberta Manuela; Messi, Elio; Marelli, Marina Montagnani; Fontana, Fabrizio; Anastasia, Alessia; Bani, Maria Rosa; Beretta, Giangiacomo; Limonta, Patrizia

2018-01-12

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

The plasma levels of soluble ST2 as a marker of gut mucosal damage in early HIV infection

PubMed Central

Mehraj, Vikram; Jenabian, Mohammad-Ali; Ponte, Rosalie; Lebouché, Bertrand; Costiniuk, Cecilia; Thomas, Réjean; Baril, Jean-Guy; LeBlanc, Roger; Cox, Joseph; Tremblay, Cécile; Routy, Jean-Pierre

2016-01-01

Objective: Following tissue barrier breaches, interleukin-33 (IL-33) is released as an ‘alarmin’ to induce inflammation. Soluble suppression of tumorigenicity 2 (sST2), as an IL-33 decoy receptor, contributes to limit inflammation. We assessed the relationship between the IL-33/ST2 axis and markers of gut mucosal damage in patients with early (EHI) and chronic HIV infection (CHI) and elite controllers. Design: Analyses on patients with EHI and CHI were conducted to determine IL-33/sST2 changes over time. Methods: IL-33 and sST2 levels were measured in plasma. Correlations between sST2 levels and plasma viral load, CD4+ and CD8+ T-cell counts, expression of T-cell activation/exhaustion markers, gut mucosal damage, microbial translocation and inflammation markers, as well as kynurenine/tryptophan ratio were assessed. Results: Plasma sST2 levels were elevated in EHI compared with untreated CHI and uninfected controls, whereas IL-33 levels were comparable in all groups. In EHI, sST2 levels were positively correlated with the CD8+ T-cell count and the percentage of T cells expressing activation and exhaustion markers, but not with viral load or CD4+ T-cell count. Plasma sST2 levels also correlated with plasma levels of gut mucosal damage, microbial translocation and kynurenine/tryptophan ratio and for some markers of inflammation. Prospective analyses showed that early antiretroviral therapy had no impact on sST2 levels, whereas longer treatment duration initiated during CHI normalized sST2. Conclusion: As sST2 levels were elevated in EHI and were correlated with CD8+ T-cell count, immune activation, and microbial translocation, sST2 may serve as a marker of disease progression, gut damage and may directly contribute to HIV pathogenesis. PMID:27045377

Aripiprazole, an Antipsychotic and Partial Dopamine Agonist, Inhibits Cancer Stem Cells and Reverses Chemoresistance.

PubMed

Suzuki, Shuhei; Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Sakaki, Hirotsugu; Watarai, Hikaru; Sanomachi, Tomomi; Seino, Shizuka; Yoshioka, Takashi; Kitanaka, Chifumi

2016-10-01

There is a growing interest in repurposing antipsychotic dopamine antagonists for cancer treatment; however, antipsychotics are often associated with an increased risk of fatal events. The anticancer activities of aripiprazole, an antipsychotic drug with partial dopamine agonist activity and an excellent safety profile, remain unknown. The effects of aripiprazole alone or in combination with chemotherapeutic agents on the growth, sphere-forming ability and stem cell/differentiation/chemoresistance marker expression of cancer stem cells, serum-cultured cancer cells from which they were derived, and normal cells were examined. At concentrations non-toxic to normal cells, aripiprazole inhibited the growth of serum-cultured cancer cells and cancer stem cells. Furthermore, aripiprazole induced differentiation and inhibited sphere formation, as well as stem cell marker expression of cancer stem cells while inhibiting their survivin expression and sensitizing them to chemotherapeutic agents. Repurposing aripiprazole as an anticancer stem cell drug may merit further consideration. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

PubMed

Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

2017-10-01

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Effect of long-term treatment with melatonin on vascular markers of oxidative stress/inflammation and on the anticontractile activity of perivascular fat in aging mice.

PubMed

Agabiti-Rosei, Claudia; Favero, Gaia; De Ciuceis, Carolina; Rossini, Claudia; Porteri, Enzo; Rodella, Luigi Fabrizio; Franceschetti, Lorenzo; Maria Sarkar, Anna; Agabiti-Rosei, Enrico; Rizzoni, Damiano; Rezzani, Rita

2017-01-01

Some reports have suggested that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction by causing the disappearance of an anticontractile effect. The aim of this study was to investigate the effects of chronic melatonin treatment on the functional responses of the small mesenteric arteries and on the expression of markers of inflammation/oxidative stress in the aortas of senescence-accelerated prone mice (SAMP8), a model of age-related vascular dysfunction. We investigated seven SAMP8 and seven control senescence-accelerated resistant mice (SAMR1) treated for 10 months with melatonin, as well as equal numbers of age-matched untreated SAMP8 and SAMR1. The mesenteric small resistance arteries were dissected and mounted on a wire myograph, and the concentration-response to norepinephrine was evaluated in vessels with intact PVAT and after the removal of the PVAT. The expression of markers of oxidative stress, inflammation and aging in the aortas was evaluated by immunostaining. In addition, the adiponectin content and the expression of adiponectin receptor 1 were evaluated in the visceral adipose tissue. In untreated SAMP8 mice, we observed an overexpression of oxidative stress and inflammatory markers in the vasculature compared with the controls. No anticontractile effect of the PVAT was observed in untreated SAMP8 mice. Long-term treatment of SAMP8 mice with melatonin increased the expression of some markers of vasoprotection, decreased oxidative stress and inflammation and restored the anticontractile effect of the PVAT. Decreased expression of adiponectin and adiponectin receptor 1 was also observed in visceral fat of untreated SAMP8, whereas a significant increase was observed after melatonin treatment.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

PubMed

Schneider, Thomas; Karl, Stephan; Moore, Lee R; Chalmers, Jeffrey J; Williams, P Stephen; Zborowski, Maciej

2010-01-01

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

PubMed

Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

2014-05-01

We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of the Lipoxygenase Pathway in RSV-induced Alternatively Activated Macrophages Leading to Resolution of Lung Pathology

PubMed Central

Shirey, Kari Ann; Lai, Wendy; Pletneva, Lioubov M.; Karp, Christopher L.; Divanovic, Senad; Blanco, Jorge C. G.; Vogel, Stefanie N.

2013-01-01

Resolution of severe RSV-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mϕ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)−/− and 15-LO−/− macrophages or mice failed to elicit AA-Mϕ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO−/− mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mϕ in vitro and, conversely, treatment of 5-LO−/− macrophages with downstream products, lipoxin A4 (LXA4) and resolvin E1 (RvE1), but not leukotriene B4 (LTB4) or LTD4, partially restored expression of AA-Mϕ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO, and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also, decreased lung pathology in RSV-infected 5-LO−/− mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mϕ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mϕ differentiation by activating the 5-LO pathway. PMID:24064666

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

PubMed Central

Crozat, Karine; Guiton, Rachel; Contreras, Vanessa; Feuillet, Vincent; Dutertre, Charles-Antoine; Ventre, Erwan; Vu Manh, Thien-Phong; Baranek, Thomas; Storset, Anne K.; Marvel, Jacqueline; Boudinot, Pierre; Hosmalin, Anne; Schwartz-Cornil, Isabelle

2010-01-01

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria. PMID:20479118

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

PubMed

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; Nakayama, Takuya; Shah, Anoop; Grainger, Robert M.; White, Judith M.; DeSimone, Douglas W.

2012-01-01

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in X. tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus. PMID:22227340

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Mucosal CCR1 gene expression as a marker of molecular activity in Crohn's disease: preliminary data.

PubMed

Dobre, Maria; Mănuc, Teodora Ecaterina; Milanesi, Elena; Pleşea, Iancu Emil; Ţieranu, Eugen Nicolae; Popa, Caterina; Mănuc, Mircea; Preda, Carmen Monica; Ţieranu, Ioana; Diculescu, Mihai Mircea; Ionescu, Elena Mirela; Becheanu, Gabriel

2017-01-01

A series of mechanisms of immune response, inflammation and apoptosis have been demonstrated to contribute to the appearance and evolution of Crohn's disease (CD) through the overexpression of several cytokines and chemokines in a susceptible host. The aim of this study was to identify the differences in gene expression profiles analyzing a panel of candidate genes in the mucosa from patients with active CD (CD-A), patients in remission (CD-R), and normal controls. Nine individuals were enrolled in the study: six CD patients (three with active lesions, three with mucosal healing) and three controls without inflammatory bowel disease (IBD) seen on endoscopy. All the individuals underwent mucosal biopsy during colonoscopy. Gene expression levels of 84 genes previously associated with CD were evaluated by polymerase chain reaction (PCR) array. Ten genes out of 84 were found significantly differentially expressed in CD-A (CCL11, CCL25, DEFA5, GCG, IL17A, LCN2, REG1A, STAT3, MUC1, CCR1) and eight genes in CD-R (CASP1, IL23A, STAT1, STAT3, TNF, CCR1, CCL5, and HSP90B1) when compared to controls. A quantitative gene expression analysis revealed that CCR1 gene was more expressed in CD-A than in CD-R. Our data suggest that CCR1 gene may be a putative marker of molecular activity of Crohn's disease. Following these preliminary data, a confirmation in larger cohort studies could represent a useful method in order to identify new therapeutic targets.

Expression Patterns and Correlations with Metabolic Markers of Zinc Transporters ZIP14 and ZNT1 in Obesity and Polycystic Ovary Syndrome

PubMed Central

Maxel, Trine; Svendsen, Pernille Fog; Smidt, Kamille; Lauridsen, Jesper Krogh; Brock, Birgitte; Pedersen, Steen Bønlykke; Rungby, Jørgen; Larsen, Agnete

2017-01-01

Polycystic ovary syndrome (PCOS) is associated with infertility, increased androgen levels, and insulin resistance. In adipose tissue, zinc facilitates insulin signaling. Circulating zinc levels are altered in obesity, diabetes, and PCOS; and zinc supplementation can ameliorate metabolic disturbances in PCOS. In adipose tissue, expression of zinc influx transporter ZIP14 varies with body mass index (BMI), clinical markers of metabolic syndrome, and peroxisome proliferator-activated receptor gamma (PPARG). In this study, we investigated expression levels of ZIP14 and PPARG in subcutaneous adipose tissue of 36 PCOS women (17 lean and 19 obese women) compared with 23 healthy controls (7 lean and 16 obese women). Further, expression levels of zinc transporter ZIP9, a recently identified androgen receptor, and zinc efflux transporter ZNT1 were investigated, alongside lipid profile and markers of glucose metabolism [insulin degrading enzyme, retinol-binding protein 4 (RBP4), and glucose transporter 4 (GLUT4)]. We find that ZIP14 expression is reduced in obesity and positively correlates with PPARG expression, which is downregulated with increasing BMI. ZNT1 is upregulated in obesity, and both ZIP14 and ZNT1 expression significantly correlates with clinical markers of altered glucose metabolism. In addition, RBP4 and GLUT4 associate with obesity, but an association with PCOS as such was present only for PPARG and RBP4. ZIP14 and ZNT1 does not relate to clinical androgen status and ZIP9 is unaffected by all parameters investigated. In conclusion, our findings support the existence of a zinc dyshomeostasis in adipose tissue in metabolic disturbances including PCOS-related obesity. PMID:28303117

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

PubMed

Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

Role of oxidative stress in a rat model of radiation-induced erectile dysfunction.

PubMed

Kimura, Masaki; Rabbani, Zahid N; Zodda, Andrew R; Yan, Hui; Jackson, Isabel L; Polascik, Thomas J; Donatucci, Craig F; Moul, Judd W; Vujaskovic, Zeljko; Koontz, Bridget F

2012-06-01

Chronic oxidative stress is one of the major factors playing an important role in radiation-induced normal tissue injury. However, the role of oxidative stress in radiation-induced erectile dysfunction (ED) has not been fully investigated. Aims.  To investigate role of oxidative stress after prostate-confined irradiation in a rat model of radiation-induced ED. Fifty-four young adult male rats (10-12 weeks of age) were divided into age-matched sham radiotherapy (RT) and RT groups. Irradiated animals received prostate-confined radiation in a single 20 Gy fraction. Intracavernous pressure (ICP) measurements with cavernous nerve electrical stimulation were conducted at 2, 4, and 9 weeks following RT. The protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox4 and gp91(phox)), markers of oxidative DNA damage (8-hydroxy-2'-deoxyguanosine [8-OHdG]), lipid peroxidation (4-hydroxynonenal [4HNE]), and inflammatory response including inducible nitric oxide synthase, macrophage activation (ED-1), and nitrotyrosine, and endogenous antioxidant defense by nuclear factor erythroid 2-related factor (Nrf2) were evaluated in irradiated prostate tissue and corpora cavernosa (CC). In addition, we investigated the relationships between results of ICP/mean arterial pressure (MAP) ratios and expression level of oxidative stress markers. In the RT group, hemodynamic functional studies demonstrated a significant time-dependent decrease in ICP. Increased expression of Nox4, gp91(phox), 8-OHdG, and 4HNE were observed in the prostate and CC after RT. Similarly, expressions of inflammatory markers were significantly increased. There was a trend for increased Nrf2 after 4 weeks. ICP/MAP ratio negatively correlated with higher expression level of oxidative markers. NADPH oxidase activation and chronic oxidative stress were observed in irradiated prostate tissue and CC, which correlated with lower ICP/MAP ratio. Persistent inflammatory responses were also found in both tissues after RT. These findings suggest that oxidative stress plays a crucial role in the development of radiation-induced ED. © 2012 International Society for Sexual Medicine.

[Peptides and CCL11 and HMGB1 as molecular markers of aging: literature review and own data].

PubMed

Khavinson, V Kh; Kuznik, B I; Tarnovskaia, S I; Lin'kova, N S

2014-01-01

Cytokines CCL11 (eotaxin) and HMGB1 (alarmin1) are molecular markers of ageing and neurological, cardiovascular and immune diseases. Created in St. Petersburg Institute of Bioregulation and Gerontology short peptides are known to regulate gene expression and protein synthesis. They promote the mortality decrease and slowdown the development of pathology in the elderly. The article presents the proposed role of dipeptide vilon (Lys-Glu) and tetrapeptide epitalon (Ala-Glu-Asp-Gly) in CCL11 and HMGB1 genes regulation as activators of their expression. Geroprotective action of vilon and epitalon probably realizes in suppression of these genes.

Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

PubMed Central

Nishitani, Shinobu; Horie, Mayumi; Ishizaki, Sonoko; Yano, Hirohisa

2013-01-01

Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. PMID:24312415

Transcriptional dynamics of immune, growth and stress related genes in skeletal muscle of the fine flounder (Paralichthys adpersus) during different nutritional statuses.

PubMed

Valenzuela, Cristián A; Escobar, Daniela; Perez, Lorena; Zuloaga, Rodrigo; Estrada, Juan Manuel; Mercado, Luis; Valdés, Juan Antonio; Molina, Alfredo

2015-11-01

The effects of stress on immune activity and growth in early vertebrates have not been studied in detail. The present study used fine flounder (Paralichthys adspersus) skeletal muscle as a model to evaluate molecules involved in the stress response, including the glucocorticoid receptors, foxo1/3, and the target genes of these. Additionally, immune markers (il-1β and tnfα) and effector molecules of atrophy (bnip3, caspase-3, and lc3) were assessed. These molecules were analyzed during periods of long-term fasting and refeeding. During fasting, gene expression related to the stress response and atrophy increased; whereas immune markers were down-regulated. During refeeding, atrophy- and stress-related gene expression significantly decreased. In contrast, immune markers were up-regulated. These results provide novel insight on the control of growth in the skeletal muscle of a non-mammalian species under a stressful condition, suggesting that growth, stress, and immune activity in muscle are closely related and coordinated by orchestrated transcriptional dynamics. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Effect of Agmatine on Expression of IL-1β and TLX Which Promotes Neuronal Differentiation in Lipopolysaccharide-Treated Neural Progenitors.

PubMed

Song, Juhyun; Kumar, Bokara Kiran; Kang, Somang; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

2013-12-01

Differentiation of neural progenitor cells (NPCs) is important for protecting neural cells and brain tissue during inflammation. Interleukin-1 beta (IL-1β) is the most common pro- inflammatory cytokine in brain inflammation, and increased IL-1β levels can decrease the proliferation of NPCs. We aimed to investigate whether agmatine (Agm), a primary polyamine that protects neural cells, could trigger differentiation of NPCs by activating IL-1β in vitro. The cortex of ICR mouse embryos (E14) was dissociated to culture NPCs. NPCs were stimulated by lipopolysaccharide (LPS). After 6 days, protein expression of stem cell markers and differentiation signal factors was confirmed by using western blot analysis. Also, immunocytochemistry was used to confirm the cell fate. Agm treatment activated NPC differentiation significantly more than in the control group, which was evident by the increased expression of a neuronal marker, MAP2, in the LPS-induced, Agm-treated group. Differentiation of LPS-induced, Agm-treated NPCs was regulated by the MAPK pathway and is thought to be related to IL-1β activation and decreased expression of TLX, a transcription factor that regulates NPC differentiation. Our results reveal that Agm can promote NPC differentiation to neural stem cells by modulating IL-1β expression under inflammatory condition, and they suggest that Agm may be a novel therapeutic strategy for neuroinflammatory diseases.

Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells

PubMed Central

Tsakiris, Ioannis; Torocsik, Daniel; Gyongyosi, Adrienn; Dozsa, Aniko; Szatmari, Istvan; Szanto, Attila; Soos, Gyorgyike; Nemes, Zoltan; Igali, Laszlo; Marton, Ildiko; Takats, Zoltan; Nagy, Laszlo; Dezso, Balazs

2012-01-01

Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte–MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation. PMID:22157720

[Expression of embryonic markers in pterygium derived mesenchymal cells].

PubMed

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum▿

PubMed Central

Smulian, A. George; Gibbons, Reta S.; Demland, Jeffery A.; Spaulding, Deborah T.; Deepe, George S.

2007-01-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene. PMID:17873086

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

PubMed

Smulian, A George; Gibbons, Reta S; Demland, Jeffery A; Spaulding, Deborah T; Deepe, George S

2007-11-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

The molecular characterization of porcine egg precursor cells

PubMed Central

Tsai, Te-Sha; Johnson, Jacqueline; White, Yvonne; John, Justin C.

2017-01-01

Female-factor infertility can be caused by poor oocyte quality and depleted ovarian reserves. Egg precursor cells (EPCs), isolated from the ovarian cortex, have the potential to be used to overcome female infertility. We aimed to define the origins of EPCs by analyzing their gene expression profiles and mtDNA content using a mini-pig model. We characterized FAC-sorted DDX4+-derived porcine EPCs by performing RNA-sequencing and determined that they utilize pathways important for cell cycle and proliferation, which supports the existence of adult mitotically active oogonial cells. Expression of the pluripotent markers Sox2 and Oct4, and the primitive germ cell markers Blimp1 and Stella were not detected. However, Nanog and Ddx4 were expressed, as were the primitive germ cell markers Fragilis, c-Kit and Tert. Moreover, porcine EPCs expressed self-renewal and proliferation markers including Myc, Esrrb, Id2, Klf4, Klf5, Stat3, Fgfr1, Fgfr2 and Il6st. The presence of Zp1, Zp2, Zp3 and Nobox were not detected, indicating that porcine EPCs are not indicative of mature primordial oocytes. We performed mitochondrial DNA Next Generation Sequencing and determined that one mtDNA variant harbored by EPCs was present in oocytes, preimplantation embryos and somatic tissues over three generations in our mini-pig model indicating the potential germline origin of EPCs. PMID:28969006

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

PubMed Central

Carvidi, Alexander B.; Smith, Louis C. B.; Khan, Shahzada; Trapecar, Martin; Stoddart, Cheryl A.; Kuritzkes, Daniel R.

2018-01-01

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection. PMID:29470552

Over-expression of thymosin β4 in granulomatous lung tissue with active pulmonary tuberculosis.

PubMed

Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Yoo, Young-Bin; Chun, Bong-Kwon; Oak, Chul-Ho; Cha, Hee-Jae

2014-05-01

Recent studies have shown that thymosin β4 (Tβ4) stimulates angiogenesis by inducing vascular endothelial growth factor (VEGF) expression and stabilizing hypoxia inducible factor-1α (HIF-1α) protein. Pulmonary tuberculosis (TB), a type of granulomatous disease, is accompanied by intense angiogenesis and VEGF levels have been reported to be elevated in serum or tissue inflamed by pulmonary tuberculosis. We investigated the expression of Tβ4 in granulomatous lung tissues at various stages of active pulmonary tuberculosis, and we also examined the expression patterns of VEGF and HIF-1α to compare their Tβ4 expression patterns in patients' tissues and in the tissue microarray of TB patients. Tβ4 was highly expressed in both granulomas and surrounding lymphocytes in nascent granulomatous lung tissue, but was expressed only surrounding tissues of necrotic or caseous necrotic regions. The expression pattern of HIF-1α was similar to that of Tβ4. VEGF was expressed in both granulomas and blood vessels surrounding granulomas. The expression pattern of VEGF co-localized with CD31 (platelet endothelial cell adhesion molecule, PECAM-1), a blood endothelial cell marker, and partially co-localized with Tβ4. However, the expression of Tβ4 did not co-localize with alveolar macrophages. Stained alveolar macrophages were present surrounding regions of granuloma highly expressing Tβ4. We also analyzed mRNA expression in the sputum of 10 normal and 19 pulmonary TB patients. Expression of Tβ4 was significantly higher in patients with pulmonary tuberculosis than in normal controls. These data suggest that Tβ4 is highly expressed in granulomatous lung tissue with active pulmonary TB and is associated with HIF-1α- and VEGF-mediated inflammation and angiogenesis. Furthermore, the expression of Tβ4 in the sputum of pulmonary tuberculosis patients can be used as a potential marker for diagnosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Testosterone Plus Low-Intensity Physical Training in Late Life Improves Functional Performance, Skeletal Muscle Mitochondrial Biogenesis, and Mitochondrial Quality Control in Male Mice

PubMed Central

Guo, Wen; Wong, Siu; Li, Michelle; Liang, Wentao; Liesa, Marc; Serra, Carlo; Jasuja, Ravi; Bartke, Andrzej; Kirkland, James L.; Shirihai, Orian; Bhasin, Shalender

2012-01-01

Testosterone supplementation increases muscle mass in older men but has not been shown to consistently improve physical function and activity. It has been hypothesized that physical exercise is required to induce the adaptations necessary for translation of testosterone-induced muscle mass gain into functional improvements. However, the effects of testosterone plus low intensity physical exercise training (T/PT) on functional performance and bioenergetics are unknown. In this pilot study, we tested the hypothesis that combined administration of T/PT would improve functional performance and bioenergetics in male mice late in life more than low-intensity physical training alone. 28-month old male mice were randomized to receive T/PT or vehicle plus physical training (V/PT) for 2 months. Compare to V/PT control, administration of T/PT was associated with improvements in muscle mass, grip strength, spontaneous physical movements, and respiratory activity. These changes were correlated with increased mitochondrial DNA copy number and expression of markers for mitochondrial biogenesis. Mice receiving T/PT also displayed increased expression of key elements for mitochondrial quality control, including markers for mitochondrial fission-and-fusion and mitophagy. Concurrently, mice receiving T/PT also displayed increased expression of markers for reduced tissue oxidative damage and improved muscle quality. Conclusion: Testosterone administered with low-intensity physical training improves grip strength, spontaneous movements, and respiratory activity. These functional improvements were associated with increased muscle mitochondrial biogenesis and improved mitochondrial quality control. PMID:23240002

Immunomodulatory/inflammatory effects of geopropolis produced by Melipona fasciculata Smith in combination with doxorubicin on THP-1 cells.

PubMed

Oliveira, Lucas Pires Garcia; Conte, Fernanda Lopes; Cardoso, Eliza de Oliveira; Conti, Bruno José; Santiago, Karina Basso; Golim, Marjorie de Assis; Cruz, Maria Teresa; Sforcin, José Maurício

2016-12-01

Geopropolis (GEO) in combination with doxorubicin (DOX) reduced HEp-2 cells viability compared to GEO and DOX alone. A possible effect of this combination on the innate immunity could take place, and its effects were analysed on THP-1 cell - a human leukaemia monocytic cell line used as a model to study monocyte activity and macrophage activity, assessing cell viability, expression of cell markers and cytokine production. THP-1 cells were incubated with GEO, DOX and their combination. Cell viability was assessed by MTT assay, cell markers expression by flow cytometry and cytokine production by ELISA. GEO + DOX did not affect cell viability. GEO alone or in combination increased TLR-4 and CD80 but not HLA-DR and TLR-2 expression. GEO stimulated TNF-α production while DOX alone or in combination did not affect it. GEO alone or in combination inhibited IL-6 production. GEO exerted a pro-inflammatory profile by increasing TLR-4 and CD80 expression and TNF-α production, favouring the activation of the immune/inflammatory response. GEO + DOX did not affect cell viability and presented an immunomodulatory action. Lower concentrations of DOX combined to GEO could be used in cancer patients, avoiding side effects and benefiting from the biological properties of GEO. © 2016 Royal Pharmaceutical Society.

The PBX1 lupus susceptibility gene regulates CD44 expression

PubMed Central

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A.; Choi, Seung-Chul; Morel, Laurence

2017-01-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4+ T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and show that the lupus-associated isoform PBX1-d has unique molecular functions. PMID:28257976

R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

PubMed

Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

PubMed Central

Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos. PMID:24918770

Mesenchymal-to-Epithelial Transition Contributes to Endometrial Regeneration Following Natural and Artificial Decidualization

PubMed Central

Patterson, Amanda L.; Zhang, Ling; Arango, Nelson A.; Teixeira, Jose

2013-01-01

Despite being a histologically dynamic organ, mechanisms coordinating uterine regeneration during the menstrual/estrous cycle and following parturition are poorly understood. In the current study, we hypothesized that endometrial epithelial tissue regeneration is accomplished, in part, by mesenchymal-to-epithelial transition (MET). To test this hypothesis, fate mapping studies were completed using a double transgenic (Tg) reporter strain, Amhr2-Cre; Rosa26-Stopfl/fl-EYFP (i.e., flox-stop EYFP reporter). EYFP expression was observed in Müllerian duct mesenchyme-derived stroma and myometrium, but not epithelia in young and peripubertal double Tg female mice. However, mosaic EYFP expression was observed in epithelia of double Tg mice after parturition. To ensure the observed epithelial EYFP expression was not due to leaky Amhr2 promoter activity, resulting in aberrant Cre expression, transgenic mice expressing LacZ under the control of the Amhr2 promoter (Amhr2-LacZ) were used to monitor β-galactosidase (β-Gal) activity within the uterus. β-Gal activity was not detected in luminal or glandular epithelia regardless of age, reproductive status, or degree of damage incurred within the uterus. Lastly, a unique population of transitional cells was identified that expressed the epithelial cell marker, pan-cytokeratin, and the stromal cell marker, vimentin. These cells localized predominantly to the regeneration zone in the mesometrial region of the endometrium. These findings suggest a previously unappreciated role for MET in endometrial regeneration and have important implications for proliferative diseases of the endometrium such as endometriosis. PMID:23216285

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

PubMed

Orfila, C; Lepert, J C; Alric, L; Carrera, G; Beraud, M; Vinel, J P; Pipy, B

1999-10-01

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu Qisheng; Valverde, Paloma; Chen, Jake

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase,more » bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.« less

Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

PubMed Central

Zhang, Yuanyuan; George, Jasmine; Li, Yun; Olufade, Rebecca; Zhao, Xueying

2015-01-01

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway. PMID:25849723

Cytokine Expression, Natural Killer Cell Activation, and Phenotypic Changes in Lymphoid Cells from Rhesus Macaques during Acute Infection with Pathogenic Simian Immunodeficiency Virus

PubMed Central

Giavedoni, Luis D.; Velasquillo, M. Cristina; Parodi, Laura M.; Hubbard, Gene B.; Hodara, Vida L.

2000-01-01

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3−CD16+ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4+ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4+ T cells and absent in peripheral blood leukocyte (PBL) CD4+ T cells, was down-regulated in LN CD4+ T cells and up-regulated in PBL CD4+ T cells immediately after infection. CD8+ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4+ T cells but not in LN CD4+ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection. PMID:10644334

Human IgG2- and IgG4-expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age.

PubMed

de Jong, Britt G; IJspeert, Hanna; Marques, Lemelinda; van der Burg, Mirjam; van Dongen, Jacques Jm; Loos, Bruno G; van Zelm, Menno C

2017-10-01

The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass-specific antibodies was performed on purified IgG + memory B-cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream-located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27 + IgG + memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27 - IgG + memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2-expressing B cells as markers for efficient generation of memory responses.

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

PubMed

Shekhani, Mohammed Talha; Forde, Toni S; Adilbayeva, Altynai; Ramez, Mohamed; Myngbay, Askhat; Bexeitov, Yergali; Lindner, Volkhard; Adarichev, Vyacheslav A

2016-07-19

The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01). CTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.

PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

PubMed

Cenciarelli, Carlo; Marei, Hany E; Felsani, Armando; Casalbore, Patrizia; Sica, Gigliola; Puglisi, Maria Ausiliatrice; Cameron, Angus J M; Olivi, Alessandro; Mangiola, Annunziato

2016-08-16

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

PubMed

Wong, Marcy; Siegrist, Mark; Goodwin, Kelly

2003-10-01

Endochondral ossification is regulated by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte maturation. Mathematical models of endochondral ossification have suggested that tension (or shear stress) can accelerate the formation of endochondral bone, while hydrostatic stress preserves the cartilage phenotype. The goal of this study was to test this hypothesis by examining the expression of hypertrophic chondrocyte markers (transcription factor Cbfa1, MMP-13, type X collagen, VEGF, CTGF) and cartilage matrix proteins under cyclic tension and cyclic hydrostatic pressure. Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers. Mammalian chondrocytes appear to have evolved complex mechanoresponsive mechanisms, the effects of which can be observed in the histomorphologic establishment of the cartilaginous skeleton during development and maturation.

Effect of Chinese herbal compound Naofucong () on the inflammatory process induced by high glucose in BV-2 cells.

PubMed

Jing, Guang-Chan; Zhang, Meng-Ren; Ji, Chao; Zuo, Ping-Ping; Liu, Yu-Qin; Gu, Bei

2016-11-01

To determine the effect of medicated serum of Chinese herbal compound Naofucong (, NFC) on the microglia BV-2 cells viability and the transcription and expression of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia BV-2 cells to further explore the mechanisms underlying the protective effect of NFC on inflammatory process induced by high glucose. The microglia BV-2 cells incubated in vitro were divided into different groups: the control group (25 mmol/L glucose), the model group (75 mmol/L glucose), high glucose media containing different dose medicated serum of NFC. After being cultured for 24 h, changes in IL-6 and TNF-α were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The expression of surface marker CD11b of activated microglia was measured by confocal laser scanning microscope and Western blot. Nuclear factor-κB (NF-κB) p-p65 expression was analyzed by Western blot. The model group obviously increased the expression of microglial surface marker CD11b and NF-κB p-p65 (all P<0.01), induced a signifificant up-regulation of release and the mRNA expression of IL-6 and TNF-α (P<0.01 or P<0.05). The medicated serum of NFC could obviously down-regulate the transcription and expression of surface marker CD11 b and NF-κB p-p65 (all P<0.01), and inhibit the mRNA and protein expression (P<0.01 or P<0.05) of inflflammatory cytokines, such as IL-6 and TNF-α, in microglia BV-2 cells cultured with high glucose for 24 h. The inhibition of microglial activation and IL-6 and TNF-α expression induced by high glucose may at least partly explain NFC therapeutic effects on diabetes-associated cognitive decline diseases. Its underlying mechanism could probably be related to the inhibition of NFC on NF-κB phosphorylation.

ERα inhibited myocardin-induced differentiation in uterine fibroids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, Xing-Hua, E-mail: xinghualiao@hotmail.com; Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education and Tianjin, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457; Li, Jun-Yan

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smoothmore » muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.« less

COX-2 and SCD, markers of inflammation and adipogenesis, are related to disease activity in Graves' ophthalmopathy.

PubMed

Vondrichova, Tereza; de Capretz, Annika; Parikh, Hemang; Frenander, Christofer; Asman, Peter; Aberg, Magnus; Groop, Leif; Hallengren, Bengt; Lantz, Mikael

2007-06-01

Inflammation and adipogenesis are two parallel processes with increased activity in severe Graves' ophthalmopathy. The aim of this work was to define target genes for therapeutic intervention in adipogenesis and inflammation in Graves' ophthalmopathy. Orbital tissue was obtained from patients with ophthalmopathy in acute or chronic phase undergoing orbital surgery to study gene expression followed by the study of potential intervention mechanisms in preadipocytes. Clinic of Endocrinology, University Hospital, Malmö, Sweden. Patients in acute severe or in chronic phase of ophthalmopathy. Lateral orbital decompression in acute phase and restorative surgery in chronic phase. In vitro treatment of preadipocytes with rosiglitazone and diclofenac. Gene expression in intraorbital tissue or preadipocytes and differentiation of preadipocytes. A marker of adipose tissue, stearoyl-coenzyme A desaturase (SCD), and the proinflammatory gene, cyclooxygenase-2 (COX-2), were overexpressed in patients in active phase compared to the chronic phase of ophthalmopathy. In growth-arrested preadipocytes stimulated with rosiglitazone, COX-2 expression increased temporarily within 1 hour and decreased to undetectable levels after 48 hours. In contrast, SCD and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression increased continuously from day 2 to day 7 during adipogenesis. Diclofenac, an inhibitor of cyclooxygenases with antagonistic effects on PPAR-gamma, reduced the number of mature adipocytes by approximately 50%. We conclude that inflammation and adipogenesis decrease with a decrease in activity of ophthalmopathy and that the nonsteroidal antiinflammatory drug diclofenac inhibits adipogenesis. This may represent a putative future treatment of endocrine ophthalmopathy.

Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function.

PubMed

Murphy, Karen J; Chronopoulos, Andriana K; Singh, Indu; Francis, Maureen A; Moriarty, Helen; Pike, Marilyn J; Turner, Alan H; Mann, Neil J; Sinclair, Andrew J

2003-06-01

Flavonoids may be partly responsible for some health benefits, including antiinflammatory action and a decreased tendency for the blood to clot. An acute dose of flavanols and oligomeric procyanidins from cocoa powder inhibits platelet activation and function over 6 h in humans. This study sought to evaluate whether 28 d of supplementation with cocoa flavanols and related procyanidin oligomers would modulate human platelet reactivity and primary hemostasis and reduce oxidative markers in vivo. Thirty-two healthy subjects were assigned to consume active (234 mg cocoa flavanols and procyanidins/d) or placebo (< or = 6 mg cocoa flavanols and procyanidins/d) tablets in a blinded parallel-designed study. Platelet function was determined by measuring platelet aggregation, ATP release, and expression of activation-dependent platelet antigens by using flow cytometry. Plasma was analyzed for oxidation markers and antioxidant status. Plasma concentrations of epicatechin and catechin in the active group increased by 81% and 28%, respectively, during the intervention period. The active group had significantly lower P selectin expression and significantly lower ADP-induced aggregation and collagen-induced aggregation than did the placebo group. Plasma ascorbic acid concentrations were significantly higher in the active than in the placebo group (P < 0.05), whereas plasma oxidation markers and antioxidant status did not change in either group. Cocoa flavanol and procyanidin supplementation for 28 d significantly increased plasma epicatechin and catechin concentrations and significantly decreased platelet function. These data support the results of acute studies that used higher doses of cocoa flavanols and procyanidins.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon.

PubMed

Kouzai, Yusuke; Kimura, Mamiko; Yamanaka, Yurie; Watanabe, Megumi; Matsui, Hidenori; Yamamoto, Mikihiro; Ichinose, Yuki; Toyoda, Kazuhiro; Onda, Yoshihiko; Mochida, Keiichi; Noutoshi, Yoshiteru

2016-03-02

Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots.

Variable expression of podocyte-related markers in the glomeruloid bodies in Wilms tumor.

PubMed

Kanemoto, Katsuyoshi; Takahashi, Shori; Shu, Yujing; Usui, Joichi; Tomari, Shinsuke; Yan, Kunimasa; Hamazaki, Yutaka; Nagata, Michio

2003-09-01

Several podocyte-related markers are organized to express in glomerular differentiation. However, whether expression of them is virtually synchronized and a reliable indicator of the state of differentiation is unknown. The present study investigated, by immunohistochemistry, the divergent expression of several podocyte markers in the improperly differentiated glomeruloid bodies from four cases of Wilms tumors. The glomeruloid bodies were classified into immature (IGB) or mature forms (MGB) based on morphology and epithelial features. Podocytes in IGB expressed WT1, synaptopodin, podocalyxin, and nephrin, and their expression was stronger in MGB. In contrast, Pax2 was strong in IGB and diminished in MGB. p27 was first expressed in MGB. The expression pattern in each molecule mimics normal glomerulogenesis. Podocytes in MGB showed persistent expression of bcl-2 and cytokeratin with synaptopodin, podocalyxin, and nephrin by serial section, a finding unusual for normal glomerulogenesis. Moreover, parietal cells in MGB also occasionally expressed these podocyte markers. The ultrastructure revealed that podocytes in MGB showed tight junctions without foot process formations, which indicated incomplete differentiation. These results suggest that a set of podocyte differentiation markers are occasionally diversely expressed, and raise the possibility that expression of these markers is insufficient to determine the state of terminal differentiation in podocytes.

Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

PubMed Central

Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

2006-01-01

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22α and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription. PMID:17030628

Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

PubMed

Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

2006-12-01

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

PubMed Central

Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

Cellular Profile and Expression of Immunologic Markers in Chronic Apical Periodontitis from HIV-infected Patients Undergoing Highly Active Antiretroviral Therapy.

PubMed

Gama, Túlio Gustavo Veiga; Pires, Fabio Ramoa; Armada, Luciana; Gonçalves, Lucio Souza

2016-06-01

This study tested the hypothesis that the inflammatory cell profile (CD3-, CD4-, CD8-, CD20-, and CD68-positive cells) and the expression of immunologic markers (tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-18) in chronic apical periodontitis are the same between non-HIV-infected patients and HIV-infected patients undergoing highly active antiretroviral therapy (HAART). Thirty-four surgically excised chronic apical periodontitis lesions were sampled from 34 patients (17 HIV-infected and 17 non-HIV-infected). The lesions were extracted from teeth with no previous endodontic treatment. All HIV-infected patients were undergoing HAART. The specimens were submitted to histopathologic and immunohistochemical analyses by using an optical microscope. Immunoexpression was graded into 2 levels, focal to weak and moderate to strong. The χ(2), Fisher exact, and Mann-Whitney tests were used to analyze all significant differences between groups. Periapical cysts represented 70.6% and 52.9% of the lesions in the HIV-infected and non-HIV-infected groups, respectively; however, no statistically significant difference was observed (P = .481). There were no statistically significant differences between groups for the inflammatory cell profile and for any of the immunologic markers (P > .05). There are no statistically significant differences of the cellular profile and expression of immunologic markers in chronic apical periodontitis between non-HIV-infected patients and HIV-infected patients undergoing HAART. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Daisuke; Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp; Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2013-05-17

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present inmore » esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.« less

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients.

PubMed

Markiewicz, Aleksandra; Nagel, Anna; Szade, Jolanta; Majewska, Hanna; Skokowski, Jaroslaw; Seroczynska, Barbara; Stokowy, Tomasz; Welnicka-Jaskiewicz, Marzena; Zaczek, Anna J

2018-06-01

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)-enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Silymarin prevents NLRP3 inflammasome activation and protects against intracerebral hemorrhage.

PubMed

Yuan, Raorao; Fan, Hengyi; Cheng, Shiqi; Gao, WeiWei; Xu, Xin; Lv, Shigang; Ye, Minhua; Wu, Miaojing; Zhu, Xingen; Zhang, Yan

2017-09-01

Inflammatory response mediates secondary injury during intracerebral hemorrhage (ICH). In the present study, we determined oxidative stress and involvement of NLRP3 in ICH injury and analyzed whether silymarin might offer protective effect against ICH injury. Post 24h after ICH injury there was increased oxidative stress markers (reactive oxygen species (ROS) and lipid peroxides) compared to sham group. Silymarin (200mg/kg) treatment 30 mins post ICH injury prevented increase in oxidative stress markers and up-regulated antioxidant status. Further, there was significant increase in nuclear levels of NF-κB-p65 and pro-inflammatory cytokine expressions post ICH injury. NLRP3 inflammasome activation and downstream targets such as caspase-1 and IL-1β expressions were significantly up regulated in ICH injury. Silymarin treatment significantly down regulated the inflammatory responses by suppressing NF-κB-p65 levels and inflammasome-mediated caspase-1/IL-1β expressions. Further, treatment with silymarin post ICH injury increased Nrf-2/HO-1 and thereby improved overall cytoprotection. These findings together show that silymarin acts as neuroprotective compound by preventing inflammatory activation and up regulating Nrf-2/HO-1 signaling post ICH injury. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

[Comparative immunophenotypic characterization of human and monkey permanent lymphoid culture cells].

PubMed

Agrba, V Z; Lapin, B A; Medvedeva, N M; Ignatova, I E; Karal-Ogly, D D

2007-01-01

The aim of the study was to define the comparative immunophenotypic characteristics ofwidely spread lymphoid cell cultures, derived from Burkett's lymphoma named as Raji and P3HR-1 in comparison with analogous monkey cultures. It has been shown that P3HR-1 culture consists of similar type cells - activated B-lymphocytes CD23 with k phenotype, which demonstrates its monoclonality. Raji culture includes cells with markers of immature B-lymphocytes CD10 and CD24, as well as elements expressing CD10 antigens. T-cell markers were found in none of the cultures. In contrast to human cells, monkey lymphoid culture expressed both B- and T-cell markers. Moreover, in one of them, obtained from a green monkey, T-cells of suppressor type (CD8) prevailed. The immunophenotypic characteristics of primate lymphoid cell cultures, revealed by the study, are of great importance for their proper application to medical and biological studies.

Paired box 7 inhibits differentiation in 3T3-L1 preadipocytes.

PubMed

Izumi, Wakana; Takuma, Yuko; Ebihara, Ryo; Mizunoya, Wataru; Tatsumi, Ryuichi; Nakamura, Mako

2018-06-13

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets. © 2018 Japanese Society of Animal Science.

The Potential Protective Effect of Physalis peruviana L. against Carbon Tetrachloride-Induced Hepatotoxicity in Rats Is Mediated by Suppression of Oxidative Stress and Downregulation of MMP-9 Expression

PubMed Central

Al-Olayan, Ebtisam M.; El-Khadragy, Manal F.; Aref, Ahmed M.; Othman, Mohamed S.; Kassab, Rami B.; Abdel Moneim, Ahmed E.

2014-01-01

The active constituent profile in Cape gooseberry (Physalis peruviana L.) juice was determined by GC-MS. Quercetin and kaempferol were active components in the juice. In this study we have evaluated its potential protective effect on hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). Twenty-eight rats divided into 4 groups: Group I served as control group, and Group II received weekly i.p. injection of 2 mL CCl4/kg bwt for 12 weeks. Group III were supplemented with Physalis juice via the drinking water. The animals of Group IV received Physalis juice as Group III and also were intraperitoneally injected weekly with 2 mL CCl4/kg bwt for 12 weeks. Hepatoprotective effect was evaluated by improvement in liver enzymes serum levels, reduction in collagen areas, downregulation in expression of the fibrotic marker MMP-9, reduction in the peroxidative marker malonaldehyde and the inflammatory marker nitric oxide, and restoration of the activity of antioxidant enzymatic and nonenzymatic systems, namely, glutathione content, superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, and glutathione reductase activities. The results show that the potential hepatoprotective effects of Physalis peruviana may be due to physalis acts by promotion of processes that restore hepatolobular architecture and through the inhibition of oxidative stress pathway. PMID:24876910

The potential protective effect of Physalis peruviana L. against carbon tetrachloride-induced hepatotoxicity in rats is mediated by suppression of oxidative stress and downregulation of MMP-9 expression.

PubMed

Al-Olayan, Ebtisam M; El-Khadragy, Manal F; Aref, Ahmed M; Othman, Mohamed S; Kassab, Rami B; Abdel Moneim, Ahmed E

2014-01-01

The active constituent profile in Cape gooseberry (Physalis peruviana L.) juice was determined by GC-MS. Quercetin and kaempferol were active components in the juice. In this study we have evaluated its potential protective effect on hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). Twenty-eight rats divided into 4 groups: Group I served as control group, and Group II received weekly i.p. injection of 2 mL CCl4/kg bwt for 12 weeks. Group III were supplemented with Physalis juice via the drinking water. The animals of Group IV received Physalis juice as Group III and also were intraperitoneally injected weekly with 2 mL CCl4/kg bwt for 12 weeks. Hepatoprotective effect was evaluated by improvement in liver enzymes serum levels, reduction in collagen areas, downregulation in expression of the fibrotic marker MMP-9, reduction in the peroxidative marker malonaldehyde and the inflammatory marker nitric oxide, and restoration of the activity of antioxidant enzymatic and nonenzymatic systems, namely, glutathione content, superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, and glutathione reductase activities. The results show that the potential hepatoprotective effects of Physalis peruviana may be due to physalis acts by promotion of processes that restore hepatolobular architecture and through the inhibition of oxidative stress pathway.

RNA sequencing from neural ensembles activated during fear conditioning in the mouse temporal association cortex

PubMed Central

Cho, Jin-Hyung; Huang, Ben S.; Gray, Jesse M.

2016-01-01

The stable formation of remote fear memories is thought to require neuronal gene induction in cortical ensembles that are activated during learning. However, the set of genes expressed specifically in these activated ensembles is not known; knowledge of such transcriptional profiles may offer insights into the molecular program underlying stable memory formation. Here we use RNA-Seq to identify genes whose expression is enriched in activated cortical ensembles labeled during associative fear learning. We first establish that mouse temporal association cortex (TeA) is required for remote recall of auditory fear memories. We then perform RNA-Seq in TeA neurons that are labeled by the activity reporter Arc-dVenus during learning. We identify 944 genes with enriched expression in Arc-dVenus+ neurons. These genes include markers of L2/3, L5b, and L6 excitatory neurons but not glial or inhibitory markers, confirming Arc-dVenus to be an excitatory neuron-specific but non-layer-specific activity reporter. Cross comparisons to other transcriptional profiles show that 125 of the enriched genes are also activity-regulated in vitro or induced by visual stimulus in the visual cortex, suggesting that they may be induced generally in the cortex in an experience-dependent fashion. Prominent among the enriched genes are those encoding potassium channels that down-regulate neuronal activity, suggesting the possibility that part of the molecular program induced by fear conditioning may initiate homeostatic plasticity. PMID:27557751

Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1

PubMed Central

Zhu, Wen; Carney, Karen E.; Pigott, Victoria M.; Falgoust, Lindsay M.; Clark, Paul A.; Kuo, John S.; Sun, Dandan

2016-01-01

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na+/H+ exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia–glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1+ microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma–microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation. PMID:27287871

Histopathological and molecular prognostic markers in medulloblastoma: c-myc, N-myc, TrkC, and anaplasia.

PubMed

Eberhart, Charles G; Kratz, John; Wang, Yunyue; Summers, Krista; Stearns, Duncan; Cohen, Kenneth; Dang, Chi V; Burger, Peter C

2004-05-01

Several molecular and histopathological prognostic markers have been proposed for the therapeutic stratification of medulloblastoma patients. Amplification of the c-myc oncogene, elevated levels of c-myc mRNA, or tumor anaplasia have been associated with worse clinical outcomes. In contrast, high TrkC mRNA expression generally presages longer survival. The goal of this study was to evaluate the prognostic value of c-myc, N-myc and TrkC expression in medulloblastomas and compare them to histopathological classification. We used in situ hybridization to measure expression of these molecular markers. c-myc mRNA was detected in 18 of 59 (31%) cases, and was significantly associated with shorter patient survival times on both univariate and multivariate analyses (p = 0.04). The presence of c-myc mRNA was also significantly associated with tumor anaplasia. While survival rates were higher for patients with low N-myc or high TrkC expression, these differences were not statistically significant. The group of patients with either moderate or severely anaplastic tumors showed only a trend towards shorter survival (p = 0.11). However, severe anaplasia alone was significantly prognostic (p = 0.002). Given the prognostic import of c-myc, we investigated 2 potential mechanisms by which its expression might be regulated: Wnt signaling and Mxi-1 mutation. Nuclear translocation of beta-catenin, a marker of Wnt pathway activation, was more common in medulloblastomas with high c-myc than in tumors overall, but the difference was not statistically significant. No Mxi-1 mutations were detected in the 22 cases examined. The association we describe between c-myc expression, tumor anaplasia, and worse clinical outcomes provides further evidence for the importance of this oncogene in medulloblastoma pathobiology.

Rosmarinic Acid Activates AMPK to Inhibit Metastasis of Colorectal Cancer

PubMed Central

Han, Yo-Han; Kee, Ji-Ye; Hong, Seung-Heon

2018-01-01

Rosmarinic acid (RA) has been used as an anti-inflammatory, anti-diabetic, and anti-cancer agent. Although RA has also been shown to exert an anti-metastatic effect, the mechanism of this effect has not been reported to be associated with AMP-activated protein kinase (AMPK). The aim of this study was to elucidate whether RA could inhibit the metastatic properties of colorectal cancer (CRC) cells via the phosphorylation of AMPK. RA inhibited the proliferation of CRC cells through the induction of cell cycle arrest and apoptosis. In several metastatic phenotypes of CRC cells, RA regulated epithelial–mesenchymal transition (EMT) through the upregulation of an epithelial marker, E-cadherin, and the downregulation of the mesenchymal markers, N-cadherin, snail, twist, vimentin, and slug. Invasion and migration of CRC cells were inhibited and expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were decreased by RA treatment. Adhesion and adhesion molecules such as ICAM-1 and integrin β1 expressions were also reduced by RA treatment. In particular, the effects of RA on EMT and MMPs expressions were due to the activation of AMPK. Moreover, RA inhibited lung metastasis of CRC cells by activating AMPK in mouse model. Collectively, these results proved that RA could be potential therapeutic agent against metastasis of CRC. PMID:29459827

Epithelial expression of cytokeratins 15 and 19 in vitiligo.

PubMed

Saleh, Fatma Y; Awad, Sherif S; Nasif, Ghada A; Halim, Christein

2016-12-01

Cytokeratins (CK) belong to the family of intermediate filament proteins, and among them specific epithelial keratins are considered markers for stem cells activation. This study aims to investigate the expression of CK15 and CK19 as possible stem cell markers in vitiligo during phototherapy. The study was conducted on vitiligo patients receiving narrow-band ultraviolet therapy. Immunohistochemical staining for CK15 and CK19 was carried out, and clinical follow-up continued for 4 weeks. Of 28 patients, CK15 expression was demonstrated in 17 cases (61%) while CK19 expression was demonstrated in 11 cases (39%). Cells expressing positive staining were demonstrated in follicular and interfollicular epithelium. Expression was clearly demonstrated in patients younger than 20 years old, with shorter disease duration, with disease stability, and with normally pigmented hairs. Expression of cytokeratins was significantly correlated to improvement of vitiligo lesions. CK15 and CK19 are expressed in vitiligo during UV repigmentation in the follicular and interfollicular epithelium. This expression of cytokeratins was significantly correlated to improvement and can be considered valuable tool to monitor stem cells stimulation for the sake of the repigmentation process in vitiligo. © 2016 Wiley Periodicals, Inc.

Over-expression of IQGAP1 indicates poor prognosis in head and neck squamous cell carcinoma.

PubMed

Wu, Cong-Cong; Li, Hao; Xiao, Yao; Yang, Lei-Lei; Chen, Lei; Deng, Wei-Wei; Wu, Lei; Zhang, Wen-Feng; Sun, Zhi-Jun

2018-05-30

IQ-domain GTPase-activating protein 1 (IQGAP1) is associated with the development and progression of many human cancers. We aimed to investigate the expression and clinicopathological significances of IQGAP1 in head and neck squamous cell carcinoma (HNSCC). In this study, immunohistochemical staining of IQGAP1, co-inhibitory immune checkpoint molecules and macrophage markers were performed in human HNSCC samples to analyze the expression and correlation with clinicopathological characteristics. Immunoreactivity of IQGAP1 was also detected in immunocompetent mouse HNSCC tissue. We found that IQGAP1 expression level was significantly increased in human HNSCC compared with dysplasia and normal mucosa, and the expression of IQGAP1 in HNSCC was positively associated with advanced lymph node status. Besides, our data indicated that patients with higher IQGAP1 expression exhibited poor overall survival compared with patients with lower IQGAP1 expression. Furthermore, this study demonstrated that IQGAP1 expression was positively associated with TIM3, Galectin-9 (TIM3 ligand), B7H4, macrophage markers CD68 and CD163. In conclusion, these findings suggest that over-expression of IQGAP1 in human HNSCC may indicate poor prognosis.

Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness

PubMed Central

Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko

2013-01-01

The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches. PMID:24009080

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture.

PubMed

Xu, Yichun; Yao, Hui; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Teng, Haijun; Guo, Zhiliang; Zhao, Huiqing; Hou, Gang

2018-01-01

An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

PubMed Central

Lee, Mi-Heon; Kachroo, Puja; Pagano, Paul C; Yanagawa, Jane; Wang, Gerald; Walser, Tonya C; Krysan, Kostyantyn; Sharma, Sherven; John, Maie St.; Dubinett, Steven M; Lee, Jay M

2015-01-01

Background The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. Objective The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. Methods and Results Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. Conclusion Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway. PMID:26523208

Zinc supplementation alleviates the progression of diabetic nephropathy by inhibiting the overexpression of oxidative-stress-mediated molecular markers in streptozotocin-induced experimental rats.

PubMed

Barman, Susmita; Pradeep, Seetur R; Srinivasan, Krishnapura

2018-04-01

Zinc deficiency during diabetes projects a role for zinc nutrition in the management of diabetic nephropathy. The current study explored whether zinc supplementation protects against diabetic nephropathy through modulation of kidney oxidative stress and stress-induced expression related to the inflammatory process in streptozotocin-induced diabetic rats. Groups of hyperglycemic rats were exposed to dietary interventions for 6 weeks with zinc supplementation (5 times and 10 times the normal level). Supplemental-zinc-fed diabetic groups showed a significant reversal of increased kidney weight and creatinine clearance. There was a significant reduction in hyperlipidemic condition along with improved PUFA:SFA ratio in the renal tissue. Expression of the lipid oxidative marker and expression of inflammatory markers, cytokines, fibrosis factors and apoptotic regulatory proteins observed in diabetic kidney were beneficially modulated by zinc supplementation, the ameliorative effect being concomitant with elevated antiapoptosis. There was a significant reduction in advanced glycation, expression of the receptor of the glycated products and oxidative stress markers. Zinc supplementation countered the higher activity and expression of polyol pathway enzymes in the kidney. Overexpression of the glucose transporters, as an adaptation to the increased need for glucose transport in diabetic condition, was minimized by zinc treatment. The pathological abnormalities in the renal architecture of diabetic animals were corrected by zinc intervention. Thus, dietary zinc supplementation has a significant beneficial effect in the control of diabetic nephropathy. This was exerted through a protective influence on oxidative-stress-induced cytokines, inflammatory proliferation and consequent renal injury. Copyright © 2017 Elsevier Inc. All rights reserved.

Fatty acid synthase as a tumor marker: its extracellular expression in human breast cancer.

PubMed

Wang, Young Y; Kuhajda, Francis P; Li, Jinong; Finch, Teia T; Cheng, Paul; Koh, Clare; Li, Tianwei; Sokoll, Lori J; Chan, Daniel W

2004-07-01

Overexpression of fatty acid synthase (FAS EC 2.3.1.85) is associated with certain cancers and therefore is a putative tumor marker. The presence of FAS in patients with breast, prostate, colon, ovarian, and other cancers has been reported. The mechanism of FAS overexpression in malignancies remains unknown. Here, we show that FAS is released into the extracellular space in cancer cells. The extracellular FAS are present in various immunoreactive forms, and show different expression patterns in various cancer cells. In serum of breast cancer patients, the FAS is a small molecule similar to the form in breast cancer cell lysate but not conditioned medium of cultured cells. The extracellular expression of FAS in breast cancer cells is time dependent and may be hormone independent. These results indicate that the FAS are an ordered cellular response of a living cell and actively exclude excess intracellular FAS molecules from the cell. This phenomenon is up-regulated in breast and may be in other cancer cells as well. Significant elevation of FAS was detected in serum of breast cancer patients compared to healthy subjects. In comparison with CA27.29, no correlation between these two tumor markers was found. Thus, the extracellular FAS may serve as a potential diagnostic and prognostic marker.

[Tumor markers p53, sFAS, FASL, CEA and CA 19-9 in evaluating the effectiveness of surgical and pharmaco nutritional treatment of patients with gastric cancer].

PubMed

Bluvshteĭn, G A; Lysenko, V G; Zakharova, N B; Kitaev, I V

2012-01-01

The objective of this study is to develop a marker panel of abnormalities of immune regulatory systems and cell genome in patients with gastric cancer for assessment of efficacy of surgical treatment in combination with pharmaco-nutritional therapy in early postoperative period. Expression of p53, sFAS, FASL, CEA, and CA 19-9, nutritial status, as well as incidence of purulent-septic complications in early postoperative period were determined in 40 patients with gastric cancer (21 patients--the test group and 19 patients--the comparative group) prior to a curative surgical intervention, postoperative days 1 and 7, while the pharmaco-nutritional therapy (the test group) or partial parenteral nutrition (the comparative group) has been performed. The pharmaco-nutritional therapy significantly decreases in activity of tumor suppressing genome, lymphocyte apoptosis, expression of oncology-associated markers, incidence of purulent-septic complications, as well as exacerbation risk for hypotrophy in patients with gastric cancer in early postoperative period. To assess the efficacy of the surgical intervention performed in patients with gastric cancer, an expression of mutant p53 and markers of lymphocyte apoptosis (sFAS/FASL) is reasonable to be evaluated together with determination of oncomarkers (CEA and CA 19-9).

LIN9, a Subunit of the DREAM Complex, Regulates Mitotic Gene Expression and Proliferation of Embryonic Stem Cells

PubMed Central

Esterlechner, Jasmina; Reichert, Nina; Iltzsche, Fabian; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

2013-01-01

The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. PMID:23667535

Cyp1b1 deletion and retinol deficiency coordinately suppress mouse liver lipogenic genes and hepcidin expression during post-natal development

PubMed Central

Maguire, Meghan; Larsen, Michele Campaigne; Foong, Yee Hoon; Tanumihardjo, Sherry; Jefcoate, Colin R.

2018-01-01

Cyp1b1 deletion and gestational vitamin A deficiency (GVAD) redirect adult liver gene expression. A matched sufficient pre- and post-natal diet, which has high carbohydrate and normal iron content (LF12), increased inflammatory gene expression markers in adult livers that were suppressed by GVAD and Cyp1b1 deletion. At birth on the LF12 diet, Cyp1b1 deletion and GVAD each suppress liver expression of the iron suppressor, hepcidin (Hepc), while increasing stellate cell activation markers and suppressing post-natal increases in lipogenesis. Hepc was less suppressed in Cyp1b1−/− pups with a standard breeder diet, but was restored by iron supplementation of the LF12 diet. Conclusions The LF12 diet delivered low post-natal iron and attenuated Hepc. Hepc decreases in Cyp1b1−/− and GVAD mice resulted in stellate activation and lipogenesis suppression. Endothelial BMP6, a Hepc stimulant, is a potential coordinator and Cyp1b1 target. These neonatal changes in Cyp1b1−/− mice link to diminished adult obesity and liver inflammation. PMID:28583802

Expression profiling of various genes during the fruit development and ripening of mango.

PubMed

Pandit, Sagar S; Kulkarni, Ram S; Giri, Ashok P; Köllner, Tobias G; Degenhardt, Jörg; Gershenzon, Jonathan; Gupta, Vidya S

2010-06-01

Mango (Mangifera indica L. cv. Alphonso) development and ripening are the programmed processes; conventional indices and volatile markers help to determine agronomically important stages of fruit life (fruit-setting, harvesting maturity and ripening climacteric). However, more and precise markers are required to understand this programming; apparently, fruit's transcriptome can be a good source of such markers. Therefore, we isolated 18 genes related to the physiology and biochemistry of the fruit and profiled their expression in developing and ripening fruits, flowers and leaves of mango using relative quantitation PCR. In most of the tissues, genes related to primary metabolism, abiotic stress, ethylene response and protein turnover showed high expression as compared to that of the genes related to flavor production. Metallothionin and/or ethylene-response transcription factor showed highest level of transcript abundance in all the tissues. Expressions of mono- and sesquiterpene synthases and 14-3-3 lowered during ripening; whereas, that of lipoxygenase, ethylene-response factor and ubiquitin-protein ligase increased during ripening. Based on these expression profiles, flower showed better positive correlation with developing and ripening fruits than leaf. Most of the genes showed their least expression on the second day of harvest, suggesting that harvesting signals significantly affect the fruit metabolism. Important stages in the fruit life were clearly indicated by the significant changes in the expression levels of various genes. These indications complemented those from the previous analyses of fruit development, ripening and volatile emission, revealing the harmony between physiological, biochemical and molecular activities of the fruit.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

PubMed

Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

Nuclear factor one B (NFIB) encodes a subtype-specific tumour suppressor in glioblastoma

PubMed Central

Stringer, Brett W.; Bunt, Jens; Day, Bryan W.; Barry, Guy; Jamieson, Paul R.; Ensbey, Kathleen S.; Bruce, Zara C.; Goasdoué, Kate; Vidal, Hélène; Charmsaz, Sara; Smith, Fiona M.; Cooper, Leanne T.; Piper, Michael

2016-01-01

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects. PMID:27083054

Statin-Induced Increases in Atrophy Gene Expression Occur Independently of Changes in PGC1α Protein and Mitochondrial Content

PubMed Central

Zacharewicz, Evelyn; Lee-Young, Robert S.; Snow, Rod J.; Russell, Aaron P.; McConell, Glenn K.

2015-01-01

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg-1·day-1) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles. PMID:26020641

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

PubMed

Kang, H W; Weissleder, R; Bogdanov, A

2002-01-01

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schechter, Melissa E.; Andrade, Bruno B.; He, Tianyu

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. Thus, a better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidencemore » of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor–α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)–related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.« less

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

DOE PAGES

Schechter, Melissa E.; Andrade, Bruno B.; He, Tianyu; ...

2017-08-30

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. Thus, a better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidencemore » of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor–α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)–related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.« less

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies. PMID:22723265

Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli

PubMed Central

Rask, C; Adlerberth, I; Berggren, A; Ahrén, I L; Wold, A E

2013-01-01

Probiotics are live microorganisms which have beneficial effects on the host when ingested in adequate amounts. Probiotic bacteria may stimulate immune effector functions in a strain-specific manner. In this blind placebo-controlled trial, we investigated the effects on the immune system following daily intake of six different strains of lactobacilli or the Gram-negative bacterium Pseudomonas lundensis for 2 or 5 weeks. Blood lymphocyte subsets were quantified by fluorescence activated cell sorter and the expression of activation and memory markers was determined. The bacterial strains were also examined for their capacity to adhere to human intestinal cells and to be phagocytosed by human peripheral blood mononuclear cells. Intake of Lactobacillus plantarum strain 299v increased the expression of the activation marker CD25 (P = 0·01) on CD8+ T cells and the memory cell marker CD45RO on CD4+ T cells (P = 0·03), whereas intake of L. paracasei tended to expand the natural killer T (NK T) cell population (P = 0·06). The phagocytic activity of granulocytes was increased following intake of L. plantarum 299v, L. plantarum HEAL, L. paracasei or L. fermentum. In contrast, ingestion of L. rhamnosus decreased the expression of CD25 and CD45RO significantly within the CD4+ cell population. The observed immune effects after in-vivo administration of the probiotic bacteria could not be predicted by either their adherence capacity or the in-vitro-induced cytokine production. The stimulation of CD8+ T cells and NK T cells suggests that intake of probiotic bacteria may enhance the immune defence against, e.g. viral infections or tumours. PMID:23574328

Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli.

PubMed

Rask, C; Adlerberth, I; Berggren, A; Ahrén, I L; Wold, A E

2013-05-01

Probiotics are live microorganisms which have beneficial effects on the host when ingested in adequate amounts. Probiotic bacteria may stimulate immune effector functions in a strain-specific manner. In this blind placebo-controlled trial, we investigated the effects on the immune system following daily intake of six different strains of lactobacilli or the Gram-negative bacterium Pseudomonas lundensis for 2 or 5 weeks. Blood lymphocyte subsets were quantified by fluorescence activated cell sorter and the expression of activation and memory markers was determined. The bacterial strains were also examined for their capacity to adhere to human intestinal cells and to be phagocytosed by human peripheral blood mononuclear cells. Intake of Lactobacillus plantarum strain 299v increased the expression of the activation marker CD25 (P = 0·01) on CD8(+) T cells and the memory cell marker CD45RO on CD4(+) T cells (P = 0·03), whereas intake of L. paracasei tended to expand the natural killer T (NK T) cell population (P = 0·06). The phagocytic activity of granulocytes was increased following intake of L. plantarum 299v, L. plantarum HEAL, L. paracasei or L. fermentum. In contrast, ingestion of L. rhamnosus decreased the expression of CD25 and CD45RO significantly within the CD4(+) cell population. The observed immune effects after in-vivo administration of the probiotic bacteria could not be predicted by either their adherence capacity or the in-vitro-induced cytokine production. The stimulation of CD8(+) T cells and NK T cells suggests that intake of probiotic bacteria may enhance the immune defence against, e.g. viral infections or tumours. © 2012 British Society for Immunology.

Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation

PubMed Central

Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping

2016-01-01

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45−/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45−/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45−/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. PMID:27269414

Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation.

PubMed

Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping

2016-09-01

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45-/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45-/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45-/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

PubMed

Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

2009-07-31

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

Organ- and species-specific biological activity of rosmarinic acid.

PubMed

Iswandana, R; Pham, B T; van Haaften, W T; Luangmonkong, T; Oosterhuis, D; Mutsaers, H A M; Olinga, P

2016-04-01

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition*

PubMed Central

Rico-Leo, Eva M.; Alvarez-Barrientos, Alberto; Fernandez-Salguero, Pedro M.

2013-01-01

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR+/+ and AhR−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells. PMID:23382382

Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Raman Deep, E-mail: Takhter.Ramandeep@mayo.edu; Schroeder, Andreas S.; Scheffer, Luana

Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins thatmore » bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily localize to lipid rafts and recruit cholesterol into protrusions and away from caveolae, leading to increased phosphorylation of caveolin-1, which inhibits Cdc42-dependent endocytosis. This study provides a new insight for the role for prominins in the regulation of PM lipid organization.« less

Nestin-expressing cells in the pancreatic islets of Langerhans.

PubMed

Hunziker, E; Stein, M

2000-04-29

The pancreatic islets of Langerhans produce several peptide hormones, predominantly the metabolically active hormones insulin and glucagon, which are critical for maintaining normal fuel homeostasis. Some evidence exists that pancreatic endocrine cells turn over at a slow rate and can regenerate in certain conditions. This could be due to the presence of pluripotent cells residing in the pancreas. Recently the intermediate filament protein nestin has been identified to be a marker for a multipotent stem cell in the central nervous system. Given the similarity between the pancreatic islets and neuronal cells, we hypothesized that stem cells expressing nestin might be present in the pancreas. Here we present evidence that a subset of cells in the pancreatic islets express the stem cell marker nestin. These cells might serve as precursors of differentiated pancreatic endocrine cells. Copyright 2000 Academic Press.

Adipokinetic hormone-induced antioxidant response in Spodoptera littoralis.

PubMed

Večeřa, Josef; Krishnan, Natraj; Mithöfer, Axel; Vogel, Heiko; Kodrík, Dalibor

2012-03-01

The antioxidative potential of the Manduca sexta adipokinetic hormone (Manse-AKH) in the last instar larvae of Spodoptera littoralis (Noctuidae, Lepidoptera) was demonstrated after exposure to oxidative stress (OS) elicited by feeding on artificial diet containing tannic acid (TA). Determination of protein carbonyls (PCs) and reduced glutathione (GSH) levels, monitoring of activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferases (GSTs), as well as measuring of the mRNA expression of CAT and SOD were used as markers of the OS. Injection of the Manse-AKH (5 pmol per individual) reversed the OS status by mitigation of PCs formation and by stimulation of glutathione-S-transferases (GSTs) activity. The CAT and SOD mRNA expression was significantly suppressed after the Manse-AKH injection while activity of these enzymes was not affected. These results indicate that diminishing of OS after the AKH injection might be a result of activation of specific enzymatic pathway possibly at the post-translational level rather than a direct effect on regulation of antioxidant marker genes at the transcriptional level. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation and maturation of bone marrow-derived DCs under serum-free conditions.

PubMed

Kim, Sung Jung; Diamond, Betty

2007-06-30

Standard protocols for the generation of murine dendritic cells (DCs) employ medium supplemented with heat-inactivated fetal calf serum (FCS). Recently, several attempts have been made to avoid serum exposure during DC culture. The impetus for these efforts has been a desire to generate DCs for clinical use, as preclinical data have demonstrated their efficacy in immune activation and in immune suppression both in vitro and in vivo. However, these protocols have resulted in contradictory outcomes with respect to DC survival in culture and activation status. In this report, we compared several serum-free culture conditions with respect to survival, differentiation, activation, and cytokine profile of murine DC progenitors. DC progenitors can survive only in some serum-free conditions. Surprisingly, DCs grown in serum-free medium display a higher expression of activation markers upon stimulation. They produce increased IL-12 and decreased IL-6 following stimulation. Furthermore, DCs derived under serum-free conditions may express unusual surface markers, B220 and Ly6C/G, implying an increased differentiation to plasmacytoid DCs (pDCs).

Lysosomal isoenzyme profiles used to classify a case of acute undifferentiated leukaemia.

PubMed

Eden, O B; Darbyshire, P; Simpson, R M; Besley, G T; Moss, S; Gentle, T

1985-01-01

Lysosomal enzyme activities and isoenzyme profiles were measured in lymphoid and non-lymphoid leukaemic cells from childhood patients. High activities, especially of beta-hexosaminidase and alpha-mannosidase, were associated with leukaemic cells of myeloid or monocytic origin. Leukaemic cells from two children with acute myeloid leukaemia had a relative reduction in the B isoenzyme of beta-hexosaminidase activity, whereas in patients with non T, non B cell acute lymphoblastic leukaemia, intermediate beta-hexosaminidase isoenzymes were expressed. A patient is described on whom conventional marker studies were either negative or equivocal, but lysosomal enzyme markers were consistent with a myeloid leukaemia. This observation was supported by the clinical course of this patient.

Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells

PubMed Central

2009-01-01

Introduction Advanced glycation end products (AGEs) have been introduced to be involved in the pathogenesis of osteoarthritis (OA). The influence of AGEs on osteoarthritic fibroblast-like synovial cells (FLS) has been incompletely understood as yet. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on cell growth, and on the expression of proinflammatory and osteoclastogenic markers in cultured FLS. Methods FLS were established from OA joints and stimulated with AGE-BSA. The mRNA expression of p27Kip1, RAGE (receptor for AGEs), nuclear factor kappa B subunit p65 (NFκB p65), tumor necrosis factor alpha (TNF-α, interleukin-6 (IL-6), receptor activator of NFκB ligand (RANKL) and osteoprotegerin was measured by real-time PCR. The respective protein expression was evaluated by western blot analysis or ELISA. NFκB activation was investigated by luciferase assay and electrophoretic mobility shift assay (EMSA). Cell cycle analysis, cell proliferation and markers of necrosis and early apoptosis were assessed. The specificity of the response was tested in the presence of an anti-RAGE antibody. Results AGE-BSA was actively taken up into the cells as determined by immunohistochemistry and western blots. AGE-induced p27Kip1 mRNA and protein expression was associated with cell cycle arrest and an increase in necrotic, but not apoptotic cells. NFκB activation was confirmed by EMSAs including supershift experiments. Anti-RAGE antibodies attenuated all AGE-BSA induced responses. The increased expression of RAGE, IL-6 and TNF-α together with NFκB activation indicates AGE-mediated inflammation. The decreased expression of RANKL and osteoprotegerin may reflect a diminished osteoclastogenic potential. Conclusions The present study demonstrates that AGEs modulate growth and expression of genes involved in the pathophysiological process of OA. This may lead to functional and structural impairment of the joints. PMID:19735566

The role of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in mediating autophagy in myositis skeletal muscle: A potential non-immune mechanism of muscle damage

PubMed Central

Alger, Heather M.; Raben, Nina; Pistilli, Emidio; Francia, Dwight; Rawat, Rashmi; Getnet, Derese; Ghimbovschi, Svetlana; Chen, Yi-Wen; Lundberg, Ingrid E.; Nagaraju, Kanneboyina

2011-01-01

Objective Multinucleated cells are relatively resistant to classical apoptosis, and the factors initiating cell-death and damage in myositis are not well defined. We hypothesized that non-immune autophagic cell death may play a role in muscle fiber damage. Recent literature indicates that tumor necrosis factor-alpha-related apoptosis inducing ligand (TRAIL) may induce both NFκB (nuclear factor kappa-light chain enhancer of activated B cells) activation and autophagic cell death in other systems. Here, we have investigated its role in cell death and pathogenesis in vitro and in vivo using myositis (human and mouse) muscle tissues. Methods Gene expression profiling indicated that expression of TRAIL and several autophagy markers was specifically upregulated in myositis muscle tissue; these results were confirmed by immunohistochemistry and immunoblotting. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NFκB activation in vitro in cultured cells. Results TRAIL was expressed predominantly in muscle fibers of myositis, but not in biopsies from normal or other dystrophic-diseased muscle. Autophagy markers were upregulated in human and mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NFκB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but undergo autophagic cell death. Conclusion Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NFκB and autophagic cell death in myositis. Thus, this non-immune pathway may be an attractive target for therapeutic intervention in myositis. PMID:21769834

Combination of tauroursodeoxycholic acid and N-acetylcysteine exceeds standard treatment for acetaminophen intoxication.

PubMed

Paridaens, Annelies; Raevens, Sarah; Colle, Isabelle; Bogaerts, Eliene; Vandewynckel, Yves-Paul; Verhelst, Xavier; Hoorens, Anne; van Grunsven, Leo A; Van Vlierberghe, Hans; Geerts, Anja; Devisscher, Lindsey

2017-05-01

Acetaminophen overdose in mice is characterized by hepatocyte endoplasmic reticulum stress, which activates the unfolded protein response, and centrilobular hepatocyte death. We aimed at investigating the therapeutic potential of tauroursodeoxycholic acid, a hydrophilic bile acid known to have anti-apoptotic and endoplasmic reticulum stress-reducing capacities, in experimental acute liver injury induced by acetaminophen overdose. Mice were injected with 300 mg/kg acetaminophen, 2 hours prior to receiving tauroursodeoxycholic acid, N-acetylcysteine or a combination therapy, and were euthanized 24 hours later. Liver damage was assessed by serum transaminases, liver histology, terminal deoxynucleotidyl transferase dUTP nick end labelling staining, expression profiling of inflammatory, oxidative stress, unfolded protein response, apoptotic and pyroptotic markers. Acetaminophen overdose resulted in a significant increase in serum transaminases, hepatocyte cell death, unfolded protein response activation, oxidative stress, NLRP3 inflammasome activation, caspase 1 and pro-inflammatory cytokine expressions. Standard of care, N-acetylcysteine and, to a lesser extent, tauroursodeoxycholic treatment were associated with significantly lower transaminase levels, hepatocyte death, unfolded protein response activation, oxidative stress markers, caspase 1 expression and NLRP3 levels. Importantly, the combination of N-acetylcysteine and tauroursodeoxycholic acid improved serum transaminase levels, reduced histopathological liver damage, UPR-activated CHOP, oxidative stress, caspase 1 expression, NLRP3 levels, IL-1β levels and the expression of pro-inflammatory cytokines and this to a greater extend than N-acetylcysteine alone. These findings indicate that a combination strategy of N-acetylcysteine and tauroursodeoxycholic acid surpasses the standard of care in acetaminophen-induced liver injury in mice and might represent an attractive therapeutic opportunity for acetaminophen-intoxicated patients. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

PubMed Central

Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

2017-01-01

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic. PMID:29250067

Galectin-3 is essential for proper bone cell differentiation and activity, bone remodeling and biomechanical competence in mice.

PubMed

Iacobini, Carla; Fantauzzi, Claudia Blasetti; Bedini, Rossella; Pecci, Raffaella; Bartolazzi, Armando; Amadio, Bruno; Pesce, Carlo; Pugliese, Giuseppe; Menini, Stefano

2018-02-09

Galectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3 -/- ) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals. Long bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/β-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. Lgals3 -/- mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p < 0.01 vs. WT at 3 months) and reduced bone strength (p < 0.01 vs. WT at 3 months). These abnormalities were accompanied by a steady inflammatory state, as revealed by higher bone expression of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6 (p < 0.001 vs. WT at 3 months), increased content of osteal macrophages (p < 0.01 vs. WT at 3 months), and reduced expression of markers of alternative (M2) macrophage activation. Lgals3 -/- osteoblasts and osteoclasts showed impaired terminal differentiation, reduced mineralization capacity (p < 0.01 vs. WT cells) and resorption activity (p < 0.01 vs. WT cells). Mechanistically, impaired differentiation and function of Lgals3 -/- osteoblasts was associated with altered WNT/β-catenin signaling (p < 0.01 vs. WT cells). These data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling. Copyright © 2018. Published by Elsevier Inc.

Protective effect of Ac-SDKP on alveolar epithelial cells through inhibition of EMT via TGF-β1/ROCK1 pathway in silicosis in rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Haijing; Xu, Hong; Zhang, Xianghong

The epithelial–mesenchymal transition (EMT) is a critical stage during the development of silicosis fibrosis. In the current study, we hypothesized that the anti-fibrotic tetrapeptide, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) may exert its anti-fibrotic effects via activation of the TGF-β1/ROCK1 pathway, leading to inhibition of EMT. To address this hypothesis, we first examined the effect of Ac-SDKP upon EMT using an in vivo rat silicosis model, as well as in an in vitro model of TGF-β1-induced EMT. Confocal laser scanning microscopy was used to examine colocalization of surfactant protein A (SP-A), fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) in vivo. Western blotmore » analysis was used to examine for changes in the protein levels of E-cadherin (E-cad) and SP-A (epithelial cell markers), vimentin (mesenchymal cell marker), α-SMA (active myofibroblast marker), and collagen I and III in both in vivo and in vitro experiments. Secondly, we utilized Western blot analysis and confocal laser scanning microscopy to examine the protein expression of TGF-β1 and ROCK1 in in vivo and in vitro studies. The results revealed that Ac-SDKP treatment prevented increases in the expression of mesenchymal markers as well as TGF-β1, ROCK1, collagen I and III. Furthermore, Ac-SDKP treatment prevented decreases in the expression of epithelial cell markers in both in vivo and in vitro experiments. Based on the results, we conclude that Ac-SDKP inhibits the transition of epithelial cell-myofibroblast in silicosis via activation of the TGF-β1/ROCK1 signaling pathway, which may serve as a novel mechanism by which it exerts its anti-fibrosis properties. - Highlights: • EMT is a critical stage during the development of silicosis fibrosis. • Ac-SDKP inhibits the EMT process in silicosis both in vivo and in vitro. • Ac-SDKP inhibits the EMT process in silicosis via TGF-β1/ROCK1 pathway.« less

Optimal Geometrical Set for Automated Marker Placement to Virtualized Real-Time Facial Emotions

PubMed Central

Maruthapillai, Vasanthan; Murugappan, Murugappan

2016-01-01

In recent years, real-time face recognition has been a major topic of interest in developing intelligent human-machine interaction systems. Over the past several decades, researchers have proposed different algorithms for facial expression recognition, but there has been little focus on detection in real-time scenarios. The present work proposes a new algorithmic method of automated marker placement used to classify six facial expressions: happiness, sadness, anger, fear, disgust, and surprise. Emotional facial expressions were captured using a webcam, while the proposed algorithm placed a set of eight virtual markers on each subject’s face. Facial feature extraction methods, including marker distance (distance between each marker to the center of the face) and change in marker distance (change in distance between the original and new marker positions), were used to extract three statistical features (mean, variance, and root mean square) from the real-time video sequence. The initial position of each marker was subjected to the optical flow algorithm for marker tracking with each emotional facial expression. Finally, the extracted statistical features were mapped into corresponding emotional facial expressions using two simple non-linear classifiers, K-nearest neighbor and probabilistic neural network. The results indicate that the proposed automated marker placement algorithm effectively placed eight virtual markers on each subject’s face and gave a maximum mean emotion classification rate of 96.94% using the probabilistic neural network. PMID:26859884

Optimal Geometrical Set for Automated Marker Placement to Virtualized Real-Time Facial Emotions.

PubMed

Maruthapillai, Vasanthan; Murugappan, Murugappan

2016-01-01

In recent years, real-time face recognition has been a major topic of interest in developing intelligent human-machine interaction systems. Over the past several decades, researchers have proposed different algorithms for facial expression recognition, but there has been little focus on detection in real-time scenarios. The present work proposes a new algorithmic method of automated marker placement used to classify six facial expressions: happiness, sadness, anger, fear, disgust, and surprise. Emotional facial expressions were captured using a webcam, while the proposed algorithm placed a set of eight virtual markers on each subject's face. Facial feature extraction methods, including marker distance (distance between each marker to the center of the face) and change in marker distance (change in distance between the original and new marker positions), were used to extract three statistical features (mean, variance, and root mean square) from the real-time video sequence. The initial position of each marker was subjected to the optical flow algorithm for marker tracking with each emotional facial expression. Finally, the extracted statistical features were mapped into corresponding emotional facial expressions using two simple non-linear classifiers, K-nearest neighbor and probabilistic neural network. The results indicate that the proposed automated marker placement algorithm effectively placed eight virtual markers on each subject's face and gave a maximum mean emotion classification rate of 96.94% using the probabilistic neural network.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

PubMed

Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

2016-06-01

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Antiretroviral therapy in HIV-1-infected individuals with CD4 count below 100 cells/mm3 results in differential recovery of monocyte activation

PubMed Central

Patro, Sean C.; Azzoni, Livio; Joseph, Jocelin; Fair, Matthew G.; Sierra-Madero, Juan G.; Rassool, Mohammed S.; Sanne, Ian; Montaner, Luis J.

2016-01-01

Reversal of monocyte and macrophage activation and the relationship to viral suppression and T cell activation are unknown in patients with advanced HIV-1 infection, initiating antiretroviral therapy. This study aimed to determine whether reduction in biomarkers of monocyte and macrophage activation would be reduced in conjunction with viral suppression and resolution of T cell activation. Furthermore, we hypothesized that the addition of CCR5 antagonism (by maraviroc) would mediate greater reduction of monocyte/macrophage activation markers than suppressive antiretroviral therapy alone. In the CCR5 antagonism to decrease the incidence of immune reconstitution inflammatory syndrome study, antiretroviral therapy-naïve patients received maraviroc or placebo in addition to standard antiretroviral therapy. PBMCs and plasma from 65 patients were assessed during 24 wk of antiretroviral therapy for biomarkers of monocyte and macrophage activation. Markers of monocyte and macrophage activation were reduced significantly by 24 wk, including CD14++CD16+ intermediate monocytes (P < 0.0001), surface CD163 (P = 0.0004), CD169 (P < 0.0001), tetherin (P = 0.0153), and soluble CD163 (P < 0.0001). A change in CD38+, HLA-DR+ CD8 T cells was associated with changes in CD169 and tetherin expression. Maraviroc did not affect biomarkers of monocyte/macrophage activation but resulted in greater percentages of CCR5-positive monocytes in PBMC. HIV-1 suppression after 24 wk of antiretroviral therapy, with or without maraviroc, demonstrates robust recovery in monocyte subset activation markers, whereas soluble markers of activation demonstrate minimal decrease, qualitatively differentiating markers of monocyte/macrophage activation in advanced disease. PMID:26609048

The Effect of Regular Intake of Dry-Cured Ham Rich in Bioactive Peptides on Inflammation, Platelet and Monocyte Activation Markers in Humans

PubMed Central

Martínez-Sánchez, Sara María; Minguela, Alfredo; Prieto-Merino, David; Zafrilla-Rentero, María Pilar; Abellán-Alemán, José; Montoro-García, Silvia

2017-01-01

Background and aims: Dietary studies have shown that active biopeptides provide protective health benefits, although the mediating pathways are somewhat uncertain. To throw light on this situation, we studied the effects of consuming Spanish dry-cured ham on platelet function, monocyte activation markers and the inflammatory status of healthy humans with pre-hypertension. Methods: Thirty-eight healthy volunteers with systolic blood pressure of >125 mmHg were enrolled in a two-arm crossover randomized controlled trial. Participants received 80 g/day dry-cured pork ham of >11 months proteolysis or 100 g/day cooked ham (control product) for 4 weeks followed by a 2-week washout before “crossing over” to the other treatment for 4 more weeks. Soluble markers and cytokines were analyzed by ELISA. Platelet function was assessed by measuring P-selectin expression and PAC-1 binding after ADP (adenosine diphosphate) stimulation using whole blood flow cytometry. Monocyte markers of the pathological status (adhesion, inflammatory and scavenging receptors) were also measured by flow cytometry in the three monocyte subsets after the interventional period. Results: The mean differences between dry-cured ham and cooked ham followed by a time period adjustment for plasmatic P-selectin and interleukin 6 proteins slightly failed (p = 0.062 and p = 0.049, respectively), notably increased for MCP-1 levels (p = 0.023) while VCAM-1 was not affected. Platelet function also decreased after ADP stimulation. The expression of adhesion and scavenging markers (ICAM1R, CXCR4 and TLR4) in the three subsets of monocytes was significantly higher (all p < 0.05). Conclusions: The regular consumption of biopeptides contained in the dry-cured ham but absent in cooked ham impaired platelet and monocyte activation and the levels of plasmatic P-selectin, MCP-1 and interleukin 6 in healthy subjects. This study strongly suggests the existence of a mechanism that links dietary biopeptides and beneficial health effects. PMID:28333093

Impaired autophagy activity is linked to elevated ER-stress and inflammation in aging adipose tissue.

PubMed

Ghosh, Amiya Kumar; Mau, Theresa; O'Brien, Martin; Garg, Sanjay; Yung, Raymond

2016-10-24

Adipose tissue dysfunction in aging is associated with inflammation, metabolic syndrome and other diseases. We propose that impaired protein homeostasis due to compromised lysosomal degradation (micro-autophagy) might promote aberrant ER stress response and inflammation in aging adipose tissue. Using C57BL/6 mouse model, we demonstrate that adipose tissue-derived stromal vascular fraction (SVF) cells from old (18-20 months) mice have reduced expression of autophagy markers as compared to the younger (4-6 months) cohort. Elevated expressions of ER-stress marker CHOP and autophagy substrate SQSTM1/p62 are observed in old SVFs compared to young, when treated with either vehicle or with thapsigargin (Tg), an ER stress inducer. Treatment with bafilomycin A1 (Baf), a vacuolar-type H (+)-ATPase, or Tg elevated expressions of CHOP, and SQSTM1/p62 and LC-3-II, in 3T3-L1-preadipocytes. We also demonstrate impaired autophagy activity in old SVFs by analyzing increased accumulation of autophagy substrates LC3-II and p62. Compromised autophagy activity in old SVFs is correlated with enhanced release of pro-inflammatory cytokines IL-6 and MCP-1. Finally, SVFs from calorie restricted old mice (CR-O) have shown enhanced autophagy activity compared to ad libitum fed old mice (AL-O). Our results support the notion that diminished autophagy activity with aging contributes to increased adipose tissue ER stress and inflammation.

Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB.

PubMed

Mao, Yulong; Wang, Baikui; Xu, Xin; Du, Wei; Li, Weifen; Wang, Youming

2015-01-01

The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization.

BRCA1/p220 loss triggers BRCA1-IRIS overexpression via mRNA stabilization in breast cancer cells

PubMed Central

Shimizu, Yoshiko; Mullins, Nicole; Blanchard, Zannel; ElShamy, Wael M.

2012-01-01

BRCA1/p220-assocaited and triple negative/basal-like (TN/BL) tumors are aggressive and incurable breast cancer diseases that share among other features the no/low BRCA1/p220 expression. Here we show that BRCA1/p220 silencing in normal human mammary epithelial (HME) cells reduces expression of two RNA-destabilizing proteins, namely AUF1 and pCBP2, both proteins bind and destabilize BRCA1-IRIS mRNA. BRCA1-IRIS overexpression in HME cells triggers expression of several TN/BL markers, e.g., cytokeratins 5 and 17, p-cadherin, EGFR and cyclin E as well as expression and activation of the pro-survival proteins; AKT and survivin. BRCA1-IRIS silencing in the TN/BL cell line, SUM149 or restoration of BRCA1/p220 expression in the mutant cell line, HCC1937 reduced expression of TN/BL markers, AKT, survivin, and induced cell death. Collectively, we propose that BRCA1/p220 loss of expression or function triggers BRCA1-IRIS overexpression through a post-transcriptional mechanism, which in turn promotes formation of aggressive and invasive breast tumors by inducing expression of TN/BL and survival proteins. PMID:22431556

Low-dose pterostilbene, but not resveratrol, is a potent neuromodulator in aging and Alzheimer's disease.

PubMed

Chang, Jaewon; Rimando, Agnes; Pallas, Merce; Camins, Antoni; Porquet, David; Reeves, Jennifer; Shukitt-Hale, Barbara; Smith, Mark A; Joseph, James A; Casadesus, Gemma

2012-09-01

Recent studies have implicated resveratrol and pterostilbene, a resveratrol derivative, in the protection against age-related diseases including Alzheimer's disease (AD). However, the mechanism for the favorable effects of resveratrol in the brain remains unclear and information about direct cross-comparisons between these analogs is rare. As such, the purpose of this study was to compare the effectiveness of diet-achievable supplementation of resveratrol to that of pterostilbene at improving functional deficits and AD pathology in the SAMP8 mouse, a model of accelerated aging that is increasingly being validated as a model of sporadic and age-related AD. Furthermore we sought to determine the mechanism of action responsible for functional improvements observed by studying cellular stress, inflammation, and pathology markers known to be altered in AD. Two months of pterostilbene diet but not resveratrol significantly improved radial arm water maze function in SAMP8 compared with control-fed animals. Neither resveratrol nor pterostilbene increased sirtuin 1 (SIRT1) expression or downstream markers of sirtuin 1 activation. Importantly, markers of cellular stress, inflammation, and AD pathology were positively modulated by pterostilbene but not resveratrol and were associated with upregulation of peroxisome proliferator-activated receptor (PPAR) alpha expression. Taken together our findings indicate that at equivalent and diet-achievable doses pterostilbene is a more potent modulator of cognition and cellular stress than resveratrol, likely driven by increased peroxisome proliferator-activated receptor alpha expression and increased lipophilicity due to substitution of hydroxy with methoxy group in pterostilbene. Copyright © 2012 Elsevier Inc. All rights reserved.

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation.

PubMed

Siebelt, M; van der Windt, A E; Groen, H C; Sandker, M; Waarsing, J H; Müller, C; de Jong, M; Jahr, H; Weinans, H

2014-04-01

Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in in vitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D in vitro cultures, and its therapeutic effects in an in vivo rat model of OA. Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated in vitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed ex vivo using contrast enhanced microCT and histology. FK506 treatment of osteoarthritic chondrocytes in vitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity in vivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. FK506 protected cartilage matrix integrity in vitro and in vivo. Additionally, FK506 treatment in vivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Mammalian target of rapamycin inhibitors, temsirolimus and torin 1, attenuate stemness-associated properties and expression of mesenchymal markers promoted by phorbol-myristate-acetate and oncostatin-M in glioblastoma cells.

PubMed

Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

2017-03-01

The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

4-Methoxylonchocarpin attenuates inflammation by inhibiting lipopolysaccharide binding to Toll-like receptor of macrophages and M1 macrophage polarization.

PubMed

Jang, Hyo-Min; Kang, Geum-Dan; Van Le, Thi Kim; Lim, Su-Min; Jang, Dae-Sik; Kim, Dong-Hyun

2017-04-01

The roots of Abrus precatorius (AP, Fabaceae) have traditionally been used in Vietnam and China for the treatment of inflammatory diseases such as stomatitis, asthma, bronchitis, and hepatitis. Therefore, in this study, we isolated 4-methoxylonchocarpin (ML), an anti-inflammatory compound present in AP, and studied its anti-inflammatory effects in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. In lipopolysaccharide (LPS)-stimulated macrophages, ML was found to inhibit nuclear factor (NF)-κB activation and tumor necrosis factor (TNF) and interleukin (IL)-6 expression by inhibiting LPS binding to Toll-like receptor 4 (TLR4) in vitro. Oral administration of ML in mice with TNBS-induced colitis suppressed colon shortening and colonic myeloperoxidase activity. ML treatment significantly inhibited the activation of nuclear factor (NF)-κB and phosphorylation of transforming growth factor β-activated kinase 1 in the colon. Treatment with ML also inhibited TNBS-induced expression of IL-1β, IL-17A, and TNF. While ML reduced the TNBS-induced expression of M1 macrophage markers such as arginase-2 and TNF, it was found to increase the expression of M2 macrophage markers such as arginase-1 and IL-10. In conclusion, oral administration of ML attenuated colitis in mice by inhibiting the binding of LPS to TLR4 on immune cells and increasing the polarization of M1 macrophages to M2 macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-13C]glutamine

NASA Astrophysics Data System (ADS)

Cabella, C.; Karlsson, M.; Canapè, C.; Catanzaro, G.; Colombo Serra, S.; Miragoli, L.; Poggi, L.; Uggeri, F.; Venturi, L.; Jensen, P. R.; Lerche, M. H.; Tedoldi, F.

2013-07-01

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized 13C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-13C]glutamine, which provides a highly sensitive 13C MR marker. With this preparation of hyperpolarized [5-13C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-13C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

CITED1 Expression in Liver Development and Hepatoblastoma12

PubMed Central

Murphy, Andrew J; de Caestecker, Christian; Pierce, Janene; Boyle, Scott C; Ayers, Gregory D; Zhao, Zhiguo; Libes, Jaime M; Correa, Hernan; Walter, Teagan; Huppert, Stacey S; Perantoni, Alan O; de Caestecker, Mark P; Lovvorn, Harold N

2012-01-01

Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF) and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1), a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT). In this current study, CITED1 expression is detected in mouse embryonic liver initially on post-coitum day 10.5 (e10.5), begins to taper by e14.5, and is undetectable in e18.5 and adult livers. CITED1 expression is detected in regenerating murine hepatocytes following liver injury by partial hepatectomy and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Importantly, while CITED1 is undetectable in normal human adult livers, 36 of 41 (87.8%) hepatoblastoma specimens express CITED1, where it is enriched in EMEF specimens compared to specimens of pure fetal histology. CITED1 overexpression in Hep293TT human hepatoblastoma cells induces cellular proliferation and upregulates the Wnt inhibitors Kringle containing transmembrane protein 1 (KREMEN1) and CXXC finger protein 4 (CXXC4). CITED1 mRNA expression correlates with expression of CXXC4 and KREMEN1 in clinical hepatoblastoma specimens. These data show that CITED1 is expressed during a defined time course of liver development and is no longer expressed in the adult liver but is upregulated in regenerating hepatocytes following liver injury. Moreover, as in WT, this embryonic marker is reexpressed in hepatoblastoma and correlates with embryonal histology. These findings identify CITED1 as a novel marker of hepatic progenitor cells that is re-expressed following liver injury and in embryonic liver tumors. PMID:23308048

Dexamethasone Stimulated Gene Expression in Peripheral Blood is a Sensitive Marker for Glucocorticoid Receptor Resistance in Depressed Patients

PubMed Central

Menke, Andreas; Arloth, Janine; Pütz, Benno; Weber, Peter; Klengel, Torsten; Mehta, Divya; Gonik, Mariya; Rex-Haffner, Monika; Rubel, Jennifer; Uhr, Manfred; Lucae, Susanne; Deussing, Jan M; Müller-Myhsok, Bertram; Holsboer, Florian; Binder, Elisabeth B

2012-01-01

Although gene expression profiles in peripheral blood in major depression are not likely to identify genes directly involved in the pathomechanism of affective disorders, they may serve as biomarkers for this disorder. As previous studies using baseline gene expression profiles have provided mixed results, our approach was to use an in vivo dexamethasone challenge test and to compare glucocorticoid receptor (GR)-mediated changes in gene expression between depressed patients and healthy controls. Whole genome gene expression data (baseline and following GR-stimulation with 1.5 mg dexamethasone p.o.) from two independent cohorts were analyzed to identify gene expression pattern that would predict case and control status using a training (N=18 cases/18 controls) and a test cohort (N=11/13). Dexamethasone led to reproducible regulation of 2670 genes in controls and 1151 transcripts in cases. Several genes, including FKBP5 and DUSP1, previously associated with the pathophysiology of major depression, were found to be reliable markers of GR-activation. Using random forest analyses for classification, GR-stimulated gene expression outperformed baseline gene expression as a classifier for case and control status with a correct classification of 79.1 vs 41.6% in the test cohort. GR-stimulated gene expression performed best in dexamethasone non-suppressor patients (88.7% correctly classified with 100% sensitivity), but also correctly classified 77.3% of the suppressor patients (76.7% sensitivity), when using a refined set of 19 genes. Our study suggests that in vivo stimulated gene expression in peripheral blood cells could be a promising molecular marker of altered GR-functioning, an important component of the underlying pathology, in patients suffering from depressive episodes. PMID:22237309

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

PubMed

Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

2017-03-01

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Fibroblast Growth Factor signaling regulates the expansion of A6-expressing hepatocytes in association with AKT-dependent β-catenin activation

PubMed Central

Utley, Sarah; James, David; Mavila, Nirmala; Nguyen, Marie V.; Vendryes, Christopher; Salisbury, S. Michael; Phan, Jennifer; Wang, Kasper S.

2014-01-01

Background & Aims Fibroblast Growth Factors (FGFs) promote the proliferation and survival of hepatic progenitor cells (HPCs) via AKT-dependent β-catenin activation. Moreover, the emergence of hepatocytes expressing the HPC marker A6 during 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury is mediated partly by FGF and β-catenin signaling. Herein, we investigate the role of FGF signaling and AKT-mediated β-catenin activation in acute DDC liver injury. Methods Transgenic mice were fed DDC chow for 14 days concurrent with either Fgf10 over-expression or inhibition of FGF signaling via expression of soluble dominant-negative FGF Receptor (R)-2IIIb. Results After 14 days of DDC treatment, there was an increase in periportal cells expressing FGFR1, FGFR2, and AKT-activated phospho-Serine 552 (pSer552) β-CATENIN in association with up-regulation of genes encoding FGFR2IIIb ligands, Fgf7, Fgf10, and Fgf22. In response to Fgf10 over-expression, there was an increase in the number of pSer552-β-CATENIN(positive)+ive periportal cells as well as cells co-positive for A6 and hepatocyte marker, Hepatocyte Nuclear Factor-4α (HNF4α). A similar expansion of A6+ive cells was observed after Fgf10 over-expression with regular chow and after partial hepatectomy during ethanol toxicity. Inhibition of FGF signaling increased the periportal A6+iveHNF4α+ive cell population while reducing centrolobular A6+ive HNF4α+ive cells. AKT inhibition with Wortmannin attenuated FGF10-mediated A6+iveHNF4α+ive cell expansion. In vitro analyses using FGF10 treated HepG2 cells demonstrated AKT-mediated β-CATENIN activation but not enhanced cell migration. Conclusion During acute DDC treatment, FGF signaling promotes the expansion of A6-expressing liver cells partly via AKT-dependent activation of β-CATENIN expansion of A6+ive periportal cells and possibly by reprogramming of centrolobular hepatocytes. PMID:24365171

Comparison of inducible nitric oxide synthase expression in the brains of Listeria monocytogenes-infected cattle, sheep, and goats and in macrophages stimulated in vitro.

PubMed

Jungi, T W; Pfister, H; Sager, H; Fatzer, R; Vandevelde, M; Zurbriggen, A

1997-12-01

The expression of inducible nitric oxide synthase (iNOS) was studied in the brains of cattle, sheep, and goat that succumbed to a natural infection with Listeria monocytogenes. The lesions in infected brains are characterized by microabscesses, perivascular cuffs, gliosis, glial nodules, and large areas of malacia. Using immunocytochemistry, we detected bacteria in microabscesses, particularly in sheep and goats, and in areas without signs of inflammation, but not in perivascular infiltrates. iNOS was expressed by macrophage (Mphi)-type cells of microabscesses and glial nodules but rarely by Mphi in areas of malacia, as determined by immunohistochemistry with iNOS-specific antibodies. iNOS was not detected in perivascular cuffs. Major histocompatibility complex class II molecules (MHC-II), another marker of cell activation, showed a different pattern of distribution. Perivascular cuffs contained high numbers of MHC-II-positive cells, including some with Mphi characteristics. Microabscesses in sheep and goats showed low expression of MHC-II, particularly in iNOS-expressing cells. In cattle, the expression of markers for activated or recruited phagocytes, the calcium-binding proteins S100A8 and S100A9 (formerly called MRP-8 and MRP-14, respectively), was largely restricted to cells showing weak or undetectable iNOS expression; iNOS-positive Mphi showed a low expression of S100A8 and S100A9. Thus, iNOS is expressed by a restricted subset of Mphi in listeric encephalitis. In cultured sheep and goat Mphi, a low proportion of cells expressed iNOS upon activation by L. monocytogenes and gamma interferon, resulting in nitrite generation at least 1 order of magnitude lower than that in similarly treated cattle Mphi. Since these species differences were much less obvious in vivo, it appears that the well-known species variation in iNOS expression by Mphi could reflect an in vitro phenomenon.

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Li, Yiwei; Ahmad, Aamir; Banerjee, Sanjeev; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes 1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment. PMID:21463919

Children with postsurgical capillary leak syndrome can be distinguished by antigen expression on neutrophils and monocytes

NASA Astrophysics Data System (ADS)

Tarnok, Attila; Pipek, Michal; Valet, Guenter; Richter, Jacqueline; Hambsch, Joerg; Schneider, Peter

1999-04-01

Our initial studies indicate that children who develop post- operative capillary leak syndrome (CLS) following cardiac surgery with cardiopulmonary bypass (CPB) can be distinguished based on their pre-operative level of circulating cytokines an adhesion molecules. We tested flow cytometric analysis of surface antigen expression as a potential assay for risk assessment of CLS. 24th preoperative blood samples were stained with monoclonal antibodies for the adhesion molecules ICAM-1, LFA1, MAC1, (beta) -integrin, activation markers CD25, CD54, CD69, HLA- DR, CD14 or CD4. Cells were measured on a dual-laser flow cytometer calibrated with microbeads. Antigen expression was detected as mean fluorescence intensity. The data indicate, that neutrophils of CLS patients express preoperatively higher levels of LFA1 and monocytes higher levels of HLA-DR and activation markers thus are in a state of activation. This could in combination with surgical trauma and CPB lead to their additional stimulation and migration into sites of inflammation and induce postoperative CLS. It is planned to set up a Flow-Classification program for individual risk assessment. By discriminate analysis over 80 percent of the patients were correctly classified. Our preliminary study indicates that flow cytometry with its low samples requirements and rapid access of the results could be a powerful tool to perform risk assessment prior to pediatric open heart surgery.

Action of the photosensitizer QLT0074 upon human T lymphocytes in vitro

NASA Astrophysics Data System (ADS)

Hunt, David W. C.; Jiang, Huijun; Salmon, Ruth A.; Granville, David J.; North, John R.; Richter, Anna M.

2001-04-01

A new photosensitizer, presently designated QLT0074, may be useful for the treatment of skin conditions, particularly those mediated by T lymphocytes, with photodynamic therapy (PDT). QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. Low concentrations of QLT0074 and blue light were sufficient to induce apoptosis in peripheral blood T cells or Jurkat T lymphoma cells as indicated by expression of the apoptosis-associated mitochondrial 7A6 marker, annexin-V labeling or activation of capsase-3 and cleavage of the capsase substrate poly(ADP-ribose)polymerase (PARP). Flow cytometry studies performed following PDT showed that peripheral blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater degree than T cells with low CD25 levels. This effect of PDT was also shown by the reduction in the percentage of T cells that expressed other activation-associated markers such as very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). In the case of T cells that remained viable following PDT, CD25 expression was lower while CD54, CD95 and HLA-DR levels were unchanged. Thus, PDT with QLT0074 has selective, dose-dependent effects on T cells in vitro.

Propylthiouracil, independent of its antithyroid effect, promotes vascular smooth muscle cells differentiation via PTEN induction.

PubMed

Chen, Wei-Jan; Pang, Jong-Hwei S; Lin, Kwang-Huei; Lee, Dany-Young; Hsu, Lung-An; Kuo, Chi-Tai

2010-01-01

Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have an antiatherosclerotic effect. The aim of this study is to determine the impact of PTU on phenotypic modulation of vascular smooth muscle cells (VSMCs), as phenotypic modulation may contribute to the growth of atherosclerotic lesions and neointimal formation after arterial injury. Propylthiouracil reduced neointimal formation in balloon-injured rat carotid arteries. In vitro, PTU may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins, including calponin and smooth muscle (SM)-myosin heavy chain (SM-MHC). Transient transfection studies in VSMCs demonstrated that PTU induced the activity of SMC marker genes (calponin and SM-MHC) promoters, indicating that PTU up-regulates these genes expression predominantly at the transcriptional level. Furthermore, PTU enhanced the expression of PTEN and inhibition of PTEN by siRNA knockdown blocked PTU-induced activation of contractile proteins expression and promoter activity. In the rat carotid injury model, PTU reversed the down-regulation of contractile proteins and up-regulated PTEN in the neointima induced by balloon injury. Propylthiouracil promotes VSMC differentiation, at lest in part, via induction of the PTEN-mediated pathway. These findings suggest a possible mechanism by which PTU may contribute to its beneficial effects on atherogenesis and neointimal formation after arterial injury.

Release of active TGF-β1 from the latent TGF-β1/GARP complex on T regulatory cells is mediated by integrin β8.

PubMed

Edwards, Justin P; Thornton, Angela M; Shevach, Ethan M

2014-09-15

Activated T regulatory cells (Tregs) express latent TGF-β1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin β8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-β1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Thus, integrin αvβ8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-β1 from the latent TGF-β1/GARP complex on the surface of tTregs.

СD44+/CD24- markers of cancer stem cells in patients with breast cancer of different molecular subtypes.

PubMed

Chekhun, S V; Zadvorny, T V; Tymovska, Yu O; Anikusko, M F; Novak, O E; Polishchuk, L Z

2015-03-01

To determine frequency of tumors with immunohistochemical markers of cancer stem cells (CSC) CD44+/CD24- in patients with breast cancer (BC) of different molecular subtype and to evaluate their prognostic value. Surgical material of 132 patients with BC stage I-II, age from 23 to 75 years, mean age - 50.2 ± 3.1 years was studied. Clinical, immunohistochemical (expression CD44+/CD24-), morphological, statistical. BC is characterized by heterogeneity of molecular subtypes and expression of markers (CD44+/CD24-). Immunohistochemical study of expression of CSC markers in surgical material has detected their expression in 34 (25.4%) patients with BC of different molecular subtypes. The highest frequency of cells with expression of CSC marker was observed in patients with basal molecular subtype (44.8% patients). Most of BC patients with phenotype CD44+/CD24 had stage I of tumor process (34.3%). Statistical processing of data has showen that Yule colligation coefficient equaled 0.28 (р > 0.05) that argues poor correlation between stage of tumor process and number of tumors with positive expression of CSC markers. Statistical processing of data has showen high correlation between presence of cells with expression of CSC markers and metastases of BC in regional lymph nodes (Yule colligation coefficient equals 0.943; р < 0.5). Difference in overall survival of patients with BC of basal molecular subtype depending on expression of CSC CD44+/CD24- markers was detected. Survival of patients with basal BC was reliably higher at lack in tumors of cells with CSC markers CD44+/CD24- and, correspondingly, lower at presence of such cells (р < 0.05). In patients with BC of luminal (A and B), HER-2-positive subtypes, significant change in survival of patients depending on expression of CSC markers was not determined (р > 0.05). Significance of tumor cells with markers CD44+/CD24- within the limits of molecular subtype of BC may be additional criterion for advanced biological characteristic of BC, and in patients with BC of basal molecular subtype - for predictive evaluation of individual potential of tumor to aggressive clinical course.

The XX sex chromosome complement in mice is associated with increased spontaneous lupus compared with XY.

PubMed

Sasidhar, Manda V; Itoh, Noriko; Gold, Stefan M; Lawson, Gregory W; Voskuhl, Rhonda R

2012-08-01

Many autoimmune diseases are characterised by a female predominance. This may be caused by sex hormones, sex chromosomes or both. This report uses a transgenic mouse model to investigate how sex chromosome complement, not confounded by differences in gonadal type, might contribute to lupus pathogenesis. Transgenic NZM2328 mice were created by deletion of the Sry gene from the Y chromosome, thereby separating genetic from gonadal sex. Survival, renal histopathology and markers of immune activation were compared in mice carrying the XX versus the XY(-) sex chromosome complement, with each genotype being ovary bearing. Mice with XX sex chromosome complement compared with XY(-) exhibited poorer survival rates and increased kidney pathology. Splenic T lymphocytes from XX mice demonstrated upregulated X-linked CD40 ligand expression and higher levels of activation markers ex vivo. Increased MMP, TGF and IL-13 production was found, while IL-2 was lower in XX mice. An accumulation of splenic follicular B cells and peritoneal marginal zone B cells was observed, coupled with upregulated costimulatory marker expression on B cells in XX mice. These data show that the XX sex chromosome complement, compared with XY(-), is associated with accelerated spontaneous lupus.

Biomarkers of Coordinate Metabolic Reprogramming in Colorectal Tumors in Mice and Humans

PubMed Central

Manna, Soumen K.; Tanaka, Naoki; Krausz, Kristopher W.; Haznadar, Majda; Xue, Xiang; Matsubara, Tsutomu; Bowman, Elise D.; Fearon, Eric R.; Harris, Curtis C.; Shah, Yatrik M.; Gonzalez, Frank J.

2014-01-01

BACKGROUND & AIMS There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis. METHODS Mass spectrometry-based metabolomic analyses of urine and tissues from wild-type C57BL/6J and ApcMin/+ mice, as well as from mice with azoxymethane-induced tumors, was employed in tandem with gene expression analysis. Metabolomics profiles were also determined on colon tumor and adjacent non-tumor tissues from 39 patients. The effects of β-catenin activity on metabolic profiles were assessed in mice with colon-specific disruption of Apc. RESULTS Thirteen markers were found in urine associated with development of colorectal tumors in ApcMin/+ mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice reflected the observed changes in metabolic products detected in urine; similar changes were observed in mice with azoxymethane-induced tumors and mice with colon-specific activation of β-catenin. The metabolic alterations indicated by markers in urine therefore appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 12 metabolites associated with the same metabolic pathways identified in mice (including amino acid metabolism and polyamine metabolism). Ten metabolites that were increased in tumor tissues, compared with non-tumor tissues (proline, threonine, glutamic acid, arginine, N1-acetylspermidine, xanthine, uracil, betaine, symmetric dimethylarginine, and asymmetric-dimethylarginine), were also increased in urine from tumor-bearing mice. CONCLUSIONS Gene expression and metabolomic profiles of urine and tissue samples from mice with colorectal tumors and of colorectal tumor samples from patients revealed metabolites associated with specific metabolic changes that are indicative of early-stage tumor development. These urine and tissue markers might be used in early detection of colorectal cancer. PMID:24440673

A comparative analysis of gene-expression data of multiple cancer types.

PubMed

Xu, Kun; Cui, Juan; Olman, Victor; Yang, Qing; Puett, David; Xu, Ying

2010-10-27

A comparative study of public gene-expression data of seven types of cancers (breast, colon, kidney, lung, pancreatic, prostate and stomach cancers) was conducted with the aim of deriving marker genes, along with associated pathways, that are either common to multiple types of cancers or specific to individual cancers. The analysis results indicate that (a) each of the seven cancer types can be distinguished from its corresponding control tissue based on the expression patterns of a small number of genes, e.g., 2, 3 or 4; (b) the expression patterns of some genes can distinguish multiple cancer types from their corresponding control tissues, potentially serving as general markers for all or some groups of cancers; (c) the proteins encoded by some of these genes are predicted to be blood secretory, thus providing potential cancer markers in blood; (d) the numbers of differentially expressed genes across different cancer types in comparison with their control tissues correlate well with the five-year survival rates associated with the individual cancers; and (e) some metabolic and signaling pathways are abnormally activated or deactivated across all cancer types, while other pathways are more specific to certain cancers or groups of cancers. The novel findings of this study offer considerable insight into these seven cancer types and have the potential to provide exciting new directions for diagnostic and therapeutic development.

Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration.

PubMed

Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

2010-12-01

Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). C57BL/6J mice were fed diets with or without ABA (100mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with downregulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4(+) and CD8(+) T-lymphocytes in blood and MLN and regulatory T cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration

PubMed Central

Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

2010-01-01

Background & Aims Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). Methods C57BL/6J mice were fed diets with or without ABA (100 mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. Results ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with down-regulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4+ and CD8+ T-lymphocytes in blood and MLN and regulatory T-cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. Conclusions We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. PMID:20236740

ERα inhibited myocardin-induced differentiation in uterine fibroids.

PubMed

Liao, Xing-Hua; Li, Jun-Yan; Dong, Xiu-Mei; Wang, Xiuhong; Xiang, Yuan; Li, Hui; Yu, Cheng-Xi; Li, Jia-Peng; Yuan, Bai-Yin; Zhou, Jun; Zhang, Tong-Cun

2017-01-01

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids. Copyright © 2016 Elsevier Inc. All rights reserved.

Eluted zinc ions stimulate osteoblast differentiation and mineralization in human dental pulp stem cells for bone tissue engineering.

PubMed

Yusa, Kazuyuki; Yamamoto, Osamu; Iino, Mitsuyoshi; Takano, Hiroshi; Fukuda, Masayuki; Qiao, Zhiwei; Sugiyama, Toshihiro

2016-11-01

Zinc is an essential element for proliferation, differentiation and survival in various cell types. In a previous study, we found that zinc ions released from zinc-modified titanium surfaces (eluted zinc ions; EZ) stimulate cell viability, osteoblast marker gene expression and calcium deposition in human bone marrow-derived mesenchymal cells (hBMCs). The aim of the present study was to investigate the effects of EZ on osteoblast differentiation among dental pulp stem cells (DPSCs) in vitro. In this study, we evaluated the effects of EZ on osteogenesis in DPSCs. Osteoblast and osteoclast marker gene expression was evaluated by real-time PCR. We also evaluated alkaline phosphatase (ALP) staining and calcium deposition. We found that EZ stimulated osteoblast marker gene (type I collagen, alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2) expression, vascular endothelial growth factor A (VEGF-A), and TGF-beta signaling pathway-related gene expression after 7days of incubation. Osteoclastogenesis occurs in a receptor for activated nuclear-factor kappa B ligand (RANKL)/osteoprotegerin (OPG)-independent manner. Real-time PCR analysis revealed that EZ did not affect RANKL or OPG mRNA expression. It was also revealed that EZ induced alkaline phosphatase (ALP) staining and calcium deposition in DPSCs. Collectively, these results demonstrate the potential for clinical application to prospective treatment of bone diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.

PubMed

Koenig, Sarah; Probst, Irmelin; Becker, Heinz; Krause, Petra

2006-12-01

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (alpha-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells

PubMed Central

Chen, Zhuo; Evans, W. Howard; Pflugfelder, Stephen C.; Li, De-Quan

2010-01-01

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells. PMID:16424398

Suppressed heat shock protein response in the kidney of exercise-trained diabetic rats.

PubMed

Lappalainen, J; Oksala, N K J; Laaksonen, D E; Khanna, S; Kokkola, T; Kaarniranta, K; Sen, C K; Atalay, M

2018-07-01

Impaired expression of heat shock proteins (HSPs) and increased oxidative stress may contribute to the pathophysiology of diabetes by disrupted tissue protection. Acute exercise induces oxidative stress, whereas exercise training up-regulates endogenous antioxidant defenses and HSP expression. Although diabetic nephropathy is a major contributor to diabetic morbidity, information regarding the effect of HSPs on kidney protection is limited. This study evaluated the effects of eight-week exercise training on kidney HSP expression and markers of oxidative stress at rest and after acute exercise in rats with or without streptozotocin-induced diabetes. Induction of diabetes increased DNA-binding activity of heat shock factor-1, but decreased the expression of HSP72, HSP60, and HSP90. The inflammatory markers IL-6 and TNF-alpha were increased in the kidney tissue of diabetic animals. Both exercise training and acute exercise increased HSP72 and HSP90 protein levels only in non-diabetic rats. On the other hand, exercise training appeared to reverse the diabetes-induced histological changes together with decreased expression of TGF-beta as a key inducer of glomerulosclerosis, and decreased levels of IL-6 and TNF-alpha. Notably, HSP72 and TGF-beta were negatively correlated. In conclusion, impaired HSP defense seems to contribute to kidney injury vulnerability in diabetes and exercise training does not up-regulate kidney HSP expression despite the improvements in histopathological and inflammatory markers. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ezrin Inhibition Up-regulates Stress Response Gene Expression*

PubMed Central

Çelik, Haydar; Bulut, Gülay; Han, Jenny; Graham, Garrett T.; Minas, Tsion Z.; Conn, Erin J.; Hong, Sung-Hyeok; Pauly, Gary T.; Hayran, Mutlu; Li, Xin; Özdemirli, Metin; Ayhan, Ayşe; Rudek, Michelle A.; Toretsky, Jeffrey A.; Üren, Aykut

2016-01-01

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes. PMID:27137931

The neonatal ventral hippocampal lesion model of schizophrenia: effects on dopamine and GABA mRNA markers in the rat midbrain.

PubMed

Lipska, Barbara K; Lerman, Daniel N; Khaing, Zin Z; Weinberger, Daniel R

2003-12-01

The neonatal ventral hippocampal lesion in the rat has been used as a model of schizophrenia, a human disorder associated with changes in markers of dopamine and gamma-aminobutyric acid (GABA) circuits in various regions of the brain. We investigated whether alterations in mRNA markers related to the activity of midbrain dopaminergic and GABAergic neurons are associated with this model. We used in situ hybridization histochemistry to assess expression of mRNAs for dopamine transporter (DAT), tyrosine hydroxylase (TH) and glutamate decarboxylase-67 (GAD67) in the midbrain of adult rats with neonatal and adult ibotenic acid lesions of the ventral hippocampus. Neonatally lesioned rats showed in adulthood significantly reduced expression of DAT mRNA in the substantia nigra and the ventral tegmental area but no changes in the expression of TH and GAD67 mRNAs in these midbrain regions. Adult lesioned rats showed no changes in the expression of any of these genes. As the neonatal ventral hippocampal lesion reproduces many aspects of schizophrenia and is used as an animal model of this disorder, these results suggest that the reduction in DAT mRNA could result from developmental neuropathology in the ventral hippocampus and may thus represent a molecular substrate of the disease process.

A novel dual-marker expression panel for easy and accurate risk stratification of patients with gastric cancer.

PubMed

Kanda, Mitsuro; Murotani, Kenta; Tanaka, Haruyoshi; Miwa, Takashi; Umeda, Shinichi; Tanaka, Chie; Kobayashi, Daisuke; Hayashi, Masamichi; Hattori, Norifumi; Suenaga, Masaya; Yamada, Suguru; Nakayama, Goro; Fujiwara, Michitaka; Kodera, Yasuhiro

2018-05-07

Development of specific biomarkers is necessary for individualized management of patients with gastric cancer. The aim of this study was to design a simple expression panel comprising novel molecular markers for precise risk stratification. Patients (n = 200) who underwent gastrectomy for gastric cancer were randomly assigned into learning and validation sets. Tissue mRNA expression levels of 15 candidate molecular markers were determined using quantitative PCR analysis. A dual-marker expression panel was created according to concordance index (C-index) values of overall survival for all 105 combinations of two markers in the learning set. The reproducibility and clinical significance of the dual-marker expression panel were evaluated in the validation set. The patient characteristics of the learning and validation sets were well balanced. The C-index values of combinations were significantly higher compared with those of single markers. The panel with the highest C-index (0.718) of the learning set comprised SYT8 and MAGED2, which clearly stratified patients into low-, intermediate-, and high-risk groups. The reproducibility of the panel was demonstrated in the validation set. High expression scores were significantly associated with larger tumor size, vascular invasion, lymph node metastasis, peritoneal metastasis, and advanced disease. The dual-marker expression panel provides a simple tool that clearly stratifies patients with gastric cancer into low-, intermediate-, and high risk after gastrectomy. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

PubMed

Orellana, Renan; Kato, Sumie; Erices, Rafaela; Bravo, María Loreto; Gonzalez, Pamela; Oliva, Bárbara; Cubillos, Sofía; Valdivia, Andrés; Ibañez, Carolina; Brañes, Jorge; Barriga, María Isabel; Bravo, Erasmo; Alonso, Catalina; Bustamente, Eva; Castellon, Enrique; Hidalgo, Patricia; Trigo, Cesar; Panes, Olga; Pereira, Jaime; Mezzano, Diego; Cuello, Mauricio A; Owen, Gareth I

2015-04-15

An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis1[W

PubMed Central

Bedoya, Leonor C.; Martínez, Fernando; Orzáez, Diego; Daròs, José-Antonio

2012-01-01

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method. PMID:22238422

Prognostic relevance of Centromere protein H expression in esophageal carcinoma.

PubMed

Guo, Xian-Zhi; Zhang, Ge; Wang, Jun-Ye; Liu, Wan-Li; Wang, Fang; Dong, Ju-Qin; Xu, Li-Hua; Cao, Jing-Yan; Song, Li-Bing; Zeng, Mu-Sheng

2008-08-13

Many kinetochore proteins have been shown to be associated with human cancers. The aim of the present study was to clarify the expression of Centromere protein H (CENP-H), one of the fundamental components of the human active kinetochore, in esophageal carcinoma and its correlation with clinicopathological features. We examined the expression of CENP-H in immortalized esophageal epithelial cells as well as in esophageal carcinoma cells, and in 12 cases of esophageal carcinoma tissues and the paired normal esophageal tissues by RT-PCR and Western blot analysis. In addition, we analyzed CENP-H protein expression in 177 clinicopathologically characterized esophageal carcinoma cases by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. The level of CENP-H mRNA and protein were higher in the immortalized cells, cancer cell lines and most cancer tissues than in normal control tissues. Immunohistochemistry showed that CENP-H was expressed in 127 of 171 ESCC cases (74.3%) and in 3 of 6 esophageal adenocarcinoma cases (50%). Statistical analysis of ESCC cases showed that there was a significant difference of CENP-H expression in patients categorized according to gender (P = 0.013), stage (P = 0.023) and T classification (P = 0.019). Patients with lower CENP-H expression had longer overall survival time than those with higher CENP-H expression. Multivariate analysis suggested that CENP-H expression was an independent prognostic marker for esophageal carcinoma patients. A prognostic value of CENP-H was also found in the subgroup of T3 approximately T4 and N0 tumor classification. Our results suggest that CENP-H protein is a valuable marker of esophageal carcinoma progression. CENP-H might be used as a valuable prognostic marker for esophageal carcinoma patients.

UV-Induced Triggering of a Biomechanical Initiation Switch Within Collagen Promotes Development of a Melanoma-Permissive Microenvironment in the Skin

DTIC Science & Technology

2014-11-01

associated with basement membranes, these M21 melanoma tumors were co- stained for fibroblasts expressing fibroblast activation protein ( FAP ) and a well...characterized marker of blood vessels CD-31 (5,6). As shown in figure 7B below, elevated levels of FAP -expressing -tumor associated fibroblasts were...by staining with Mab D93 in frozen section of tumor tissue from each condition. B). Example of co-distribution of FAP expressing cancer associated

Suppression of dendritic cells' maturation and functions by daidzein, a phytoestrogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yum, Min Kyu; Jung, Mi Young; Cho, Daeho

2011-12-15

Isoflavones are ubiquitous compounds in foods and in the environment in general. Daidzein and genistein, the best known of isoflavones, are structurally similar to 17{beta}-estradiol and known to exert estrogenic effects. They also evidence a broad variety of biological properties, including antioxidant, anti-carcinogenic, anti-atherogenic and anti-osteoporotic activities. Previously, daidzein was reported to increase the phagocytic activity of peritoneal macrophages and splenocyte proliferation, and to inhibit nitric oxide (NO) production in macrophages. However, its potential impacts on immune response in dendritic cells (DCs), antigen-presenting cells that link innate and adaptive immunity, have yet to be clearly elucidated. In this study, wemore » evaluated the effects of isoflavones on the maturation and activation of DCs. Isoflavones (formononetin, daidzein, equol, biochanin A, genistein) were found to differentially affect the expression of CD86, a costimulatory molecule, on lipopolysaccharide (LPS)-stimulated DCs. In particular, daidzein significantly and dose-dependently inhibited the expression levels of maturation-associated cell surface markers including CD40, costimulatory molecules (CD80, CD86), and major histocompatibility complex class II (I-A{sup b}) molecule on LPS-stimulated DCs. Daidzein also suppressed pro-inflammatory cytokine production such as IL-12p40, IL-6 and TNF-{alpha}, whereas it didn't affect IL-10 and IL-1{beta} expression. Furthermore, daidzein enhanced endocytosis and inhibited the allo-stimulatory ability of LPS-stimulated DCs on T cells, indicating that daidzein treatment can inhibit the functional maturation of DCs. These results demonstrate that daidzein may exhibit immunosuppressive activity by inhibiting the maturation and activation of DCs. -- Highlights: Black-Right-Pointing-Pointer Daidzein inhibited expression of maturation-associated cell surface markers in DCs. Black-Right-Pointing-Pointer Daidzein suppressed expression of pro-inflammatory cytokines in LPS-stimulated DCs. Black-Right-Pointing-Pointer Daidzein enhanced endocytosis and inhibited allo-stimulatory ability of DCs. Black-Right-Pointing-Pointer Daidzein exhibited immunosuppressive activity by inhibiting the activation of DCs.« less

Analysis of immune activation and clinical events in acute infectious mononucleosis.

PubMed

Williams, Hilary; Macsween, Karen; McAulay, Karen; Higgins, Craig; Harrison, Nadine; Swerdlow, Anthony; Britton, Kate; Crawford, Dorothy

2004-07-01

The symptoms of infectious mononucleosis (IM) are thought to be caused by T cell activation and cytokine production. Surface lymphocyte activation marker (SLAM)-associated protein (SAP) regulates lymphocyte activation via signals from cell-surface CD244 (2B4) and SLAM (CD150). We followed T cell activation via this SAP/SLAM/CD244 pathway in IM and analyzed whether the results were associated with clinical severity. At diagnosis, SAP, SLAM, and CD244 were significantly up-regulated on CD4 and CD8 T cells; expression decreased during IM, but CD244 and SLAM levels remained higher on CD8 cells 40 days later. There were significantly more lymphocytes expressing CD8 and CD244/CD8 in patients with severe sore throat. The expression of CD8 alone and CD244 on CD8 cells correlated with increased virus load. We suggest that T cells expressing CD244 and SLAM are responsible for the clinical features of IM but that the control of activation is maintained by parallel increased expression of SAP.

Classical and alternative macrophage activation in the lung following ozone-induced oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunil, Vasanthi R., E-mail: sunilva@pharmacy.rutgers.edu; Patel-Vayas, Kinal; Shen, Jianliang

Ozone is a pulmonary irritant known to cause oxidative stress, inflammation and tissue injury. Evidence suggests that macrophages play a role in the pathogenic response; however, their contribution depends on the mediators they encounter in the lung which dictate their function. In these studies we analyzed the effects of ozone-induced oxidative stress on the phenotype of alveolar macrophages (AM). Exposure of rats to ozone (2 ppm, 3 h) resulted in increased expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), as well as heme oxygenase-1 (HO-1) in AM. Whereas 8-OHdG was maximum at 24 h, expression of HO-1 was biphasic increasing after 3 h andmore » 48–72 h. Cleaved caspase-9 and beclin-1, markers of apoptosis and autophagy, were also induced in AM 24 h post-ozone. This was associated with increased bronchoalveolar lavage protein and cells, as well as matrix metalloproteinase (MMP)-2 and MMP-9, demonstrating alveolar epithelial injury. Ozone intoxication resulted in biphasic activation of the transcription factor, NFκB. This correlated with expression of monocyte chemotactic protein‐1, inducible nitric oxide synthase and cyclooxygenase‐2, markers of proinflammatory macrophages. Increases in arginase-1, Ym1 and galectin-3 positive anti-inflammatory/wound repair macrophages were also observed in the lung after ozone inhalation, beginning at 24 h (arginase-1, Ym1), and persisting for 72 h (galectin-3). This was associated with increased expression of pro-surfactant protein-C, a marker of Type II cell proliferation and activation, important steps in wound repair. These data suggest that both proinflammatory/cytotoxic and anti-inflammatory/wound repair macrophages are activated early in the response to ozone-induced oxidative stress and tissue injury. -- Highlights: ► Lung macrophages are highly sensitive to ozone induced oxidative stress. ► Ozone induces autophagy and apoptosis in lung macrophages. ► Proinflammatory and wound repair macrophages are activated early after ozone. ► Oxidative stress may contribute to regulating macrophage phenotype and function.« less

The molecular basis of cytotoxicity of α-spinasterol from Ganoderma resinaceum: Induction of apoptosis and overexpression of p53 in breast and ovarian cancer cell lines.

PubMed

Sedky, Nada K; El Gammal, Zaynab H; Wahba, Amir E; Mosad, Eman; Waly, Zahraa Y; El-Fallal, Amira Ali; Arafa, Reem K; El-Badri, Nagwa

2018-05-01

Despite advances in therapy of breast and ovarian cancers, they still remain among the most imperative causes of cancer death in women. The first can be considered one of the most widespread diseases among females, while the latter is more lethal and needs prompt treatment. Thus, the research field can still benefit from discovery of new compounds that can be of potential use in management of these grave illnesses. We hereby aimed to assess the antitumor activity of the phytosterol α-spinasterol isolated from Ganoderma resinaceum mushroom on human breast cancer cell lines (MCF-7, MDA-MB-231), as well as, on human ovarian cancer cell line (SKOV-3). The anti-tumor activity of α-spinasterol, isolated from the mycelial extract of the Egyptian G. resinaceum, on human breast and ovarian cancer cell lines was evaluated by MTT cell viability assay and AnnexinV/propidium iodide apoptosis assay. The molecular mechanism underlying this effect was assessed by the relative expression of the following markers; tumor suppressor (p53, BRCA1, BRCA2), apoptotic marker (Bax) and cell cycle progression markers (cyclin dependent kinases cdk4/6) using real-time PCR. Cell cycle analysis was performed for the three investigated cancer cell lines to explore the effect on cell cycle progression. Our findings showed that α-spinasterol exhibited a higher antitumor activity on MCF-7 cells relative to SKOV-3 cells, while its lowest antitumor activity was against MDA-MB-231 cells. A significant increase in the expression of p53 and Bax was observed in cells treated with α-spinasterol, while cdk4/6 were significantly down-regulated upon exposure to α-spinasterol. Cell cycle analysis of α-spinasterol treated cells showed a G 0 -G 1 arrest. In conclusion, α-spinasterol isolated from G. resinaceum mushroom exerts a potent inhibitory activity on breast and ovarian cancer cell lines in a time- and dose-dependent manner. This can be reasonified in lights of the compound's ability to increase p53 and Bax expressions, and to lower the expression of cdk4/6. © 2017 Wiley Periodicals, Inc.

Alterations in expression of senescence marker protein-30 gene by 3,3',5-triiodo-L-thyronine (T3).

PubMed

Sar, Pranati; Rath, Bandita; Subudhi, Umakanta; Chainy, Gagan Bihari Nityananda; Supakar, Prakash Chandra

2007-09-01

Thyroid hormone (T3) is essential for normal development, differentiation, and metabolic balance of the body. A toxic dose of T(3) in animals increases the basal metabolic rate and reactive oxygen species production, resulting more oxidative stress through Ca(2+) influx to cytoplasm. Senescence Marker Protein-30 (SMP30) is preferentially expressed in the liver and protects cells against various injuries by enhancement of Ca(2+) efflux to either extra cellular space or intraorganellar spaces through membrane Ca(2+) pump activity. In this paper we report an alteration in the level of SMP30 gene expression using RT-PCR and western blot analysis in T(3) treated female Wistar rats. The results indicate that there is an induction of SMP30 expression during early hours of T(3 )treatment and it declines in severe hyperthyroidism. Therefore, we speculate that SMP30 is regulated by T(3) and might play a protective role in hyperthyroidism.

Transcription of PR3 and Related Myelopoiesis Genes in Peripheral Blood Mononuclear Cells in Active Wegener's Granulomatosis

PubMed Central

Cheadle, Chris; Berger, Alan E.; Andrade, Felipe; James, Regina; Johnson, Kristen; Watkins, Tonya; Park, Jin Kyun; Chen, Yu-Chi; Ehrlich, Eva; Mullins, Marissa; Chrest, Francis; Barnes, Kathleen C.; Levine, Stuart M.

2010-01-01

Objective Wegener's granulomatosis (WG) is a systemic inflammatory disease causing substantial morbidity. This study seeks to understand the biology underlying WG, and to discover markers of disease activity useful in prognosis and treatment guidance. Methods Gene expression profiling was performed using total RNA from PBMC and granulocyte fractions from 41 WG patients and 23 healthy controls. Gene set enrichment analysis (GSEA) was performed to search for candidate WG-associated molecular pathways and disease activity biomarkers. Principal component analysis (PCA) was used to visualize relationships between subgroups of WG patients and controls. Longitudinal changes in PR3 expression were evaluated using RT-PCR, and clinical outcomes including remission status and disease activity were determined using the BVAS-WG. Results We identified 86 genes significantly up-regulated in WG PBMCs and 40 in WG PMNs relative to controls. Genes up-regulated in WG PBMCs were involved in myeloid differentiation, and included the WG autoantigen, PR3. The coordinated regulation of myeloid differentiation genes was confirmed by gene set analysis. Median expression values of the 86 WG PBMC genes were associated with disease activity (p=1.3 × 10−4), and patients expressing these genes at a lower level were only modestly different from healthy controls (p=0.07). PR3 transcription was significantly up-regulated in the PBMCs (p=1.3 ×10−5, FDR=0.002), but not in the PMNs (p=0.03, FDR=0.28) of WG patients, and changes in BVAS-WG tracked with PBMC PR3 RNA levels in a preliminary longitudinal analysis. Conclusion Transcription of PR3 and related myeloid differentiation genes in PBMCs may represent novel markers of disease activity in WG. PMID:20155833

PAI-1, CAIX and VEGFA expressions as prognosis markers in oral squamous cell carcinoma.

PubMed

Peterle, Gabriela Tonini; Maia, Lucas Lima; Trivilin, Leonardo Oliveira; de Oliveira, Mayara Mota; Dos Santos, Joaquim Gasparini; Mendes, Suzanny Oliveira; Stur, Elaine; Agostini, Lidiane Pignaton; Rocha, Lília Alves; Moysés, Raquel Ajub; Cury, Patrícia Maluf; Nunes, Fábio Daumas; Louro, Iúri Drumond; Dos Santos, Marcelo; da Silva, Adriana Madeira Álvares

2018-04-25

In oral squamous cell carcinoma (OSCC), the HIF-1 complex promotes the expression of genes involved in specific mechanisms of cell survival under hypoxic conditions, such as plasminogen activator inhibitor-1 (PAI-1), carbonic anhydrase 9 (CAIX) and vascular endothelial growth factor A (VEGFA). The study aimed to investigate the presence and prognostic value of PAI-1, CAIX, and VEGFA in OSCC. Immunohistochemistry was used to analyze the expressions of these proteins in 52 tumoral tissue samples of patients with OSCC, surgically treated and followed by a minimum of 24 months after surgery. The correlations between proteins expressions and clinicopathological parameters and prognosis were analyzed. Positive PAI-1 membrane expression was significantly associated with local disease relapse (p=0.027). Multivariate analysis revealed that the positive PAI-1 membrane expression is an independent marker for local disease relapse, with approximately 14-fold increased risk when compared to negative expression (OR=14.49; CI=1.40-150.01, p=0.025). Strong PAI-1 cytoplasmic expression was significantly associated with the less differentiation grade (p=0.027). Strong CAIX membrane expression was significantly associated with local disease-free survival (p=0.038). Positive CAIX cytoplasmic expression was significantly associated with lymph node affected (p=0.025) and with disease-specific survival (p=0.022). Multivariate analysis revealed that the positive CAIX cytoplasmic expression is an independent risk factor for disease-related death, increasing their risk approximately 3-fold when compared to negative expression (HR=2.84; CI=1.02-7.87, p=0.045). Positive VEGFA cytoplasmic expression was significantly associated with less differentiation grade (p=0.035). Our results suggest a potential role for these expressions profiles as tumor prognostic markers in OSCC patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Brain c-fos expression patterns induced by emotional stressors differing in nature and intensity.

PubMed

Úbeda-Contreras, Jesús; Marín-Blasco, Ignacio; Nadal, Roser; Armario, Antonio

2018-06-01

Regardless of its particular nature, emotional stressors appear to elicit a widespread and roughly similar brain activation pattern as evaluated by c-fos expression. However, their behavioral and physiological consequences may strongly differ. Here we addressed in adult male rats the contribution of the intensity and the particular nature of stressors by comparing, in a set of brain areas, the number of c-fos expressing neurons in response to open-field, cat odor or immobilization on boards (IMO). These are qualitatively different stressors that are known to differ in terms of intensity, as evaluated by biological markers. In the present study, plasma levels of the adrenocorticotropic hormone (ACTH) demonstrated that intensity increases in the following order: open-field, cat odor and IMO. Four different c-fos activation patterns emerged among all areas studied: (i) positive relationship with intensity (posterior-dorsal medial amygdala, dorsomedial hypothalamus, lateral septum ventral and paraventricular nucleus of the hypothalamus), (ii) negative relationship with intensity (cingulate cortex 1, posterior insular cortex, dorsal striatum, nucleus accumbens and some subdivisions of the hippocampal formation); (iii) activation not dependent on the intensity of the stressor (prelimbic and infralimbic cortex and lateral and basolateral amygdala); and (iv) activation specifically associated with cat odor (ventromedial amygdala and ventromedial hypothalamus). Histone 3 phosphorylation at serine 10, another neuronal activation marker, corroborated c-fos results. Summarizing, deepest analysis of the brain activation pattern elicit by emotional stressor indicated that, in spite of activating similar areas, each stressor possess their own brain activation signature, mediated mainly by qualitative aspects but also by intensity.

Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

PubMed

Fukuda, Tatsuya; Fukuchi, Tomokazu; Yagi, Shinomi; Shiojiri, Nobuyoshi

2016-05-20

The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48-72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

Role of P-glycoprotein on CD69+CD4+ cells in the pathogenesis of proliferative lupus nephritis and non-responsiveness to immunosuppressive therapy

PubMed Central

Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

Introduction P-glycoprotein (P-gp) expression on activated lymphocytes in systemic lupus erythematosus (SLE) plays a role in active efflux of intracellular drugs, resulting in drug resistance. The role of P-gp-expressing lymphocytes in the pathogenesis of SLE remains unclear. The aim of this study was to determine the importance of P-gp+CD4+ cells in organ manifestations in refractory SLE. Methods The proportion of P-gp+CD4+ cells was determined by flow cytometry in peripheral blood of patients with SLE (n=116) and healthy adults (n=10). Renal biopsy specimens were examined by immunohistochemistry for P-gp expression. Results CD69 is a marker of CD4 cell activation. The proportion of both P-gp-expressing CD4+ cells and CD69-expressing CD4+ cells in peripheral blood was higher in SLE than control. The proportion of P-gp+CD69+CD4+ cells correlated with Systemic Lupus Erythematosus Disease Activity Index and was higher in poor responders to corticosteroids. Furthermore, the proportion of P-gp+CD69+CD4+ cells was significantly higher in proliferative lupus nephritis (LN) with poor response to corticosteroids. The efficacy of immunosuppressive therapy depended on the regulation of the proportion of P-gp+CD69+CD4+ cells. Marked accumulation of P-gp+CD4+ cells in renal interstitial tissue and high proportion of peripheral P-gp+CD69+CD4+ cells were noted in patients with proliferative LN. Conclusions The results showed high proportion of P-gp+CD69+CD4+ cells in peripheral blood and their accumulation in renal tissue in patients with proliferative LN refractory to CS therapy, suggesting that P-gp expression on activated CD4+ T cells is a potentially useful marker for refractoriness to treatment and a novel target for treatment. PMID:29225917

Role of P-glycoprotein on CD69+CD4+ cells in the pathogenesis of proliferative lupus nephritis and non-responsiveness to immunosuppressive therapy.

PubMed

Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

P-glycoprotein (P-gp) expression on activated lymphocytes in systemic lupus erythematosus (SLE) plays a role in active efflux of intracellular drugs, resulting in drug resistance. The role of P-gp-expressing lymphocytes in the pathogenesis of SLE remains unclear. The aim of this study was to determine the importance of P-gp + CD4 + cells in organ manifestations in refractory SLE. The proportion of P-gp + CD4 + cells was determined by flow cytometry in peripheral blood of patients with SLE (n=116) and healthy adults (n=10). Renal biopsy specimens were examined by immunohistochemistry for P-gp expression. CD69 is a marker of CD4 cell activation. The proportion of both P-gp-expressing CD4 + cells and CD69-expressing CD4 + cells in peripheral blood was higher in SLE than control. The proportion of P-gp + CD69 + CD4 + cells correlated with Systemic Lupus Erythematosus Disease Activity Index and was higher in poor responders to corticosteroids. Furthermore, the proportion of P-gp + CD69 + CD4 + cells was significantly higher in proliferative lupus nephritis (LN) with poor response to corticosteroids. The efficacy of immunosuppressive therapy depended on the regulation of the proportion of P-gp + CD69 + CD4 + cells. Marked accumulation of P-gp + CD4 + cells in renal interstitial tissue and high proportion of peripheral P-gp + CD69 + CD4 + cells were noted in patients with proliferative LN. The results showed high proportion of P-gp + CD69 + CD4 + cells in peripheral blood and their accumulation in renal tissue in patients with proliferative LN refractory to CS therapy, suggesting that P-gp expression on activated CD4 + T cells is a potentially useful marker for refractoriness to treatment and a novel target for treatment.

Effects of selenium supplementation on the oxidative state of acute heat stress-exposed quails.

PubMed

Del Vesco, A P; Gasparino, E; Zancanela, V; Grieser, D O; Stanquevis, C E; Pozza, P C; Oliveira Neto, A R

2017-02-01

This study aimed to evaluate the effect of heat stress (HS) and selenium supplementation on markers of stress, meat quality and gene expression. For this, meat quails of 42 days of age were fed a diet that either met [0.33 mg/kg, nutritional demand for selenium (SS)] or did not meet [0.11 mg/kg, selenium deficient (SD)] the nutritional demands for selenium during the 7 days of evaluation. In addition, the animals were kept at either a thermal comfort temperature (25 °C) or exposed to HS (38 °C for 24 h). Glutathione synthetase (GSS), glutathione reductase (GSR) and uncoupling protein (UCP) gene expression were influenced by the interaction between temperature and diet. Animals subjected to HS and fed the SS diet exhibited the highest GSS and GSR gene expression. In terms of UCP gene expression, the lowest values were observed in HS animals on the SD diet. Glutathione peroxidase 7 (GPX7) gene expression, body temperature (BT) and creatine kinase (CK) activity were influenced by both selenium supplementation and HS. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and creatinine content all were influenced by the diet/environment interaction. The highest AST activity, ALT activity and creatinine levels were observed in animals that were both on the SD diet and exposed to HS. HS animals also exhibited an increased heterophil/lymphocyte ratio and lower triiodothyronine (T3) hormone levels than birds that remained at the comfortable temperature. Animals subjected to HS and fed with selenium supplemented diet showed better results regarding gene expression and, thus, better results for the activities of enzymes used as stress markers, which could be due to the higher antioxidant capacity provided by the action of the studied genes. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Targeting Therapy Resistant Tumor Vessels

DTIC Science & Technology

2007-05-01

Porkka K, Laakko- nen P, Ruoslahti E. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell...anti-angiogenic therapy. Markers of such vessels will be useful in developing strategies for complete destruction of breast cancer vasculature, and in...express specific markers , and that these lymphatic markers are tumor type specific and distinct from blood vessel markers in the same tumors. The

Network-based expression analyses and experimental validations revealed high co-expression between Yap1 and stem cell markers compared to differentiated cells.

PubMed

Dehghanian, Fariba; Hojati, Zohreh; Esmaeili, Fariba; Masoudi-Nejad, Ali

2018-05-21

The Hippo signaling pathway is identified as a potential regulatory pathway which plays critical roles in differentiation and stem cell self-renewal. Yap1 is a primary transcriptional effector of this pathway. The importance of Yap1 in embryonic stem cells (ESCs) and differentiation procedure remains a challenging question, since two different observations have been reported. To answer this question we used co-expression network and differential co-expression analyses followed by experimental validations. Our results indicate that Yap1 is highly co-expressed with stem cell markers in ESCs but not in differentiated cells (DCs). The significant Yap1 down-regulation and also translocation of Yap1 into the cytoplasm during P19 differentiation was also detected. Moreover, our results suggest the E2f7, Lin28a and Dppa4 genes as possible regulatory nuclear factors of Hippo pathway in stem cells. The present findings are actively consistent with studies that suggested Yap1 as an essential factor for stem cell self-renewal. Copyright © 2018 Elsevier Inc. All rights reserved.

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme

PubMed Central

Tejada-Romero, Belen; Carter, Jean-Michel; Mihaylova, Yuliana; Neumann, Bjoern; Aboobaker, A. Aziz

2015-01-01

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration. PMID:26062938

Murine lung eosinophil activation and chemokine production in allergic airway inflammation

PubMed Central

Rose, C Edward; Lannigan, Joanne A; Kim, Paul; Lee, James J; Fu, Shu Man; Sung, Sun-sang J

2010-01-01

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation. PMID:20622891

The ras/mitogen-activated protein kinase pathway inhibitor and likely tumor suppressor proteins, sprouty 1 and sprouty 2 are deregulated in breast cancer.

PubMed

Lo, Ting Ling; Yusoff, Permeen; Fong, Chee Wai; Guo, Ke; McCaw, Ben J; Phillips, Wayne A; Yang, He; Wong, Esther Sook Miin; Leong, Hwei Fen; Zeng, Qi; Putti, Thomas Choudary; Guy, Graeme R

2004-09-01

Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated Spry expression levels in various cancers and found that Spry1 and Spry2 were down-regulated consistently in breast cancers. Such prevalent patterns of down-regulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. Spry1 and 2 were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mutant of Spry, hSpry2Y55F resulted in enhanced cell proliferation in vitro. The hSpry2Y55F stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An in vivo nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2Y55F stable cells. The consistent down-regulation of Spry1 and 2 in breast cancer and the experimental evidence using a dominant-negative hSpry2Y55F indicate that Spry proteins may actively maintain tissue integrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development.

Improved Cognitive Performance and Reduced Monocyte Activation in Virally Suppressed Chronic HIV Following Dual CCR2 and CCR5 Antagonism.

PubMed

D'Antoni, Michelle L; Paul, Robert H; Mitchell, Brooks I; Kohorn, Lindsay; Fischer, Laurent; Lefebvre, Eric; Seyedkazemi, Star; Nakamoto, Beau K; Walker, Maegen; Kallianpur, Kalpana J; Ogata-Arakaki, Debra; Ndhlovu, Lishomwa C; Shikuma, Cecilia

2018-05-16

To evaluate changes in neuropsychological (NP) performance and in plasma and cell surface markers of peripheral monocyte activation/migration following treatment with cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and type 5 (CCR5) antagonist, in treatment-experienced, HIV-infected individuals. Single-arm, 24-week, open-label clinical trial. HIV-infected individuals on antiretroviral therapy (ART) >1 year with plasma HIV RNA <50 copies/ml and below-normal cognitive performance [defined as age, gender and education-adjusted NP performance (NPZ) <-0.5 in a single cognitive domain or in global performance] were enrolled. Changes over 24 weeks were assessed for global and domain-specific NPZ scores, plasma markers of monocyte/macrophage activation [neopterin, soluble (s)CD14 and sCD163] quantified by ELISA, and CCR2 and CCR5 expression on monocytes and T cells measured by flow cytometry. Seventeen of 20 enrolled participants completed the study. Improvements over 24 weeks were observed in global NPZ [median change (Δ)=0.24; p=0.008], and in cognitive domains of attention (Δ0.23; p=0.011) and working memory (Δ0.44; p=0.017). Plasma levels of sCD163, sCD14, and neopterin decreased significantly (p's<0.01). CCR2 and CCR5 monocyte expression remained unchanged; however, CCR5 levels on CD4 and CD8 T cells and CCR2 expression on CD4 T cells increased (p's<0.01). CVC given over 24 weeks was associated with improved NP test performance and decreased plasma markers of monocyte immune activation in virally-suppressed, HIV-infected participants. These data potentially link changes in monocyte activation to cognitive performance. Further study of CVC for HIV cognitive impairment in a randomized controlled study is warranted.

Gc-protein-derived macrophage activating factor counteracts the neuronal damage induced by oxaliplatin.

PubMed

Morucci, Gabriele; Branca, Jacopo J V; Gulisano, Massimo; Ruggiero, Marco; Paternostro, Ferdinando; Pacini, Alessandra; Di Cesare Mannelli, Lorenzo; Pacini, Stefania

2015-02-01

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce the dual effect of reduction of oxaliplatin-induced neurotoxicity, together with possible synergism in the overall anticancer effect.

A state of reversible compensated ventricular dysfunction precedes pathological remodelling in response to cardiomyocyte-specific activity of angiotensin II type-1 receptor in mice.

PubMed

Frentzou, Georgia A; Drinkhill, Mark J; Turner, Neil A; Ball, Stephen G; Ainscough, Justin F X

2015-08-01

Cardiac dysfunction is commonly associated with high-blood-pressure-induced cardiomyocyte hypertrophy, in response to aberrant renin-angiotensin system (RAS) activity. Ensuing pathological remodelling promotes cardiomyocyte death and cardiac fibroblast activation, leading to cardiac fibrosis. The initiating cellular mechanisms that underlie this progressive disease are poorly understood. We previously reported a conditional mouse model in which a human angiotensin II type-I receptor transgene (HART) was expressed in differentiated cardiomyocytes after they had fully matured, but not during development. Twelve-month-old HART mice exhibited ventricular dysfunction and cardiomyocyte hypertrophy with interstitial fibrosis following full receptor stimulation, without affecting blood pressure. Here, we show that chronic HART activity in young adult mice causes ventricular dysfunction without hypertrophy, fibrosis or cardiomyocyte death. Dysfunction correlated with reduced expression of pro-hypertrophy markers and increased expression of pro-angiogenic markers in the cardiomyocytes experiencing increased receptor load. This stimulates responsive changes in closely associated non-myocyte cells, including the downregulation of pro-angiogenic genes, a dampened inflammatory response and upregulation of Tgfβ. Importantly, this state of compensated dysfunction was reversible. Furthermore, increased stimulation of the receptors on the cardiomyocytes caused a switch in the secondary response from the non-myocyte cells. Progressive cardiac remodelling was stimulated through hypertrophy and death of individual cardiomyocytes, with infiltration, proliferation and activation of fibroblast and inflammatory cells, leading to increased angiogenic and inflammatory signalling. Together, these data demonstrate that a state of pre-hypertrophic compensated dysfunction can exist in affected individuals before common markers of heart disease are detectable. The data also suggest that there is an initial response from the housekeeping cells of the heart to signals emanating from distressed neighbouring cardiomyocytes to suppress those changes most commonly associated with progressive heart disease. We suggest that the reversible nature of this state of compensated dysfunction presents an ideal window of opportunity for personalised therapeutic intervention. © 2015. Published by The Company of Biologists Ltd.

Microglia recapitulate a hematopoietic master regulator network in the aging human frontal cortex

PubMed Central

Wehrspaun, Claudia C.; Haerty, Wilfried; Ponting, Chris P.

2015-01-01

Microglia form the immune system of the brain. Previous studies in cell cultures and animal models suggest altered activation states and cellular senescence in the aged brain. Instead, we analyzed 3 transcriptome data sets from the postmortem frontal cortex of 381 control individuals to show that microglia gene markers assemble into a transcriptional module in a gene coexpression network. These markers predominantly represented M1 and M1/M2b activation phenotypes. Expression of genes in this module generally declines over the adult life span. This decrease was more pronounced in microglia surface receptors for microglia and/or neuron crosstalk than in markers for activation state phenotypes. In addition to these receptors for exogenous signals, microglia are controlled by brain-expressed regulatory factors. We identified a subnetwork of transcription factors, including RUNX1, IRF8, PU.1, and TAL1, which are master regulators (MRs) for the age-dependent microglia module. The causal contributions of these MRs on the microglia module were verified using publicly available ChIP-Seq data. Interactions of these key MRs were preserved in a protein-protein interaction network. Importantly, these MRs appear to be essential for regulating microglia homeostasis in the adult human frontal cortex in addition to their crucial roles in hematopoiesis and myeloid cell-fate decisions during embryogenesis. PMID:26002684

LOX-1 activation by oxLDL triggers an epithelial mesenchymal transition and promotes tumorigenic potential in prostate cancer cells.

PubMed

González-Chavarría, I; Fernandez, E; Gutierrez, N; González-Horta, E E; Sandoval, F; Cifuentes, P; Castillo, C; Cerro, R; Sanchez, O; Toledo, Jorge R

2018-02-01

Obesity is related to an increased risk of developing prostate cancer with high malignancy stages or metastasis. Recent results demonstrated that LOX-1, a receptor associated with obesity and atherosclerosis, is overexpressed in advanced and metastatic prostate cancer. Furthermore, high levels of oxLDL, the main ligand for LOX-1, have been found in patients with advanced prostate cancer. However, the role of LOX-1 in prostate cancer has not been unraveled completely yet. Here, we show that LOX-1 is overexpressed in prostate cancer cells and its activation by oxLDL promotes an epithelial to mesenchymal transition, through of lowered expression of epithelial markers (E-cadherin and plakoglobin) and an increased expression of mesenchymal markers (vimentin, N-cadherin, snail, slug, MMP-2 and MMP-9). Consequently, LOX-1 activation by oxLDL promotes actin cytoskeleton restructuration and MMP-2 and MMP-9 activity inducing prostate cancer cell invasion and migration. Additionally, LOX-1 increased the tumorigenic potential of prostate cancer cells and its expression was necessary for tumor growth in nude mice. In conclusion, our results suggest that oxLDL/LOX-1 could be ones of mechanisms that explain why obese patients with prostate cancer have an accelerated tumor progression and a greater probability of developing metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups.

PubMed

Sumazin, Pavel; Chen, Yidong; Treviño, Lisa R; Sarabia, Stephen F; Hampton, Oliver A; Patel, Kayuri; Mistretta, Toni-Ann; Zorman, Barry; Thompson, Patrick; Heczey, Andras; Comerford, Sarah; Wheeler, David A; Chintagumpala, Murali; Meyers, Rebecka; Rakheja, Dinesh; Finegold, Milton J; Tomlinson, Gail; Parsons, D Williams; López-Terrada, Dolores

2017-01-01

Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large-scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk-stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high-risk tumors were characterized by up-regulated nuclear factor, erythroid 2-like 2 activity; high lin-28 homolog B, high mobility group AT-hook 2, spalt-like transcription factor 4, and alpha-fetoprotein expression; and high coordinated expression of oncofetal proteins and stem-cell markers, while low-risk tumors had low lin-28 homolog B and lethal-7 expression and high hepatic nuclear factor 1 alpha activity. Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (Hepatology 2017;65:104-121). © 2016 by the American Association for the Study of Liver Diseases.

Expression of protease-activated receptor-2 (PAR-2) is related to advanced clinical stage and adverse prognosis in ovarian clear cell carcinoma.

PubMed

Aman, Murasaki; Ohishi, Yoshihiro; Imamura, Hiroko; Shinozaki, Tomoko; Yasutake, Nobuko; Kato, Kiyoko; Oda, Yoshinao

2017-06-01

Recent studies demonstrated that protease-activated receptor-2 (PAR-2) correlates with tumor progression in various tissues. On the other hand, oxidative stress arising from endometriosis has been considered a cause of carcinogenesis in ovarian clear cell carcinoma (OCCC). We previously demonstrated that oxidative stress up-regulates PAR-2 expression, and we conducted the present study to investigate the PAR-2 expression and its relation to clinicopathological factors and oxidative stress in OCCC. We performed an immunohistochemical evaluation in 95 cases of OCCC. For the evaluation of oxidative stress markers, 31 cases of ovarian endometrioid carcinoma (OEC) were also examined. No significant differences in the expression of cyclooxygenase-2 and inducible nitric oxide synthase were observed between OCCC and OEC. Sixty-two percent of the OCCC cases showed high 8-hydroxydeoxyguanosine expression, whereas all of the OEC cases showed almost negative immunoreactivities. The presence of endometriosis did not affect the expression of these oxidative stress markers or prognosis. High PAR-2 expression was observed in 20% (14/71) of the early International Federation of Gynecology and Obstetrics (FIGO) stage cases and 58% (14/24) of the advanced FIGO stage cases. High PAR-2 expression was significantly correlated with advanced FIGO stage and shorter overall survival. We found no correlations between PAR-2 expression and oxidative stress in OCCC. Our results suggest that PAR-2 plays an important role in the progression of OCCC. The expression of 8-hydroxydeoxyguanosine is a characteristic finding of OCCC, indicating that the injury of DNA by oxidative stress may be involved in the carcinogenesis of OCCC. Copyright © 2017 Elsevier Inc. All rights reserved.

1,25-DIHYDROXYVITAMIN D3 INDUCES MONOCYTIC DIFFERENTIATION OF HUMAN MYELOID LEUKEMIA CELLS BY REGULATING C/EBPβ EXPRESSION THROUGH MEF2C

PubMed Central

Zheng, Ruifang; Wang, Xuening; Studzinski, George P.

2015-01-01

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/Enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (Activating Transcription Factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, is mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression. PMID:25448741

The PBX1 lupus susceptibility gene regulates CD44 expression.

PubMed

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A; Choi, Seung-Chul; Morel, Laurence

2017-05-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4 + T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and shows that the lupus-associated isoform PBX1-d has unique molecular functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas α1-adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na+,K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. PMID:20380860

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

PubMed

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Oxidative Injury and Iron Redistribution Are Pathological Hallmarks of Marmoset Experimental Autoimmune Encephalomyelitis.

PubMed

Dunham, Jordon; Bauer, Jan; Campbell, Graham R; Mahad, Don J; van Driel, Nikki; van der Pol, Susanne M A; 't Hart, Bert A; Lassmann, Hans; Laman, Jon D; van Horssen, Jack; Kap, Yolanda S

2017-06-01

Oxidative damage and iron redistribution are associated with the pathogenesis and progression of multiple sclerosis (MS), but these aspects are not entirely replicated in rodent experimental autoimmune encephalomyelitis (EAE) models. Here, we report that oxidative burst and injury as well as redistribution of iron are hallmarks of the MS-like pathology in the EAE model in the common marmoset. Active lesions in the marmoset EAE brain display increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p22phox, p47phox, and gp91phox) and inducible nitric oxide synthase immunoreactivity within lesions with active inflammation and demyelination, coinciding with enhanced expression of mitochondrial heat-shock protein 70 and superoxide dismutase 1 and 2. The EAE lesion-associated liberation of iron (due to loss of iron-containing myelin) was associated with altered expression of the iron metabolic markers FtH1, lactoferrin, hephaestin, and ceruloplasmin. The enhanced expression of oxidative damage markers in inflammatory lesions indicates that the enhanced antioxidant enzyme expression could not counteract reactive oxygen and nitrogen species-induced cellular damage, as is also observed in MS brains. This study demonstrates that oxidative injury and aberrant iron distribution are prominent pathological hallmarks of marmoset EAE thus making this model suitable for therapeutic intervention studies aimed at reducing oxidative stress and associated iron dysmetabolism. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Soluble soy protein peptic hydrolysate stimulates adipocyte differentiation in 3T3-L1 cells.

PubMed

Goto, Tsuyoshi; Mori, Ayaka; Nagaoka, Satoshi

2013-08-01

The molecular mechanisms underlying the potential health benefit effects of soybean proteins on obesity-associated metabolic disorders have not been fully clarified. In this study, we investigated the effects of soluble soybean protein peptic hydrolysate (SPH) on adipocyte differentiation by using 3T3-L1 murine preadipocytes. The addition of SPH increased lipid accumulation during adipocyte differentiation. SPH increased the mRNA expression levels of an adipogenic marker gene and decreased that of a preadipocyte marker gene, suggesting that SPH promotes adipocyte differentiation. SPH induced antidiabetic and antiatherogenic adiponectin mRNA expression and secretion. Moreover, SPH increased the mRNA expression levels of insulin-responsive glucose transporter 4 and insulin-stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, during adipocyte differentiation were up-regulated in 3T3-L1 cells treated with SPH, and lipid accumulation during adipocyte differentiation induced by SPH was inhibited in the presence of a PPARγ antagonist. However, SPH did not exhibit PPARγ ligand activity. These findings indicate that SPH stimulates adipocyte differentiation, at least in part, via the up-regulation of PPARγ expression levels. These effects of SPH might be important for the health benefit effects of soybean proteins on obesity-associated metabolic disorders. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inflammation associated anemia and ferritin as disease markers in SLE

PubMed Central

2012-01-01

Introduction In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia. Methods A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed. Results Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced. Conclusion Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE. PMID:22871034

AMPK does not play a requisite role in regulation of PPARGC1A gene expression via the alternative promoter in endurance-trained human skeletal muscle.

PubMed

Popov, Daniil V; Lysenko, Evgeny A; Butkov, Alexey D; Vepkhvadze, Tatiana F; Perfilov, Dmitriy V; Vinogradova, Olga L

2017-03-01

What is the central question of this study? This study was designed to investigate the role of AMPK in the regulation of PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. What is the main finding and its importance? Low-intensity exercise markedly increased the expression of PGC-1α mRNA via the alternative promoter, without increases in ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. A single dose of the AMPK activator metformin indicated that AMPK was not involved in regulating PGC-1α mRNA expression via the alternative promoter in endurance-trained human skeletal muscle. In human skeletal muscle, PGC-1α is constitutively expressed via the canonical promoter. In contrast, the expression of PGC-1α mRNA via the alternative promoter was found to be highly dependent on the intensity of exercise and to contribute largely to the postexercise increase of total PGC-1α mRNA. This study investigated the role of AMPK in regulating PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. AMPK activation and PGC-1α gene expression were assayed in skeletal muscle of nine endurance-trained men before and after low-intensity exercise (38% of maximal oxygen uptake) and with or without administration of a single dose (2 g) of the AMPK activator metformin. Low-intensity exercise markedly and significantly increased (∼100-fold, P < 0.05) the expression of PGC-1α mRNA via the alternative promoter, without increasing ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. Moreover, in contrast to placebo, metformin increased the level of ACC Ser79/222 phosphorylation immediately after exercise (2.6-fold, P < 0.05). However postexercise expression of PGC-1α gene via the alternative promoter was not affected. This study was unable to confirm that AMPK plays a role in regulating PGC-1α gene expression via the alternative promoter in endurance-trained human skeletal muscle. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

PubMed Central

Li, Yunlong; Zhang, Yong; Lu, Pei; Rayner, Simon; Chen, Shiyun

2012-01-01

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions. PMID:23185557

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production.

PubMed

Polfliet, Machteld M J; Fabriek, Babs O; Daniëls, Wouter P; Dijkstra, Christine D; van den Berg, Timo K

2006-01-01

The monoclonal antibody ED2 is widely used to define macrophages (mphi) in the rat. We have recently identified the ED2 antigen as the rat CD163 glycoprotein. CD163 is a member of the scavenger receptor cysteine-rich group B (SRCR-B) family and functions as a scavenger receptor for hemoglobin-haptoglobin complexes. Moreover, CD163 has also been indicated as a marker for alternatively activated mphi. In the current study, we identify rat CD163/ED2-antigen as a marker for mature tissue mphi. Rat CD163 is constitutively expressed on most subpopulations of mature tissue mphi, including splenic red pulp mphi, thymic cortical mphi, Kupffer cells in the liver, resident bone marrow mphi and central nervous system perivascular and meningeal mphi, but is apparently absent from monocytes. Rat CD163 expression can be promoted by glucocorticoids, and this can be further enhanced by IL4. Finally, engagement of rat CD163 on peritoneal mphi induces the production of pro-inflammatory mediators, including NO, IL-1beta, IL-6 and TNF-alpha. Collectively, our findings identify rat CD163 as a broadly expressed macrophage scavenger receptor that may play a role in the activation of mphi during hemolytic and/or inflammatory conditions.

A comprehensive gene expression analysis at sequential stages of in vitro cardiac differentiation from isolated MESP1-expressing-mesoderm progenitors

PubMed Central

den Hartogh, Sabine C.; Wolstencroft, Katherine; Mummery, Christine L.; Passier, Robert

2016-01-01

In vitro cardiac differentiation of human pluripotent stem cells (hPSCs) closely recapitulates in vivo embryonic heart development, and therefore, provides an excellent model to study human cardiac development. We recently generated the dual cardiac fluorescent reporter MESP1mCherry/wNKX2-5eGFP/w line in human embryonic stem cells (hESCs), allowing the visualization of pre-cardiac MESP1+ mesoderm and their further commitment towards the cardiac lineage, marked by activation of the cardiac transcription factor NKX2-5. Here, we performed a comprehensive whole genome based transcriptome analysis of MESP1-mCherry derived cardiac-committed cells. In addition to previously described cardiac-inducing signalling pathways, we identified novel transcriptional and signalling networks indicated by transient activation and interactive network analysis. Furthermore, we found a highly dynamic regulation of extracellular matrix components, suggesting the importance to create a versatile niche, adjusting to various stages of cardiac differentiation. Finally, we identified cell surface markers for cardiac progenitors, such as the Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4), belonging to the same subfamily of LGR5, and LGR6, established tissue/cancer stem cells markers. We provide a comprehensive gene expression analysis of cardiac derivatives from pre-cardiac MESP1-progenitors that will contribute to a better understanding of the key regulators, pathways and markers involved in human cardiac differentiation and development. PMID:26783251

Increased Circulating FGF23 Does Not Lead to Cardiac Hypertrophy in the Male Hyp Mouse Model of XLH.

PubMed

Liu, Eva S; Thoonen, Robrecht; Petit, Elizabeth; Yu, Binglan; Buys, Emmanuel S; Scherrer-Crosbie, Marielle; Demay, Marie B

2018-05-01

Serum levels of fibroblast growth factor 23 (FGF23) markedly increase with renal impairment, with FGF23 levels correlating with the presence of left ventricular hypertrophy (LVH) and mortality in patients with chronic kidney disease (CKD). FGF23 activates calcineurin/nuclear factor of activated T cell (NFAT) signaling and induces hypertrophy in murine cardiomyocytes. X-linked hypophosphatemia (XLH) is characterized by high circulating levels of FGF23 but, in contrast to CKD, is associated with hypophosphatemia. The cardiac effects of high circulating levels of FGF23 in XLH are not well defined. Thus, studies were undertaken to define the cardiac phenotype in the mouse model of XLH (Hyp mice). Echocardiographic and histological analyses demonstrated that Hyp left ventricles (LVs) are smaller than those of wild-type mice. Messenger RNA expression of cardiac hypertrophy markers was not altered in the LV or right ventricle of Hyp mice. However, the Hyp LVs had increased expression of the NFAT target genes NFATc1 and RCAN1. To determine whether phosphate alone can induce markers of hypertrophy, differentiated C2C12 myocytes were treated with phosphate. Phosphate treatment increased expression of cardiac hypertrophy markers, supporting a primary role for phosphate in inducing LVH. Although previous studies showed that increased circulating FGF23 and phosphate levels are associated with LVH, our results demonstrated that in XLH, high circulating levels of FGF23 in the setting of hypophosphatemia do not induce cardiac hypertrophy.

Cell-surface markers for colon adenoma and adenocarcinoma

PubMed Central

Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S.; Wojtkowiak, Jonathan W.; Stark, Valerie E.; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L.

2016-01-01

Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC. PMID:26894861

Cell-surface markers for colon adenoma and adenocarcinoma.

PubMed

Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S; Wojtkowiak, Jonathan W; Stark, Valerie E; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L

2016-04-05

Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC.

Cell-surface marker discovery for lung cancer

PubMed Central

Cohen, Allison S.; Khalil, Farah K.; Welsh, Eric A.; Schabath, Matthew B.; Enkemann, Steven A.; Davis, Andrea; Zhou, Jun-Min; Boulware, David C.; Kim, Jongphil; Haura, Eric B.; Morse, David L.

2017-01-01

Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan–Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients. PMID:29371917

Angiogenesis and lymphangiogenesis are downregulated in primary breast cancer

PubMed Central

Boneberg, E-M; Legler, D F; Hoefer, M M; Öhlschlegel, C; Steininger, H; Füzesi, L; Beer, G M; Dupont-Lampert, V; Otto, F; Senn, H-J; Fürstenberger, G

2009-01-01

Background: Angiogenesis and lymphangiogenesis are considered to play key roles in tumour growth, progression and metastasis. However, targeting tumour angiogenesis in clinical trials showed only modest efficacy. We therefore scrutinised the concept of tumour angiogenesis and lymphangiogenesis by analysing the expression of crucial markers involved in these processes in primary breast cancer. Methods: We analysed the expression of angiogenic, lymphangiogenic or antiangiogenic factors, their respective receptors and specific markers for endothelial and lymphendothelial cells by quantitative real-time RT-PCR in primary breast cancer and compared the expression profiles to non-cancerous, tumour-adjacent tissues and breast tissues from healthy women. Results: We found decreased mRNA amounts of major angiogenic and lymphangiogenic factors in tumour compared to healthy tissues, whereas antiangiogenic factors were upregulated. Concomitantly, angiogenic and lymphangiogenic receptors were downregulated in breast tumours. This antiangiogenic, antilymphangiogenic microenvironment was even more pronounced in aggressive tumours and accompanied by reduced amounts of endothelial and lymphatic endothelial cell markers. Conclusion: Primary breast tumours are not a site of highly active angiogenesis and lymphangiogenesis. Selection for tumour cells that survive with minimal vascular supply may account for this observation in clinical apparent tumours. PMID:19672262

CD25 is expressed by canine cutaneous mast cell tumors but not by cutaneous connective tissue mast cells.

PubMed

Meyer, A; Gruber, A D; Klopfleisch, R

2012-11-01

Canine cutaneous mast cell tumors (MCT) of different histological grades have distinct biological behaviors. However, little is known about underlying molecular mechanisms that lead to tumor development and increasing malignancy with higher tumor grade. Recent studies have identified the interleukin-2 receptor (IL-2R) subunits CD25 and CD2 as markers that distinguish nonneoplastic from neoplastic mast cells in human systemic mastocytosis. In this study, their potential as a marker for canine MCT and their possible impact on MCT carcinogenesis were evaluated. mRNA expression levels of both genes were compared between grade 1 (n = 12) and grade 3 (n = 8) MCT, and protein expression levels of CD25 were compared in 90 MCT of different tumor grades. mRNA expression levels of both CD25 and CD2 were upregulated in grade 3 MCT. In contrast, CD25 protein was expressed by fewer tumor cells and at decreased levels in grade 3 tumors, while most grade 1 MCT had strong CD25 protein expression. Moreover, CD25 was not expressed by nonneoplastic, resting cutaneous mast cells, while few presumably activated mast cells in tissue samples from dogs with allergic dermatitis had weak CD25 expression. Taken together, these findings suggest that CD25 may play a critical role in early MCT development and may be a stimulatory factor in grade 1 MCT, while grade 3 MCT seem to be less dependent on CD25. Because of the low number of CD25-positive tumor cells in high-grade tumors, the usefulness of CD25 as a tumor marker is, however, questionable.

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets

PubMed Central

Marchetto, Aruna; Gerke, Julia S.; Rubio, Rebeca Alba; Kiran, Merve M.; Musa, Julian; Knott, Maximilian M. L.; Ohmura, Shunya; Li, Jing; Akpolat, Nusret; Akatli, Ayse N.; Özen, Özlem; Dirksen, Uta; Hartmann, Wolfgang; de Alava, Enrique; Baumhoer, Daniel; Sannino, Giuseppina; Kirchner, Thomas; Grünewald, Thomas G. P.

2018-01-01

Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care. PMID:29416716

Adipose tissue macrophages in non-rodent mammals: a comparative study.

PubMed

Ampem, Grace; Azegrouz, Hind; Bacsadi, Árpád; Balogh, Lajos; Schmidt, Susanne; Thuróczy, Julianna; Röszer, Tamás

2016-02-01

The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.

Monoamine oxidase A (MAO-A): a signature marker of alternatively activated monocytes/macrophages

PubMed Central

Cathcart, Martha K.; Bhattacharjee, Ashish

2015-01-01

Monocytes/macrophages are versatile cells centrally involved in host defense and immunity. Th1 cytokines induce a classical activation program in monocytes/macrophages leading to a proinflammatory M1 macrophage phenotype while Th2 cytokines IL-4 and IL-13 promote monocyte differentiation into an alternatively activated, anti-inflammatory M2 macrophage phenotype. Although monoamine oxidase A (MAO-A) is primarily known for its action in the nervous system, several recent studies have identified MAO-A as a signature marker of alternative activation of monocytes/macrophages. In this brief review we explore the signaling pathways/molecules that regulate MAO-A expression in alternatively activated monocytes/macrophages. We further discuss the contribution of MAO-A to the resolution of inflammation and identify potential therapeutic targets for controlling inflammation. Altogether this review provides deeper insight into the role of MAO-A in alternative activation of monocytes/macrophages and their participation in the inflammatory response. PMID:26052543

Monoamine oxidase A (MAO-A): a signature marker of alternatively activated monocytes/macrophages.

PubMed

Cathcart, Martha K; Bhattacharjee, Ashish

Monocytes/macrophages are versatile cells centrally involved in host defense and immunity. Th1 cytokines induce a classical activation program in monocytes/macrophages leading to a proinflammatory M1 macrophage phenotype while Th2 cytokines IL-4 and IL-13 promote monocyte differentiation into an alternatively activated, anti-inflammatory M2 macrophage phenotype. Although monoamine oxidase A (MAO-A) is primarily known for its action in the nervous system, several recent studies have identified MAO-A as a signature marker of alternative activation of monocytes/macrophages. In this brief review we explore the signaling pathways/molecules that regulate MAO-A expression in alternatively activated monocytes/macrophages. We further discuss the contribution of MAO-A to the resolution of inflammation and identify potential therapeutic targets for controlling inflammation. Altogether this review provides deeper insight into the role of MAO-A in alternative activation of monocytes/macrophages and their participation in the inflammatory response.

Magnolol promotes thermogenesis and attenuates oxidative stress in 3T3-L1 adipocytes.

PubMed

Parray, Hilal Ahmad; Lone, Jameel; Park, Jong Pil; Choi, Jang Won; Yun, Jong Won

2018-06-01

The aim of this study was to explore the browning and antioxidative effects of magnolol in 3T3-L1 adipocytes, as recruitment of beige-like adipocytes (browning) by natural compounds is being considered as a promising strategy to fight against obesity. Magnolol-induced browning effect was evaluated by determining the expression levels of specific marker genes and proteins using real-time polymerase chain reaction and immunoblotting, respectively. Induction of thermogenesis and suppression of oxidative stress in 3T3-L1 adipocytes were further validated by immunofluorescence. Magnolol significantly enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cd137, Prdm16, Cidea, and Tbx1) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of the AMPK, PPARγ, and protein kinase A (PKA) pathways. In addition, magnolol up-regulated key fatty acid oxidation and lipolytic markers (CPT1, ACSL1, SIRT1, and PLIN) and down-regulated lipogenic markers (FAS and SREBP1). Magnolol also reduced the production and release of reactive oxygen species. The current data suggest possible roles for magnolol in browning of white adipocytes, augmentation of lipolysis, and thermogenesis, as well as repression of oxidative stress and lipogenesis. Thus, magnolol may be explored as a potentially promising therapeutic agent for the prevention of obesity and other metabolic disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

Examination of the expanding pathways for the regulation of p21 expression and activity.

PubMed

Jung, Yong-Sam; Qian, Yingjuan; Chen, Xinbin

2010-07-01

p21(Waf1/Cip1/Sdi1) was originally identified as an inhibitor of cyclin-dependent kinases, a mediator of p53 in growth suppression and a marker of cellular senescence. p21 is required for proper cell cycle progression and plays a role in cell death, DNA repair, senescence and aging, and induced pluripotent stem cell reprogramming. Although transcriptional regulation is considered to be the initial control point for p21 expression, there is growing evidence that post-transcriptional and post-translational regulations play a critical role in p21 expression and activity. This review will briefly discuss the activity of p21 and focus on current knowledge of the determinants that control p21 transcription, mRNA stability and translation, and protein stability and activity. (c) 2010 Elsevier Inc. All rights reserved.

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells.

PubMed

Raabe, Oksana; Shell, Katja; Würtz, Antonia; Reich, Christine Maria; Wenisch, Sabine; Arnhold, Stefan

2011-08-01

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.

Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line

PubMed Central

Tesfazghi, Sara; Eide, Jacob; Dammalapati, Ajitha; Korlesky, Colin; Wyche, Thomas P; Bugni, Tim S; Chen, Herbert; Jaskula-Sztul, Renata

2013-01-01

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC. Thiocoraline is a potent Notch pathway activator and an inhibitor of medullary thyroid cancer cell line (MTC-TT) cell proliferation at low nanomolar concentrations. These results provide evidence for the use of thiocoraline as a potential treatment for intractable MTC. PMID:24403239

Mass Cytometry Identifies Distinct Lung CD4+ T Cell Patterns in Löfgren’s Syndrome and Non-Löfgren’s Syndrome Sarcoidosis

PubMed Central

Kaiser, Ylva; Lakshmikanth, Tadepally; Chen, Yang; Mikes, Jaromir; Eklund, Anders; Brodin, Petter; Achour, Adnane; Grunewald, Johan

2017-01-01

Sarcoidosis is a granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4+ T cells in the lungs. Disease phenotypes Löfgren’s syndrome (LS) and “non-LS” differ in terms of clinical manifestations, genetic background, HLA association, and prognosis, but the underlying inflammatory mechanisms largely remain unknown. Bronchoalveolar lavage fluid cells from four HLA-DRB1*03+ LS and four HLA-DRB1*03− non-LS patients were analyzed by mass cytometry, using a panel of 33 unique markers. Differentially regulated CD4+ T cell populations were identified using the Citrus algorithm, and t-stochastic neighborhood embedding was applied for dimensionality reduction and single-cell data visualization. We identified 19 individual CD4+ T cell clusters differing significantly in abundance between LS and non-LS patients. Seven clusters more frequent in LS patients were characterized by significantly higher expression of regulatory receptors CTLA-4, PD-1, and ICOS, along with low expression of adhesion marker CD44. In contrast, 12 clusters primarily found in non-LS displayed elevated expression of activation and effector markers HLA-DR, CD127, CD39, as well as CD44. Hierarchical clustering further indicated functional heterogeneity and diverse origins of T cell receptor Vα2.3/Vβ22-restricted cells in LS. Finally, a near-complete overlap of CD8 and Ki-67 expression suggested larger influence of CD8+ T cell activity on sarcoid inflammation than previously appreciated. In this study, we provide detailed characterization of pulmonary T cells and immunological parameters that define separate disease pathways in LS and non-LS. With direct association to clinical parameters, such as granuloma persistence, resolution, or chronic inflammation, these results provide a valuable foundation for further exploration and potential clinical application. PMID:28955342

Aqueous Ethanolic Extract of Tinospora cordifolia as a Potential Candidate for Differentiation Based Therapy of Glioblastomas

PubMed Central

Mishra, Rachana; Kaur, Gurcharan

2013-01-01

Glioblastomas are the most aggressive primary brain tumors and their heterogeneity and complexity often renders them non responsive to various conventional treatments. Search for herbal products having potential anti-cancer activity is an active area of research in the Indian traditional system of medicine i.e., Ayurveda. Tinospora cordifolia, also named as ‘heavenly elixir’ is used in various ayurvedic decoctions as panacea to treat several body ailments. The current study investigated the anti-brain cancer potential of 50% ethanolic extract of Tinospora cordifolia (TCE) using C6 glioma cells. TCE significantly reduced cell proliferation in dose-dependent manner and induced differentiation in C6 glioma cells, resulting in astrocyte-like morphology as indicated by phase contrast images, GFAP expression and process outgrowth data of TCE treated cells which exhibited higher number and longer processes than untreated cells. Reduced proliferation of cells was accompanied by enhanced expression of senescence marker, mortalin and its translocation from perinuclear to pancytoplasmic spaces. Further, TCE showed anti-migratory and anti-invasive potential as depicted by wound scratch assay and reduced expression of plasticity markers NCAM and PSA-NCAM along with MMP-2 and 9. On analysis of the cell cycle and apoptotic markers, TCE treatment was seen to arrest the C6 cells in G0/G1 and G2/M phase, suppressing expression of G1/S phase specific protein cyclin D1 and anti-apoptotic protein Bcl-xL, thus supporting its anti-proliferative and apoptosis inducing potential. Present study provides the first evidence for the presence of anti-proliferative, differentiation-inducing and anti-migratory/anti-metastatic potential of TCE in glioma cells and possible signaling pathways involved in its mode of action. Our primary data suggests that TCE and its active components may prove to be promising phytotherapeutic interventions in gliobalstoma multiformae.  PMID:24205314

ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation through the JNK signaling pathway.

PubMed

Jeong, Byung-Chul

2018-05-15

Tumor necrosis factor (TNF)-α, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, the precise interactions between ATF3 and the TNF-α signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-α-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-α downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-α. Conversely, the enforced expression of Atf3 enhanced the TNF-α-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-α-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-α expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-α on osteoblast differentiation and that the TNF-α-activated JNK pathway is responsible for the induction of Atf3 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

Impaired autophagy activity is linked to elevated ER-stress and inflammation in aging adipose tissue

PubMed Central

Ghosh, Amiya Kumar; Mau, Theresa; O'Brien, Martin; Garg, Sanjay; Yung, Raymond

2016-01-01

Adipose tissue dysfunction in aging is associated with inflammation, metabolic syndrome and other diseases. We propose that impaired protein homeostasis due to compromised lysosomal degradation (micro-autophagy) might promote aberrant ER stress response and inflammation in aging adipose tissue. Using C57BL/6 mouse model, we demonstrate that adipose tissue-derived stromal vascular fraction (SVF) cells from old (18-20 months) mice have reduced expression of autophagy markers as compared to the younger (4-6 months) cohort. Elevated expressions of ER-stress marker CHOP and autophagy substrate SQSTM1/p62 are observed in old SVFs compared to young, when treated with either vehicle or with thapsigargin (Tg), an ER stress inducer. Treatment with bafilomycin A1 (Baf), a vacuolar-type H (+)-ATPase, or Tg elevated expressions of CHOP, and SQSTM1/p62 and LC-3-II, in 3T3-L1-preadipocytes. We also demonstrate impaired autophagy activity in old SVFs by analyzing increased accumulation of autophagy substrates LC3-II and p62. Compromised autophagy activity in old SVFs is correlated with enhanced release of pro-inflammatory cytokines IL-6 and MCP-1. Finally, SVFs from calorie restricted old mice (CR-O) have shown enhanced autophagy activity compared to ad libitum fed old mice (AL-O). Our results support the notion that diminished autophagy activity with aging contributes to increased adipose tissue ER stress and inflammation. PMID:27777379

IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla

PubMed Central

Ma, Shu; Liu, Genxia; Jin, Lin; Pang, Xiyao; Wang, Yanqiu; Wang, Zilu; Yu, Yan; Yu, Jinhua

2016-01-01

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways. PMID:27833148

Macrophage activation by glycoprotein isolated from Dioscorea batatas

PubMed Central

Huong, Pham Thi Thu

2011-01-01

We demonstrate that glycoprotein isolated from Dioscorea batatas (GDB) activates macrophage function. Analysis of the infiltration of macrophages into peritoneal cavity showed GDB treatment significantly increased the recruitment of macrophages into the peritoneal cavity. In order to further confirm and investigate the mechanism of GDB on macrophage activation, we analyzed the effects of GDB on the cytokine expression including IL-1β, TNF-α, and IL-6 in mouse peritoneal macrophages. GDB increased the expression of IL-1β, TNF-α, and IL-6. Cytokine induction by GDB was further confirmed by RT-PCR and ELISA in mouse macrophage cell line, RAW264.7 cells. Treatment of RAW264.7 cells with GDB produced strong induction of NF-κB DNA binding and MAPK phosphorylation, markers for macrophage activation and important factors for cytokine gene expression. Collectively, this series of experiments indicates that GDB stimulates macrophage activation. PMID:24278568

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway.

PubMed

Hou, Gang; Zhao, Huiqing; Teng, Haijun; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Guo, Zhiliang; Wei, Li; Yao, Hui; Xu, Yichun

2018-01-01

Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured by SA-β-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-β-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration. © 2018 The Author(s). Published by S. Karger AG, Basel.

Monoterpene limonene induces brown fat-like phenotype in 3T3-L1 white adipocytes.

PubMed

Lone, Jameel; Yun, Jong Won

2016-05-15

Several dietary compounds that are able to induce the brown fat-like phenotype in white adipocytes have been considered for treatment of obesity due to their ability to increase energy expenditure. Here, we report that limonene induces the brown fat-like phenotype in 3T3-L1 adipocytes by increasing expression of brown adipocyte-specific genes and proteins. Limonene-induced browning in white adipocytes was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR, immunoblot analysis, and immunocytochemical staining. Limonene enhanced mitochondrial biogenesis, as evidenced by increased mitochondrial content and immunofluorescent intensity. Limonene also significantly elevated protein levels of HSL, PLIN, p-AMPK, p-ACC, ACO, COX4, CPT1, and CYT C, suggesting its possible role in enhancement of lipolysis and lipid catabolism. Increased expression of PRDM16, UCP1, C/EBPβ, and other brown fat-specific markers by limonene was possibly mediated by activation of β3-adnergenic receptor (β3-AR), as inhibition of β3-AR inhibited up-regulation of brown fat-specific markers. Similarly, limonene-mediated activation of ERK and up-regulation of key brown adipocyte specific markers were eliminated by treatment with ERK antagonist. Taken together, these results suggest that limonene induces browning of 3T3-L1 adipocytes via activation of β3-AR and the ERK signaling pathway. In conclusion, our findings suggest that limonene plays a dual modulatory role in induction of the brown adipocyte-like phenotype as well as promotion of lipid metabolism and thus may have potential therapeutic implications for treatment of obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Antifibrotic Mechanism of Pinocembrin: Impact on Oxidative Stress, Inflammation and TGF-β /Smad Inhibition in Rats.

PubMed

Said, Marwa M; Azab, Samar S; Saeed, Noha M; El-Demerdash, Ebtehal

2018-03-01

The present study aimed to elucidate the potential antifibrotic effects of pinocembrin (PIN), a flavanone found abundantly in honey and propolis, by studying its effect on different oxidative stress, inflammatory and fibrosis markers in an experimental model of CCl4-induced liver fibrosis. PIN (20 mg/kg) was given orally 3 times/week for 6 consecutive weeks alternating with CCl4 (0.5 mL/kg, 1:1 mixture with corn oil, i. p.) twice weekly. Different hepatotoxicity indices, oxidative stress, inflammatory and liver fibrosis markers were assessed. PIN significantly restored liver transaminases and total cholesterol to normal levels. Also, PIN ameliorated oxidative stress injury evoked by CCl4 as evidenced by inhibition of reduced glutathione depletion and lipid peroxidation as well as elevation of antioxidant enzyme superoxide dismutase (SOD). Further, PIN upregulated the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), thereby inducing the expression and activity of the cytoprotective enzyme hemeoxygenase-1 (HO-1). Moreover, PIN alleviated pro-inflammatory cytokines such as TNF-α via inhibiting nuclear factor-κB (NF-κB) activation. As markers of fibrosis, collagen and α-SMA expression increased markedly in the CCl4 group and PIN prevented these alterations. In addition, PIN down-regulated TGFβ1 and p-Smad2/3, thereby inhibiting TGFβ1/Smad signaling pathway. These results suggest that PIN possess potent antifibrotic effects that can be explained on its antioxidant properties. It ameliorates oxidative stress and inflammation during induction of fibrogenesis via its ability to augment celular antioxidant defenses, activating Nrf2-mediated HO-1 expression and modulating NF-κB and TGF-β1/Smad signaling pathway.

PSC-derived Galectin-1 inducing epithelial-mesenchymal transition of pancreatic ductal adenocarcinoma cells by activating the NF-κB pathway

PubMed Central

Tang, Dong; Zhang, Jingqiu; Yuan, Zhongxu; Zhang, Hongpeng; Chong, Yang; Huang, Yuqin; Wang, Jie; Xiong, Qingquan; Wang, Sen; Wu, Qi; Tian, Ying; Lu, Yongdie; Ge, Xiao; Shen, Wenjing; Wang, Daorong

2017-01-01

Galectin-1 has previously been shown to be strongly expressed in activated pancreatic stellate cells (PSCs) and promote the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms by which Galectin-1 promotes the malignant behavior of pancreatic cancer cells remain unclear. In this study, we examined the effects of Galectin-1 knockdown or overexpression in PSCs co-cultured with pancreatic cancer (PANC-1) cells. Immunohistochemical analysis showed expression of epithelial-mesenchymal transition (EMT) markers and MMP9 were positively associated with the expression of Galectin-1 in 66 human PDAC tissues. In addition, our in vitro studies showed PSC-derived Galectin-1 promoted the proliferation, invasion, and survival (anti-apoptotic effects) of PANC-1 cells. We also showed PSC-derived Galectin-1 induced EMT of PANC-1 cells and activated the NF-кB pathway in vitro. Our mixed (PSCs and PANC-1 cells) mouse orthotopic xenograft model indicated that overexpression of Galectin-1 in PSCs significantly promoted the proliferation, growth, invasion, and liver metastasis of the transplanted tumor. Moreover, Galectin-1 overexpression in PSCs was strongly associated with increased expression of EMT markers in both the orthotopic xenograft tumor in the pancreas and in metastatic lesions of naked mice. We conclude that PSC-derived Galectin-1 promotes the malignant behavior of PDAC by inducing EMT via activation of the NF-κB pathway. Our results suggest that targeting Galectin-1 in PSCs could represent a promising therapeutic strategy for PDAC progression and metastasis. PMID:29156810

Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis

PubMed Central

Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia; Jiménez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Ibarretxe, Gaskon; Unda, Fernando J.

2016-01-01

Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity. Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. PMID:27066482

Antioxidant defense and apoptotic effectors in ascorbic acid and β-glycerophosphate-induced osteoblastic differentiation.

PubMed

Chaves Neto, Antonio Hernandes; Machado, Daisy; Yano, Cláudia Lumy; Ferreira, Carmen Veríssima

2011-01-01

MC3T3-E1 cells grown in the presence of ascorbic acid and β-glycerophosphate (AA/β-GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3-E1 cells are more sensitive to hydrogen peroxide-induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3-E1 cells (cell differentiation was induced by treatment with AA/β-GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S-transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/β-GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/β-GP increased the Bax/Bcl-2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress. © 2011 The Authors. Journal compilation © 2011 Japanese Society of Developmental Biologists.

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

PubMed

Kalantari, Elham; Asgari, Mojgan; Nikpanah, Seyedehmoozhan; Salarieh, Naghme; Asadi Lari, Mohammad Hossein; Madjd, Zahra

2017-10-01

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Wise, a context-dependent activator and inhibitor of Wnt signalling.

PubMed

Itasaki, Nobue; Jones, C Michael; Mercurio, Sara; Rowe, Alison; Domingos, Pedro M; Smith, James C; Krumlauf, Robb

2003-09-01

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.

PubMed

Wang, Lan; Yin, Jie; Wang, Xuefei; Shao, Miaomiao; Duan, Fangfang; Wu, Weicheng; Peng, Peike; Jin, Jing; Tang, Yue; Ruan, Yuanyuan; Sun, Yihong; Gu, Jianxin

2016-05-01

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples. CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

ACE2 activation by xanthenone prevents leptin-induced increases in blood pressure and proteinuria during pregnancy in Sprague-Dawley rats.

PubMed

Ibrahim, Hisham Saleh; Froemming, Gabrielle Ruth Anisah; Omar, Effat; Singh, Harbindar Jeet

2014-11-01

This study investigates the effect of ACE2 activation on leptin-induced changes in systolic blood pressure (SBP), proteinuria, endothelial activation and ACE2 expression during pregnancy in Sprague-Dawley rats. Pregnant rats were given subcutaneous injection of either saline, or leptin, or leptin plus xanthenone (ACE2 activator), or xanthenone (XTN) alone. SBP, serum ACE, ACE2, endothelin-1, E-selectin and ICAM-1 levels were estimated; also their gene expressions were determined in the kidney and aorta respectively. Compared to control, SBP was higher in the leptin-only treated group (P<0.001) and lower in rats treated with xanthenone alone (P<0.01). Proteinuria, markers of endothelial activation were significantly higher than controls in leptin-only treated rats (P<0.05). ACE2 activity and expression were lower in leptin-only treated rats when compared to controls (P<0.05). It seems, leptin administration during pregnancy significantly increases SBP, proteinuria, endothelial activation, but decreases ACE2 level and expression. These effects are prevented by concurrent administration of xanthenone. Copyright © 2014 Elsevier Inc. All rights reserved.

The orphan receptor ERRα interferes with steroid signaling

PubMed Central

Teyssier, Catherine; Bianco, Stéphanie; Lanvin, Olivia; Vanacker, Jean-Marc

2008-01-01

The estrogen receptor-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily that has been shown to interfere with the estrogen-signaling pathway. In this report, we demonstrate that ERRα also cross-talks with signaling driven by other steroid hormones. Treatment of human prostatic cells with a specific ERRα inverse agonist reduces the expression of several androgen-responsive genes, in a manner that does not involve perturbation of androgen receptor expression or activity. Furthermore, ERRα activates the expression of androgen response elements (ARE)-containing promoters, such as that of the prostate cancer marker PSA, in an ARE-dependent manner. In addition, promoters containing a steroid response element can be activated by all members of the ERR orphan receptor subfamily, and this, even in the presence of antisteroid compounds. PMID:18697814

Characterization of a thermostable β-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana.

PubMed

Xu, Jing; Tian, Yong-Sheng; Peng, Ri-He; Zhu, Bo; Gao, Jian-Jie; Yao, Quan-Hong

2012-09-01

TmGUSI, a gene identical to that encoding a thermostable β-glucuronidase in the hyperthermophilic anaerobe Thermotoga maritima, has been synthesized using a PCR-based two-step DNA synthesis and codon optimization for plants, and expressed in both Escherichia coli and Arabidopsis thaliana. TmGUSI expressed in transformed E. coli cells exhibited maximum hydrolytic activity at 65 °C and pH 6.5 and retained more than 80% activity after incubation at 85 °C for 30 min. TmGUSI activity in transgenic A. thaliana plants containing TmGUSI was also stable over the temperature range 65-80 °C. Our data suggest that β-glucuronidase from T. maritima can serve as a useful thermostable marker in higher plants.

Pseudomonas aeruginosa infection alters the macrophage phenotype switching process during wound healing in diabetic mice.

PubMed

Chen, Sinuo; Li, Renren; Cheng, Chun; Xu, Jing-Ying; Jin, Caixia; Gao, Furong; Wang, Juan; Zhang, Jieping; Zhang, Jingfa; Wang, Hong; Lu, Lixia; Xu, Guo-Tong; Tian, Haibin

2018-03-07

Macrophages play critical roles in wound healing process. They switch from "classically activated" (M1) phenotype in the early inflammatory phase to "alternatively activated" (M2) phenotype in the later healing phase. However, the dynamic process of macrophage phenotype switching in diabetic wounds burdened with bacteria is unclear. In this report, Pseudomonas aeruginosa, frequently detected in diabetic foot ulcers, was inoculated into cutaneous wounds of db/db diabetic mice to mimic bacterium-infected diabetic wound healing. We observed that P. aeruginosa infection impaired diabetic wound healing and quickly promoted the expression of pro-inflammatory genes (M1 macrophage markers) tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β) and il-6 in wounds. The expression of markers of M2 macrophages, including il-10, arginase-1, and ym1 were also upregulated. In addition, similar gene expression patterns were observed in macrophages isolated directly from wounds. Immunostaining showed that P. aeruginosa infection increased both the ratios of M1 and M2 macrophages in wounds compared with that in control groups, which was further confirmed by in vitro culturing macrophages with P. aeruginosa and skin fibroblast conditioned medium. However, the ratios of the expression levels of pro-inflammatory genes to anti-inflammatory gene il-10 was increased markedly in P. aeruginosa infected wounds and macrophages compared with that in control groups, and P. aeruginosa prolonged the presence of M1 macrophages in the wounds. These data demonstrated that P. aeruginosa in diabetic wounds activates a mixed M1/M2 macrophage phenotype with an excessive activation of M1 phenotype or relatively inadequate activation of M2 phenotype. © 2018 International Federation for Cell Biology.

T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers.

PubMed

Palma, Marzia; Gentilcore, Giusy; Heimersson, Kia; Mozaffari, Fariba; Näsman-Glaser, Barbro; Young, Emma; Rosenquist, Richard; Hansson, Lotta; Österborg, Anders; Mellstedt, Håkan

2017-03-01

Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3 + cells and the CD8 + subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4 + and CD8 + cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients ( P =0.0003 and P =0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4 + and CD8 + subsets, with a significantly higher PD-1 expression. Higher numbers of CD4 + and CD8 + cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67 + ) and activated (CD69 + ) CD4 + and CD8 + cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls ( P <0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. Copyright© Ferrata Storti Foundation.

p63 expression defines a lethal subset of muscle-invasive bladder cancers.

PubMed

Choi, Woonyoung; Shah, Jay B; Tran, Mai; Svatek, Robert; Marquis, Lauren; Lee, I-Ling; Yu, Dasom; Adam, Liana; Wen, Sijin; Shen, Yu; Dinney, Colin; McConkey, David J; Siefker-Radtke, Arlene

2012-01-01

p63 is a member of the p53 family that has been implicated in maintenance of epithelial stem cell compartments. Previous studies demonstrated that p63 is downregulated in muscle-invasive bladder cancers, but the relationship between p63 expression and survival is not clear. We used real-time PCR to characterize p63 expression and several genes implicated in epithelial-to-mesenchymal transition (EMT) in human bladder cancer cell lines (n = 15) and primary tumors (n = 101). We correlated tumor marker expression with stage, disease-specific (DSS), and overall survival (OS). Expression of E-cadherin and p63 correlated directly with one another and inversely with expression of the mesenchymal markers Zeb-1, Zeb-2, and vimentin. Non-muscle-invasive (Ta and T1) bladder cancers uniformly expressed high levels of E-cadherin and p63 and low levels of the mesenchymal markers. Interestingly, a subset of muscle-invasive (T2-T4) tumors maintained high levels of E-cadherin and p63 expression. As expected, there was a strongly significant correlation between EMT marker expression and muscle invasion (p<0.0001). However, OS was shorter in patients with muscle-invasive tumors that retained p63 (p = 0.007). Our data confirm that molecular markers of EMT are elevated in muscle-invasive bladder cancers, but interestingly, retention of the "epithelial" marker p63 in muscle-invasive tumors is associated with a worse outcome.

Molecular alterations in skeletal muscle in rheumatoid arthritis are related to disease activity, physical inactivity, and disability.

PubMed

Huffman, Kim M; Jessee, Ryan; Andonian, Brian; Davis, Brittany N; Narowski, Rachel; Huebner, Janet L; Kraus, Virginia B; McCracken, Julie; Gilmore, Brian F; Tune, K Noelle; Campbell, Milton; Koves, Timothy R; Muoio, Deborah M; Hubal, Monica J; Kraus, William E

2017-01-23

To identify molecular alterations in skeletal muscle in rheumatoid arthritis (RA) that may contribute to ongoing disability in RA. Persons with seropositive or erosive RA (n = 51) and control subjects matched for age, gender, race, body mass index (BMI), and physical activity (n = 51) underwent assessment of disease activity, disability, pain, physical activity and thigh muscle biopsies. Muscle tissue was used for measurement of pro-inflammatory markers, transcriptomics, and comprehensive profiling of metabolic intermediates. Groups were compared using mixed models. Bivariate associations were assessed with Spearman correlation. Compared to controls, patients with RA had 75% greater muscle concentrations of IL-6 protein (p = 0.006). In patients with RA, muscle concentrations of inflammatory markers were positively associated (p < 0.05 for all) with disease activity (IL-1β, IL-8), disability (IL-1β, IL-6), pain (IL-1β, TNF-α, toll-like receptor (TLR)-4), and physical inactivity (IL-1β, IL-6). Muscle cytokines were not related to corresponding systemic cytokines. Prominent among the gene sets differentially expressed in muscles in RA versus controls were those involved in skeletal muscle repair processes and glycolytic metabolism. Metabolic profiling revealed 46% higher concentrations of pyruvate in muscle in RA (p < 0.05), and strong positive correlation between levels of amino acids involved in fibrosis (arginine, ornithine, proline, and glycine) and disability (p < 0.05). RA is accompanied by broad-ranging molecular alterations in skeletal muscle. Analysis of inflammatory markers, gene expression, and metabolic intermediates linked disease-related disruptions in muscle inflammatory signaling, remodeling, and metabolic programming to physical inactivity and disability. Thus, skeletal muscle dysfunction might contribute to a viscous cycle of RA disease activity, physical inactivity, and disability.

Activity of the hypoxia-activated pro-drug TH-302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy.

PubMed

Saggar, Jasdeep K; Tannock, Ian F

2014-06-01

Many chemotherapy drugs have poor therapeutic activity in regions distant from tumor blood vessels because of poor tissue penetration and low cytotoxic activity against slowly-proliferating cells. The hypoxia-activated pro-drug TH-302 may have selective toxicity for hypoxic and neighboring cells in tumors. Here we characterize the spatial distribution and ability of TH-302 to selectively target hypoxic regions and complement the effect of doxorubicin and docetaxel by modifying biomarker distribution. Athymic nude mice bearing human breast MCF-7 or prostate PC-3 tumors were treated with doxorubicin or docetaxel respectively and TH-302 alone or in combination. Biomarkers of drug effect including γH2aX (a marker of DNA damage), cleaved caspase-3 or -6 (markers of apoptosis) and reduction in Ki-67 (a marker of cell proliferation) were quantified in tumor sections in relation to functional blood vessels (recognized by DiOC7) and hypoxia (recognized by EF5) using immunohistochemistry. γH2aX expression at 10 min and cleaved caspase-3 or -6 at 24 hr after doxorubicin or docetaxel decreased with increasing distance from tumor blood vessels, with minimal expression in hypoxic regions; maximum reduction in Ki67 levels was observed in regions closest to vasculature at 24 hr. TH-302 induced maximal cell damage in hypoxic and neighboring regions, but was also active in tumor regions closer to blood vessels. TH-302 given 4 hr before doxorubicin or docetaxel increased DNA damage and apoptosis throughout the tumor compared to chemotherapy alone. When given with doxorubicin or docetaxel, TH-302 complements and enhances anticancer effects in both perivascular and hypoxic regions but also increases toxicity. © 2013 UICC.

[Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

PubMed

Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

2015-09-01

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

Short Communication: Low Expression of Activation and Inhibitory Molecules on NK Cells and CD4(+) T Cells Is Associated with Viral Control.

PubMed

Taborda, Natalia A; Hernández, Juan C; Lajoie, Julie; Juno, Jennifer A; Kimani, Joshua; Rugeles, María T; Fowke, Keith R

2015-06-01

Chronic HIV-1 infection induces severe immune alterations, including hyperactivation, exhaustion, and apoptosis. In fact, viral control has been associated with low frequencies of these processes. Here, we evaluated the expression of activation and inhibitory molecules on natural killer (NK) and CD4(+) T cells and plasma levels of proinflammatory cytokines in individuals exhibiting viral control: a cohort of HIV-1-exposed-seronegative individuals (HESN) and a cohort of HIV controllers. There was lower expression of CD69, LAG-3, PD-1, and TIM-3 in both cohorts when compared to a low-risk population or HIV progressors. In addition, HIV controllers exhibited lower plasma levels of proinflamatory molecules TNF-α and IP-10. These findings suggest that individuals exhibiting viral control have lower basal expression of markers associated with cellular activation and particularly immune exhaustion.

Acute Penicillium marneffei infection stimulates host M1/M2a macrophages polarization in BALB/C mice.

PubMed

Dai, Xiaoying; Mao, Congzheng; Lan, Xiuwan; Chen, Huan; Li, Meihua; Bai, Jing; Deng, Jingmin; Liang, Qiuli; Zhang, Jianquan; Zhong, Xiaoning; Liang, Yi; Fan, Jiangtao; Luo, Honglin; He, Zhiyi

2017-08-18

Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to provide novel ideas for prevention of P. marneffei-infection.

Release of active TGF-β1 from the Latent TGF-β1/GARP complex on T regulatory cells is mediated by Integrin β81

PubMed Central

Edwards, Justin P.; Thornton, Angela M.; Shevach, Ethan M.

2014-01-01

Activated T regulatory cells (Treg) express latent TGF-β1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. Mouse CD4+Foxp3+ Treg, but not CD4+Foxp3− T cells, expressed integrin β8 (Itgb8) as detected by qRT-PCR. Itgb8 expression was a marker of thymically-derived (t)Treg, as it could not be detected on Foxp3+Helios− Tregs or on Foxp3+ T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis, but failed to provide TGF-β1 to drive Th17 or iTreg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Thus, integrin αvβ8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-β1 from the latent TGF-β1/GARP complex on the surface of tTregs. PMID:25127859

Syncytin-1, an endogenous retroviral protein, triggers the activation of CRP via TLR3 signal cascade in glial cells.

PubMed

Wang, Xiuling; Liu, Zhongchun; Wang, Peigang; Li, Shan; Zeng, Jie; Tu, Xiaoning; Yan, Qiujin; Xiao, Zheman; Pan, Mengxian; Zhu, Fan

2018-01-01

Schizophrenia is a devastating psychiatric disorder that impacts on social functioning and quality of life, and there is accumulating evidence that inflammation is a potential pathogenic mechanism of schizophrenia. However, the mechanism of inflammation possibly occurred in schizophrenia has not been well understood. The endogenous retroviral protein syncytin-1 and inflammatory marker CRP are both abnormally expressed in schizophrenia patients. CRP is one of the markers of bacterial infection generally. Less clear is whether virus or viral protein can trigger the activation of CRP. Here, we detected a robust increase of the levels of syncytin-1 and CRP in schizophrenia patients, and displayed a positive correlation and marked consistency between expressions of syncytin-1 and CRP in schizophrenia patients. Furthermore, overexpression of syncytin-1 significantly elevated the levels of CRP, TLR3, and IL-6 in both human microglia and astrocytes. TLR3 deficiency impaired the expressions of CRP and IL-6 induced by syncytin-1. Importantly, we observed a cellular co-localization and a direct interaction between syncytin-1 and TLR3. Additionally, knockdown of IL-6 inhibited the syncytin-1-induced CRP expression. Thus, the totality of these results showed that viral protein syncytin-1 could trigger the activation of CRP, which might explain the elevated CRP in sterile inflammation and exhibit a novel mechanism for regulation of inflammation by syncytin-1 in schizophrenia. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteolytic Regulation of the Intestinal Epithelial Barrier: Mechanisms and Interventions

DTIC Science & Technology

2014-09-01

DSS protocol to evaluate molecular markers of acute inflammation in the subepithelial lamina propria, including quantity and nature of immune cell...will be investigated by immunostaining of colonic segments for Ki-67, a nuclear protein preferentially expressed during active phases of the cell

Histone deacetylase inhibitor (HDACI) PCI-24781 enhances chemotherapy induced apoptosis in multidrug resistant sarcoma cell lines

PubMed Central

Yang, Cao; Choy, Edwin; Hornicek, Francis J.; Wood, Kirkham B; Schwab, Joseph H; Liu, Xianzhe; Mankin, Henry; Duan, Zhenfeng

2013-01-01

The anti-tumor activity of histone deacetylase inhibitors (HDACI) on multi-drug resistant sarcoma cell lines has never been previously described. Four multidrug resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC50 of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug resistant sarcoma cell lines. PMID:21508354

In vivo imaging of the dynamics of different variants of EGFR in glioblastomas.

PubMed

Shah, Khalid

2011-01-01

A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression.

Isolated Anxa5{sup +}/Sca-1{sup +} perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brachvogel, Bent; Pausch, Friederike; Farlie, Peter

2007-07-15

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, {alpha}SMA, PDGFR-{beta}). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2.more » PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ{sup +} PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.« less

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas.

PubMed

Würth, Roberto; Barbieri, Federica; Pattarozzi, Alessandra; Gaudenzi, Germano; Gatto, Federico; Fiaschi, Pietro; Ravetti, Jean-Louis; Zona, Gianluigi; Daga, Antonio; Persani, Luca; Ferone, Diego; Vitale, Giovanni; Florio, Tullio

2017-09-01

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro

PubMed Central

Chhor, Vibol; Le Charpentier, Tifenn; Lebon, Sophie; Oré, Marie-Virgine; Celador, Idoia Lara; Josserand, Julien; Degos, Vincent; Jacotot, Etienne; Hagberg, Henrik; Sävman, Karin; Mallard, Carina; Gressens, Pierre; Fleiss, Bobbi

2013-01-01

Microglia mediate multiple facets of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype is an appealing neurotherapeutic strategy but a comprehensive study of classical and more novel microglial phenotypic markers in vitro is lacking. The aim of this study was to outline the temporal expression of a battery of phenotype markers from polarised microglia to generate an in vitro tool for screening the immunomodulatory potential of novel compounds. We characterised expression of thirty-one macrophage/microglial phenotype markers in primary microglia over time (4, 12, 36, and 72 h), using RT-qPCR or multiplex protein assay. Firstly, we selected Interleukin-4 (IL-4) and lipopolysaccharide (LPS) as the strongest M1–M2 polarising stimuli, from six stimuli tested. At each time point, markers useful to identify that microglia were M1 included iNOS, Cox-2 and IL-6 and a loss of M2a markers. Markers useful for quantifying M2b-immunomodulatory microglia included, increased IL-1RA and SOCS3 and for M2a-repair and regeneration, included increased arginase-1, and a loss of the M1 and M2b markers were discriminatory. Additional markers were regulated at fewer time points, but are still likely important to monitor when assessing the immunomodulatory potential of novel therapies. Further, to facilitate identification of how novel immunomodulatory treatments alter the functional affects of microglia, we characterised how the soluble products from polarised microglia affected the type and rate of neuronal death; M1/2b induced increasing and M2a-induced decreasing neuronal loss. We also assessed any effects of prior activation state, to provide a way to identify how a novel compound may alter phenotype depending on the stage of injury/insult progression. We identified generally that a prior M1/2b reduced the ability of microglia to switch to M2a. Altogether, we have characterised a profile of phenotype markers and a mechanism of assessing functional outcome that we can use as a reference guide for first-line screening of novel immunomodulatory therapies in vitro in the search for viable neuroprotectants. PMID:23454862

Conservation of spermatogonial stem cell marker expression in undifferentiated felid spermatogonia.

PubMed

Vansandt, Lindsey M; Livesay, Janelle L; Dickson, Melissa Joy; Li, Lei; Pukazhenthi, Budhan S; Keefer, Carol L

2016-09-01

Spermatogonial stem cells (SSCs) are distinct in their ability to self-renew, transmit genetic information, and persist throughout the life of an individual. These characteristics make SSCs a useful tool for addressing diverse challenges such as efficient transgenic production in nonrodent, biomedical animal models, or preservation of the male genome for species in which survival of frozen-thawed sperm is low. A requisite first step to access this technology in felids is the establishment of molecular markers. This study was designed to evaluate, in the domestic cat (Felis catus), the expression both in situ and following enrichment in vitro of six genes (GFRA1, GPR125, ZBTB16, POU5F1, THY1, and UCHL1) that had been previously identified as SSC markers in other species. Antibodies for surface markers glial cell line-derived neurotrophic factor family receptor alpha 1, G protein-coupled receptor 125, and thymus cell antigen 1 could not be validated, whereas Western blot analysis of prepubertal, peripubertal, and adult cat testis confirmed protein expression for the intracellular markers ubiquitin carboxy-terminal hydrolase 1, zinc finger and BTB domain-containing protein 16, and POU domain, class 5, transcription factor 1. Colocalization of the markers by immunohistochemistry revealed that several cells within the subpopulation adjacent to the basement membrane of the seminiferous tubules and identified morphologically as spermatogonia, expressed all three intracellular markers. Studies performed on cheetah (Acinonyx jubatus) and Amur leopard (Panthera pardus orientalis) testis exhibited a conserved expression pattern in protein molecular weights, relative abundance, and localization of positive cells within the testis. The expression of the three intracellular SSC marker proteins in domestic and wild cat testes confirms conservation of these markers in felids. Enrichment of marker transcripts after differential plating was also observed. These markers will facilitate further studies in cell enrichment and IVC of felid SSCs enabling both production of transgenic domestic cats and preservation of the male genome from rare and endangered felids. Copyright © 2016 Elsevier Inc. All rights reserved.

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

PubMed

Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

2016-02-01

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

PubMed Central

2013-01-01

Background Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells. Methods STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors. Results Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype. Conclusion IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27. PMID:24274066

Upregulation of heat shock factor 1 transcription activity is associated with hepatocellular carcinoma progression.

PubMed

Li, Shulian; Ma, Wanli; Fei, Teng; Lou, Qiang; Zhang, Yaqin; Cui, Xiukun; Qin, Xiaoming; Zhang, Jun; Liu, Guangchao; Dong, Zheng; Ma, Yuanfang; Song, Zhengshun; Hu, Yanzhong

2014-11-01

Heat shock factor 1 (HSF1) is associated with tissue‑specific tumorigenesis in a number of mouse models, and has been used a as prognostic marker of cancer types, including breast and prostatic cancer. However, its role in human hepatocellular carcinoma (HCC) is not well understood. Using immunoblotting and immunohistochemical staining, it was identified that HSF1 and its serine (S) 326 phosphorylation, a biomarker of HSF1 activation, are significantly upregulated in human HCC tissues and HCC cell lines compared with their normal counterparts. Cohort analyses indicated that upregulation of the expression of HSF1 and its phospho‑S326 is significantly correlated with HCC progression, invasion and patient survival prognosis (P<0.001); however, not in the presence of a hepatitis B virus infection and the expression of alpha-fetoprotein and carcinoembryonic antigen. Knockdown of HSF1 with shRNA induced the protein expression of tumor suppressor retinoblastoma protein, resulting in attenuated plc/prf5 cell growth and colony formation in vitro. Taken together, these data markedly support that HSF1 is a potential prognostic marker and therapeutic target for the treatment of HCC.

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.

PubMed

Pinto, Alexander R; Paolicelli, Rosa; Salimova, Ekaterina; Gospocic, Janko; Slonimsky, Esfir; Bilbao-Cortes, Daniel; Godwin, James W; Rosenthal, Nadia A

2012-01-01

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

Immune activation by medium-chain triglyceride-containing lipid emulsions is not modulated by n-3 lipids or toll-like receptor 4.

PubMed

Olthof, Evelyn D; Gülich, Alexandra F; Renne, Mike F; Landman, Sija; Joosten, Leo A B; Roelofs, Hennie M J; Wanten, Geert J A

2015-10-01

Saturated medium-chain triglycerides (MCT) as part of the parenteral lipid regimen (50% MCT and 50% long chain triglycerides (LCT)) activate the immune system in vitro. Fish oil (FO)-derived n-3 fatty acids (FA) inhibit saturated FA-induced immune activation via a toll-like receptor (TLR)-4 mediated mechanism. We hypothesized that effects of parenteral MCTs on immune cells involve TLR-4 signaling and that these effects are modulated by n-3 FA that are present in FO. To test this hypothesis we assessed effects of addition of various commercially available mixed parenteral lipid emulsions, n-3 FA and of TLR-4 inhibition on MCT-induced human immune cell activation by evaluation of the expression of leukocyte membrane activation markers and reactive oxygen species (ROS) production. All MCT-containing lipid emulsions activated leukocytes by inducing changes in expression of membrane markers and stimulus induced ROS production, whereas MCT-free lipid emulsions lacked this effect. Moreover, addition of n-3 FA to LCT/MCT did not prevent MCT-induced immune activation. TLR-4 inhibitors did not distinctly modulate MCT-induced changes in immune function. Taken together, these findings suggest that leukocyte activation by parenteral MCTs does not involve TLR-4 signaling and is not modulated by n-3 FA in FO-, but is exerted via different signaling pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Omega-3 fatty acid supplement prevents development of intracranial atherosclerosis.

PubMed

Shen, Jiamei; Hafeez, Adam; Stevenson, James; Yang, Jianjie; Yin, Changbin; Li, Fengwu; Wang, Sainan; Du, Huishan; Ji, Xunming; Rafols, Jose A; Geng, Xiaokun; Ding, Yuchuan

2016-10-15

Intracranial atherosclerotic stenosis (ICAS) is one of the most common causes of stroke worldwide and, in particular, has been implicated as a leading cause of recurrent ischemic stroke. We adapted a rat model of atherosclerosis to study brain intracranial atherosclerosis, and further investigated the effect of omega-3 fatty acids (O3FA) in attenuating development of ICAS. Adult male Sprague-Dawley rats were divided into control normal-cholesterol or high-cholesterol diet groups with or without O3FA for up to 6weeks. During the first 2weeks, NG-nitro-l-arginine methyl ester (l-NAME, 3mg/mL) was added to the drinking water of the high-cholesterol groups. The rats received supplementation with O3FA (5mg/kg/day) by gavages. Blood lipids including low density lipoprotein (LDL), cholesterol (CHO), triglycerides (TG) and high density lipoprotein (HDL) were measured at 3 and 6weeks. The lumen of middle cerebral artery (MCA) and the thickness of the vessel wall were assessed. Inflammatory molecular markers were assessed by Western blot. A high-cholesterol diet exhibited a significant increase in the classic blood markers (LDL, CHO, and TG) for atherosclerosis, as well as a decrease in HDL. These markers were found to be progressively more severe with time. Lumen stenosis and intimal thickening were increased in MCA. O3FA showed attenuation of blood lipids with an absence of morphological changes. O3FA significantly reduced the inflammatory marker CD68 in MCA and prevented monocyte chemotactic protein (MCP-1) and interferon-γ (IFN-γ) expression in the brain. O3FA similarly decreased inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6), markers affiliated with monocyte activity in atherosclerosis. Furthermore, O3FA significantly inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1), a marker for endothelial activation. Lastly, O3FA increased ATP-binding cassette transporter A1 (ABCA1) protein expression via silent information regulator 1 (SIRT1) activation, thus increasing cholesterol efflux from macrophages to HDL. Long-term O3FA dietary supplementation prevents the development of intracranial atherosclerosis. This O3FA effect appears to be mediated by its prevention of macrophage infiltration into the vessel wall, therefore reducing inflammation and intimal thickening. While similar effects in humans need to be determined, O3FA dietary supplement shows promising results in the prevention of ICAS. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamazaki, Hiroto; Naito, Motohiko; Ghani, Farhana Ishrat

2012-03-16

Highlights: Black-Right-Pointing-Pointer We focused on CD24 and CD26 for further analysis of CSC properties in MM. Black-Right-Pointing-Pointer Their expressions were correlated with chemoresistance, cell growth, and invasion. Black-Right-Pointing-Pointer Their expressions were also correlated with several cancer related genes. Black-Right-Pointing-Pointer The expression of each marker was correlated with different CSC property in Meso1. Black-Right-Pointing-Pointer Phosphorylation of ERK by EGF was regulated by expression of CD26, but not CD24. -- Abstract: Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSCmore » properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.« less

Structural changes in endometrial basal glands during menstruation.

PubMed

Garry, R; Hart, R; Karthigasu, K A; Burke, C

2010-09-01

To prospectively observe the changes occurring in endometrial glandular morphology during menstrual shedding and regeneration. Prospective observational study. The academic gynaecological endoscopy unit of a university teaching hospital. Population Thirteen patients investigated for a variety of benign, non-infective gynaecological disorders during the active bleeding phase of the menstrual cycle. The morphological appearances of concurrent histological and scanning electron microscopic images of endometrium taken at different stages of the active bleeding phase of menstruation were studied and correlated with the simultaneous immunohistochemical expression of the Ki-67 proliferation marker and the CD68 marker of macrophage activity. Change in morphology of endometrial glands at various stages of menstruation. Endometrial glands within the basalis show evidence of apoptosis and associated macrophage activity immediately before and during menstruation. There is subsequent destruction and removal of old secretory glandular epithelial elements, and rapid replacement with new narrow glands lined with small epithelial cells. There is no evidence of mitotic cell division or expression of Ki-67 in the glandular cells during this replacement process, but there is evidence of marked macrophage activity prior to glandular cell loss. Early endometrial epithelial repair after menstruation is not a consequence of mitotic cell division. It occurs without evidence of Ki-67 expression. There is structural evidence of programmed cell death and intense macrophage activity associated with glandular remodelling. Dead epithelial cells are shed from the glands and accumulate within the endometrial cavity to be replaced by new small epithelial cells that appear to arise by differentiation of the surrounding stromal cells. We propose that these stromal cells are endometrial progenitor/stem cells.

Calcitriol Prevents Cardiovascular Repercussions in Puromycin Aminonucleoside-Induced Nephrotic Syndrome

PubMed Central

Roberto, Roncon-Albuquerque

2018-01-01

Puromycin aminonucleoside-induced nephrotic syndrome (PAN-NS) is characterized by cardiac remodeling and increased local inflammatory activity. Patients with NS and animal models of NS have vitamin D3 deficiency. The aim of the present study was to evaluate the influence of calcitriol on cardiac remodeling and local inflammatory state in PAN-NS rat model. Male Sprague-Dawley rats were injected with PAN or vehicle on day 0. PAN and control rats were divided into two subgroups for the administration of calcitriol (PAN-D and Ct-D groups) or the vehicle (PAN-V and Ct-V groups) during 21 days. On day 21, the renal function, metabolic balance, calcitriol and FGF-23 plasma levels, prohypertrophy and proinflammatory markers (ET-1, TGF-β1, TNF-α, and IL-1β), and calcium signaling molecules (PLB and SERCA-2a) were evaluated. Twenty-one days after injection, PAN-V group presented cardiac hypertrophy and a modulation of proinflammatory markers local expression. Calcitriol treatment of PAN rats prevented cardiac hypertrophy and was associated with marked reduction in the cardiac expression levels of proinflammatory markers. Our results suggest that vitamin D3 deficiency in PAN-NS may contribute to cardiac remodeling and to the increase in local inflammatory activity. Calcitriol treatment prevents both cardiac repercussions and local inflammatory processes in PAN-NS. PMID:29607318

Ezrin Inhibition Up-regulates Stress Response Gene Expression.

PubMed

Çelik, Haydar; Bulut, Gülay; Han, Jenny; Graham, Garrett T; Minas, Tsion Z; Conn, Erin J; Hong, Sung-Hyeok; Pauly, Gary T; Hayran, Mutlu; Li, Xin; Özdemirli, Metin; Ayhan, Ayşe; Rudek, Michelle A; Toretsky, Jeffrey A; Üren, Aykut

2016-06-17

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

PubMed

Olivier, T T; Viljoen, I M; Hofmeyr, J; Hausler, G A; Goosen, W J; Tordiffe, A S W; Buss, P; Loxton, A G; Warren, R M; Miller, M A; van Helden, P D; Parsons, S D C

2017-06-01

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON ® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions. © 2015 Blackwell Verlag GmbH.

Effect of Roux-en-Y gastric bypass on liver mitochondrial dynamics in a rat model of obesity.

PubMed

Sacks, Jessica; Mulya, Anny; Fealy, Ciaran E; Huang, Hazel; Mosinski, John D; Pagadala, Mangesh R; Shimizu, Hideharu; Batayyah, Esam; Schauer, Philip R; Brethauer, Stacy A; Kirwan, John P

2018-02-01

Bariatric surgery provides significant and durable improvements in glycemic control and hepatic steatosis, but the underlying mechanisms that drive improvements in these metabolic parameters remain to be fully elucidated. Recently, alterations in mitochondrial morphology have shown a direct link to nutrient adaptations in obesity. Here, we evaluate the effects of Roux-en-Y gastric bypass (RYGB) surgery on markers of liver mitochondrial dynamics in a diet-induced obesity Sprague-Dawley (SD) rat model. Livers were harvested from adult male SD rats 90-days after either Sham or RYGB surgery and continuous high-fat feeding. We assessed expression of mitochondrial proteins involved in fusion, fission, mitochondrial autophagy (mitophagy) and biogenesis, as well as differences in citrate synthase activity and markers of oxidative stress. Gene expression for mitochondrial fusion genes, mitofusin 1 (Mfn1; P < 0.05), mitofusin 2 (Mfn2; P < 0.01), and optic atrophy 1 (OPA1; P < 0.05) increased following RYGB surgery. Biogenesis regulators, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; P < 0.01) and nuclear respiratory factor 1 (Nrf1; P < 0.05), also increased in the RYGB group, as well as mitophagy marker, BCL-2 interacting protein 3 (Bnip3; P < 0.01). Protein expression for Mfn1 (P < 0.001), PGC1α (P < 0.05), BNIP3 (P < 0.0001), and mitochondrial complexes I-V (P < 0.01) was also increased by RYGB, and Mfn1 expression negatively correlated with body weight, insulin resistance, and fasting plasma insulin. In the RYGB group, citrate synthase activity was increased (P < 0.02) and reactive oxygen species (ROS) was decreased compared to the Sham control group (P < 0.05), although total antioxidant capacity was unchanged between groups. These data are the first to show an association between RYGB surgery and improved markers of liver mitochondrial dynamics. These observed improvements may be related to weight loss and reduced energetic demand on the liver, which could facilitate normalization of glucose homeostasis and protect against hepatic steatosis. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

[The influence of lumbar sympathetic ganglion radiofrequency thermocoagulation on the activation of microglia in rats with diabetic neuropathic pain].

PubMed

Zhang, J H; Yang, C X; Zhong, J Y; Zhang, L; Xiong, Q M; Wang, J; Wang, H B

2016-06-28

To observe the influence of lumbar sympathetic ganglion radiofrequency thermocoagulation on the activation of spinal microglia in rats with diabetic neuropathic pain (DNP). Thirty-six painful diabetic Sprague-Dawley rats induced by 60 mg/kg streptozotocin (STZ) intraperitoneal injection were randomly divided into diabetic neuropathic pain group (group DNP, n=12), Sham operation group (group Sham, n=12) and radiofrequency thermocoagulation group (group R, n=12). Meanwhile another 12 age-matched rats were allocated as normal control group (group N), rats in group N received intraperitoneal injection of equal volume of normal saline. Twenty-eight days after STZ injection, rats in group R received L3 lumbar sympathetic ganglia radiofrequency thermocoagulation on the left side under X-ray guideline after anesthesia with damage time 60 s and damage temperature 60 ℃. Rats in group Sham received puncture positioning, but not thermocoagulation therapy. The mechanical paw withdrawal threshold (PWT) were performed before STZ injection, 7, 14, 21, 28 days after STZ injection and 1, 3, 5, 7, 14 days after radiofrequency thermocoagulation, respectively. Blood glucose were performed before STZ injection, 3, 28 days after STZ injection and 1, 14days after radiofrequency thermocoagulation. After the final behavioral testing, L3-L5 spinal cord tissues were removed to exam the expression of microglia marker OX42 by Western blotting and immunofluorescence technique, and the changes in the expression of inflammation factor IL-1β, IL-6, TNF-α were detected by ELISA technique. Compared with group N, after 14, 21, 28 days of STZ injection and 1, 3, 5, 7, 14 days of radiofrequency thermocoagulation, the PWT of group DNP and group Sham decreased significantly (P<0.05); Before radiofrequency thermocoagulation, the PWT of rats in group DNP was (3.84±0.83) g, the PWT of rats in group R was (4.45±0.88) g, there was no statistically significant difference between group DNP and group R (t=1.514, P>0.05), but after radiofrequency thermocoagulation, compared with DNP group, the PWT of rats in group R increased significantly (P<0.05), and lasted to 14 d after radiofrequency thermocoagulation. The ratio of spinal microglia marker OX42 and GAPDH, the expression of inflammation factor IL-1β, IL-6, and TNF-α in group N were 0.074±0.023, (35.93±6.16) pg/ml, (92.11±13.23) pg/ml, and (169.50±22.64) pg/ml, respectively. The ratio of spinal microglia marker OX42 and GAPDH, the expression of inflammation factor IL-1β, IL-6, and TNF-α in group DNP were 1.023±0.185, (73.82±9.25) pg/ml, (155.33±21.82) pg/ml, and (298.30±33.21) pg/ml, respectively. The ratio of spinal microglia marker OX42 and GAPDH, the expression of inflammation factor IL-1β, IL-6, and TNF-α in group Sham were 0.951±0.103, (73.00±7.54) pg/ml, (151.02±24.26) pg/ml, and (294.01±36.37) pg/ml, respectively. The ratio of spinal microglia marker OX42 and GAPDH, the expression of inflammation factor IL-1β, IL-6, and TNF-α in group R were 0.563±0.019, (51.81±7.36) pg/ml, (123.24±16.13) pg/ml, and (229.23±29.16) pg/ml, respectively. Compared with group N, the expression of spinal microglia marker OX42 and inflammation factor IL-1β, IL-6, and TNF-α in group DNP, group Sham and group R increased significantly (F=7.501, 348.698, 568.021, 145.110, all P<0.05). Compared with DNP group, the expression of spinal microglia marker OX42 and inflammation factor IL-1β, IL-6, and TNF-α of group R reduced significantly (all P<0.05). The lumbar sympathetic ganglion radiofrequency thermocoagulation can alleviate diabetic neuropathic pain. The mechanism may relate with the inhibition of spinal microglia activation and the lower expression of inflammation factor.

Induction of plant defense gene expression by plant activators and Pseudomonas syringae pv. tomato in greenhouse-grown tomatoes.

PubMed

Herman, M A B; Davidson, J K; Smart, C D

2008-11-01

Plant activators provide an appealing management option for bacterial diseases of greenhouse-grown tomatoes. Two types of plant activators, one that induces systemic acquired resistance (SAR) and a second that activates induced systemic resistance (ISR), were evaluated for control of Pseudomonas syringae pv. tomato and effect on plant defense gene activation. Benzothiadiazole (BTH, SAR-inducing compound) effectively reduced bacterial speck incidence and severity, both alone and in combination with the ISR-inducing product. Application of BTH also led to elevated activation of salicylic acid and ethylene-mediated responses, based on real-time polymerase chain reaction analysis of marker gene expression levels. In contrast, the ISR-inducing product (made up of plant growth-promoting rhizobacteria) inconsistently modified defense gene expression and did not provide disease control to the same level as did BTH. No antagonism was observed by combining the two activators as control of bacterial speck was similar to or better than BTH alone.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

PubMed Central

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

PubMed

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong; Qu, Shaohong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. © 2015 American Society of Plant Biologists. All Rights Reserved.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

Immune surveillance properties of human NK cell-derived exosomes.

PubMed

Lugini, Luana; Cecchetti, Serena; Huber, Veronica; Luciani, Francesca; Macchia, Gianfranco; Spadaro, Francesca; Paris, Luisa; Abalsamo, Laura; Colone, Marisa; Molinari, Agnese; Podo, Franca; Rivoltini, Licia; Ramoni, Carlo; Fais, Stefano

2012-09-15

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

PubMed

Winkler, Tamara; Mahoney, Eric J; Sinner, Debora; Wylie, Christopher C; Dahia, Chitra Lekha

2014-01-01

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

XIAP and Ki-67: A Correlation Between Antiapoptotic and Proliferative Marker Expression in Benign and Malignant Tumours of Salivary Gland: An Immunohistochemical Study.

PubMed

Bagulkar, Bhupesh Bhayyaji; Gawande, Madhuri; Chaudhary, Minal; Gadbail, Amol Ramchandra; Patil, Swati; Bagulkar, Smita

2015-02-01

Impaired balance between cell proliferation and apoptosis is crucial to the development of malignant neoplasm. The purpose of this study was to evaluate and compare the expression of X-Linked inhibitor of apoptotic protein (XIAP) (antiapoptotic marker) and Ki-67 (proliferative marker) expression in benign and malignant salivary gland (SG) tumours. The study consisted of 40 cases of benign SG tumours and 50 cases of malignant SG tumours. The immunohistochemistry was carried out by using Ki-67 antibody (clone MIB-1) and XIAP antibody in all the groups. XIAP expression was significantly higher in malignant SG tumours than benign SG tumours (p = 0.016). Ki-67 LI was significantly higher in malignant SG tumours than benign SG tumours (p = 0.0002). Statistically significant positive correlation between Ki-67 count and XIAP expression was noted in benign and malignant SG tumours (p = 0.000). As the expression of an antiapoptotic marker (XIAP) increases, the expression of a proliferative marker (Ki-67) also increases from benign to malignant SG tumours. Thus, targeted therapy of XIAP may play a future role in the management of SG malignancy.

XIAP and Ki-67: A Correlation Between Antiapoptotic and Proliferative Marker Expression in Benign and Malignant Tumours of Salivary Gland: An Immunohistochemical Study

PubMed Central

Gawande, Madhuri; Chaudhary, Minal; Gadbail, Amol Ramchandra; Patil, Swati; Bagulkar, Smita

2015-01-01

Background: Impaired balance between cell proliferation and apoptosis is crucial to the development of malignant neoplasm. The purpose of this study was to evaluate and compare the expression of X-Linked inhibitor of apoptotic protein (XIAP) (antiapoptotic marker) and Ki-67 (proliferative marker) expression in benign and malignant salivary gland (SG) tumours. Materials and Methods: The study consisted of 40 cases of benign SG tumours and 50 cases of malignant SG tumours. The immunohistochemistry was carried out by using Ki-67 antibody (clone MIB-1) and XIAP antibody in all the groups. Results: XIAP expression was significantly higher in malignant SG tumours than benign SG tumours (p = 0.016). Ki-67 LI was significantly higher in malignant SG tumours than benign SG tumours (p = 0.0002). Statistically significant positive correlation between Ki-67 count and XIAP expression was noted in benign and malignant SG tumours (p = 0.000). Conclusion: As the expression of an antiapoptotic marker (XIAP) increases, the expression of a proliferative marker (Ki-67) also increases from benign to malignant SG tumours. Thus, targeted therapy of XIAP may play a future role in the management of SG malignancy. PMID:25859460

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

PubMed

Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

2015-05-14

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. © 2015 The Authors.

FGF signaling is required for brain left-right asymmetry and brain midline formation.

PubMed

Neugebauer, Judith M; Yost, H Joseph

2014-02-01

Early disruption of FGF signaling alters left-right (LR) asymmetry throughout the embryo. Here we uncover a role for FGF signaling that specifically disrupts brain asymmetry, independent of normal lateral plate mesoderm (LPM) asymmetry. When FGF signaling is inhibited during mid-somitogenesis, asymmetrically expressed LPM markers southpaw and lefty2 are not affected. However, asymmetrically expressed brain markers lefty1 and cyclops become bilateral. We show that FGF signaling controls expression of six3b and six7, two transcription factors required for repression of asymmetric lefty1 in the brain. We found that Z0-1, atypical PKC (aPKC) and β-catenin protein distribution revealed a midline structure in the forebrain that is dependent on a balance of FGF signaling. Ectopic activation of FGF signaling leads to overexpression of six3b, loss of organized midline adherins junctions and bilateral loss of lefty1 expression. Reducing FGF signaling leads to a reduction in six3b and six7 expression, an increase in cell boundary formation in the brain midline, and bilateral expression of lefty1. Together, these results suggest a novel role for FGF signaling in the brain to control LR asymmetry, six transcription factor expressions, and a midline barrier structure. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival.

PubMed

Rosati, Alessandra; Bersani, Samantha; Tavano, Francesca; Dalla Pozza, Elisa; De Marco, Margot; Palmieri, Marta; De Laurenzi, Vincenzo; Franco, Renato; Scognamiglio, Giosuè; Palaia, Raffaele; Fontana, Andrea; di Sebastiano, Pierluigi; Donadelli, Massimo; Dando, Ilaria; Medema, Jan Paul; Dijk, Frederike; Welling, Lieke; di Mola, Fabio Francesco; Pezzilli, Raffaele; Turco, Maria Caterina; Scarpa, Aldo

2012-11-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

FGF Signaling is Required for Brain Left-Right Asymmetry and Brain Midline Formation

PubMed Central

Neugebauer, Judith M.; Yost, H. Joseph

2014-01-01

Early disruption of FGF signaling alters left-right (LR) asymmetry throughout the embryo. Here we uncover a role for FGF signaling that specifically disrupts brain asymmetry, independent of normal lateral plate mesoderm (LPM) asymmetry. When FGF signaling is inhibited during mid-somitogenesis, asymmetrically expressed LPM markers southpaw and lefty2 are not affected. However, asymmetrically expressed brain markers lefty1 and cyclops become bilateral. We show that FGF signaling controls expression of six3b and six7, two transcription factors required for repression of asymmetric lefty1 in the brain. We found that Z0-1, atypical PKC (aPKC) and β-catenin protein distribution revealed a midline structure in the forebrain that is dependent on a balance of FGF signaling. Ectopic activation of FGF signaling leads to overexpression of six3b, loss of organized midline adherins junctions and bilateral loss of lefty1 expression. Reducing FGF signaling leads to a reduction in six3b and six7 expression, an increase in cell boundary formation in the brain midline, and bilateral expression of lefty1. Together, these results suggest a novel role for FGF signaling in the brain to control LR asymmetry, six transcription factor expression, and a midline barrier structure. PMID:24333178

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

PubMed Central

Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

2017-01-01

ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.

PubMed

Fatima, Soghra; Zhou, Sheng; Sorrentino, Brian P

2012-02-01

The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays. Copyright © 2011 AlphaMed Press.

Safety and pharmacodynamics of dalazatide, a Kv1.3 channel inhibitor, in the treatment of plaque psoriasis: A randomized phase 1b trial

PubMed Central

Tarcha, Eric J.; Probst, Peter; Peckham, David; Muñoz-Elías, Ernesto J.; Kruger, James G.; Iadonato, Shawn P.

2017-01-01

Background Dalazatide is a specific inhibitor of the Kv1.3 potassium channel. The expression and function of Kv1.3 channels are required for the function of chronically activated memory T cells, which have been shown to be key mediators of autoimmune diseases, including psoriasis. Objective The primary objective was to evaluate the safety of repeat doses of dalazatide in adult patients with mild-to-moderate plaque psoriasis. Secondary objectives were to evaluate clinical proof of concept and the effects of dalazatide on mediators of inflammation in the blood and on chronically activated memory T cell populations. Methods Patients (n = 24) were randomized 5:5:2 to receive dalazatide at 30 mcg/dose, 60 mcg/dose, or placebo twice weekly by subcutaneous injection (9 doses total). Safety was assessed on the basis of physical and neurological examination and laboratory testing. Clinical assessments included body-surface area affected, Psoriasis Area and Severity Index (PASI), and investigator and patient questionnaires. Results The most common adverse events were temporary mild (Grade 1) hypoesthesia (n = 20; 75% placebo, 85% dalazatide) and paresthesia (n = 15; 25% placebo, 70% dalazatide) involving the hands, feet, or perioral area. Nine of 10 patients in the 60 mcg/dose group had a reduction in their PASI score between baseline and Day 32, and the mean reduction in PASI score was significant in this group (P < 0.01). Dalazatide treatment reduced the plasma levels of multiple inflammation markers and reduced the expression of T cell activation markers on peripheral blood memory T cells. Limitations The study was small and drug treatment was for a short duration (4 weeks). Conclusion This study indicates that dalazatide is generally well tolerated and can improve psoriatic skin lesions by modulating T cell surface and activation marker expression and inhibiting mediators of inflammation in the blood. Larger studies of longer duration are warranted. PMID:28723914

High expression of FOXP3 in primary melanoma is associated with tumour progression.

PubMed

Gerber, A L; Münst, A; Schlapbach, C; Shafighi, M; Kiermeir, D; Hüsler, R; Hunger, R E

2014-01-01

The antitumour immune response plays an important role in the prognosis of melanoma. High numbers of circulating regulatory T cells have been associated with rapid disease progression. To assess the influence of forkhead box protein (FOXP)3, CD1a and langerin expression on the prognosis of primary melanoma. We analysed 185 primary melanomas by immunohistochemical staining for expression of the regulatory T-cell marker FOXP3 and the dendritic cell markers langerin and CD1a, and correlated marker expression with clinical outcome. Disease-free survival and overall survival were significantly longer in patients expressing low levels of FOXP3 in the primary melanoma, whereas they were associated with high expression of CD1a. The negative prognostic value of FOXP3 expression was independent of the Breslow tumour thickness. Langerin expression did not correlate with the clinical outcome. High expression of FOXP3 in the primary melanoma may be used as an additional independent prognostic marker for early tumour progression in patients with melanoma. © 2013 British Association of Dermatologists.

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system.

PubMed

Ohtani-Kaneko, Rsituko; Sato, Kenjiro; Tsutiya, Atsuhiro; Nakagawa, Yuka; Hashizume, Kazutoshi; Tazawa, Hidekatsu

2017-10-09

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a humanmore » adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPAR{alpha} activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR{gamma} agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.« less

NK cell-released exosomes: Natural nanobullets against tumors.

PubMed

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

NK cell-released exosomes

PubMed Central

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. PMID:23482694

Dual Role of Cyanidin-3-glucoside on the Differentiation of Bone Cells.

PubMed

Park, K H; Gu, D R; So, H S; Kim, K J; Lee, S H

2015-12-01

Cyanidin-3-glucoside (C3G) is one of the major components of anthocyanin, a water-soluble phytochemical. Recent studies demonstrated the chemopreventive and chemotherapeutic activities of C3G in various conditions, including cancer, although the precise effects of C3G on osteoclast and osteoblast differentiation remain unclear. Here, we investigated the role of C3G in the differentiation of bone-associated cells and its underlying mechanism. C3G inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation and formation in a dose-dependent manner and downregulated the expression of osteoclast differentiation marker genes. Pretreatment with C3G considerably reduced the induction of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated kinases activation by RANKL in osteoclast precursor cells. Furthermore, C3G dramatically inhibited the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1, which are important transcription factors for osteoclast differentiation and activation. The formation of osteoclasts in coculture of bone marrow cells and calvaria-derived osteoblasts was also inhibited by C3G treatment, although the expression of macrophage colony-stimulating factor and RANKL (master factors for osteoclast differentiation and formation) and osteoprotegerin (a decoy receptor for RANKL) on osteoblasts was unaffected. The inhibitory effect of C3G on osteoclastogenesis is therefore targeted specifically to osteoclasts but not osteoblasts. Moreover, analysis of the expression levels of osteoblast differentiation marker genes and alizarin red staining showed that osteoblast differentiation and matrix formation increased after C3G treatment. Taken together, these results strongly suggest that C3G has a dual role in bone metabolism, as an effective inhibitor of osteoclast differentiation but also as an activator of osteoblast differentiation. Therefore, C3G may be used as a potent preventive or therapeutic agent for bone-related diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis. © International & American Associations for Dental Research 2015.

E2F4 is required for early eye patterning.

PubMed

Ruzhynsky, Vladimir A; Furimsky, Marosh; Park, David S; Wallace, Valerie A; Slack, Ruth S

2009-01-01

Increasingly, studies reveal novel functions for cell cycle proteins during development. Here, we investigated the role of E2F4 in eye development. E2F4-deficient mouse embryos exhibit severe early eye patterning defects, which are evident from embryonic day 11.5 and characterized by aberrant shape of the optic cup, coloboma as well as abnormal eye pigmentation. Loss of E2F4 is associated with proximal-distal patterning defects in the optic vesicle. These defects are characterized by the expansion of optic stalk marker gene expression to the optic cup and reduced expression of ventral optic cup markers. These defects are associated with a split of Shh expression domain at the ventral midline of the forebrain and expansion of the Shh activity into the ventral optic cup. Despite these patterning defects, early neuronal differentiation and Shh expression in the retina are not affected by E2F4 deletion. Overall, the results of our studies show a novel role of E2F4 in the early eye development. 2009 S. Karger AG, Basel.

MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

PubMed

Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

2016-06-21

The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells.

PubMed

Liu, Lei; Nielsen, Frederik Mølgaard; Emmersen, Jeppe; Bath, Chris; Hjortdal, Jesper Østergaard; Riis, Simone; Fink, Trine; Pennisi, Cristian Pablo; Zachar, Vladimir

2018-05-20

Ex-vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting (FACS) and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3 (CK3). A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

PubMed

Ise, H; Sugihara, N; Negishi, N; Nikaido, T; Akaike, T

2001-07-13

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease. Copyright 2001 Academic Press.

Bisphenol A causes malformation of the head region in embryos of Xenopus laevis and decreases the expression of the ESR-1 gene mediated by Notch signaling.

PubMed

Imaoka, Susumu; Mori, Tomohiro; Kinoshita, Tsutomu

2007-02-01

Bisphenol A (BpA) is widely used in industry and dentistry. Its effects on the embryonic development of Xenopus laevis were investigated. Xenopus embryos at stage 10.5 were treated with BpA. Developmental abnormalities were observed at stage 35; malformation of the head region including eyes and scoliosis. The expression of several markers of embryonic development was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The pan-neural marker SOX-2, the neural stem cell marker nrp-1, the mesodermal marker MyoD, and the endodermal marker sox17alpha, were used. Although the expression of marker genes was not changed by treatment with BpA, that of Pax-6, a key regulator of the morphogenesis of the eyes, was decreased by BpA. Pax-6 is a downstream factor of Notch signaling. So, the expression of a typical Notch-dependent factor, ESR-1, was investigated in the presence of BpA. The expression of ESR-1 was efficiently suppressed by BpA. In whole mount in situ hybridization (WISH), Pax-6 was expressed in the central nervous system and eyes. The expression was lost completely on treatment with BpA. The expression of ESR-1 in the central nervous system and eyes also disappeared with BpA treatment. Injection of the intracellular domain of Notch efficiently recovered ESR-1 expression in the presence of BpA although injection of a ligand for notch, Delta, did not. These results suggest that BpA decreased the expression of ESR-1 by disrupting the Notch signal.

Heterogeneity in Immune Marker Expression after Acquisition of Resistance to EGFR Kinase Inhibitors: Analysis of a Case with Small Cell Lung Cancer Transformation.

PubMed

Suda, Kenichi; Murakami, Isao; Yu, Hui; Kim, Jihye; Ellison, Kim; Rivard, Christopher J; Mitsudomi, Tetsuya; Hirsch, Fred R

2017-06-01

Expression of immune markers is of scientific interest because of their potential roles as predictive biomarkers for immunotherapy. Although the microenvironment of metastatic tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers, there are few data regarding this context. A 76-year-old never-smoking female with EGFR-mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, an autopsy revealed SCLC transformation and EGFR T790M secondary mutation (T790M) as mutually exclusive resistance mechanisms occurring differently in different metastases; two liver metastases (SCLC versus AC with T790M) and two lymph node metastases (SCLC versus AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry and by an immune oncology gene expression panel. Programmed death ligand 1 (PD-L1) protein was partially expressed in tumor cells with AC lesions (T790M) but not in tumor cells with SCLC transformation. The liver metastasis with SCLC transformation showed no stromal PD-L1 expression and scant tumor-infiltrating lymphocytes, whereas the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. Data generated using an immuno-oncology gene expression panel indicated a higher level of T-cell costimulatory molecules and lower expression of type I interferon-regulated genes in lesions with SCLC transformation. These data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation and indicate that the biopsy specimen from one lesion may not be representative of immune marker status for all lesions. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Assessment of the U937 cell line for the detection of contact allergens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Python, Francois; Goebel, Carsten; Aeby, Pierre

2007-04-15

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1{beta} and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times.more » Test item-specific modulations of the chosen activation markers (CD86, IL-1{beta} and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.« less

Rapamycin protects against paraquat-induced pulmonary fibrosis: Activation of Nrf2 signaling pathway.

PubMed

Xu, Yiheng; Tai, Wenlin; Qu, Xiaoyuan; Wu, Wenjuan; Li, ZhenKun; Deng, Shuhao; Vongphouttha, Chanthasone; Dong, Zhaoxing

2017-08-19

Paraquat (PQ) is a widely used herbicide indeveloping countries worldwide, and pulmonary fibrosis is one of the most typical features of PQ poisoning. The molecular mechanism of PQ toxicity especially how to treat PQ-induced pulmonary fibrosis is still largely unknown. In animal model of pulmonary fibrosis, we used HE staining, western blotting assay and Real-time PCR assay to analyze the effects of rapamycin on the PQ-induced epithelial mesenchymal transition (EMT). We found that PQ induced the pulmonary fibrosis using HE staining and Masson's staining, and up-regulated the activity of HYP and the mRNA expressions of Collagen I and III (COL-1and COL-3) in pulmonary tissues. We also found that rapamycin down-regulated the mesenchymal cell marker Vimentin and up-regulated the epithelial cell marker E-cadherin both in mRNA and protein levels compared with PQ group. And the EMT associated transcription factor Snail was decreased by rapamycin treatment compared with PQ group. And PQ decreased the Nrf2 expression both in mRNA and protein levels, and rapamycin inhibited these effects of PQ. SFN, a activator of Nrf2, could inhibit the EMT and the expression of Snail. And knockdowon of Nrf2 could abolish the inhibitory effects of rapamycin of PQ-induced EMT. In conclusion, rapamycin protects against paraquat-induced pulmonary fibrosis by activation of Nrf2 signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

FOXQ1, a Novel Target of the Wnt Pathway and a New Marker for Activation of Wnt Signaling in Solid Tumors

PubMed Central

Christensen, Jon; Bentz, Susanne; Sengstag, Thierry; Shastri, V. Prasad; Anderle, Pascale

2013-01-01

Background The forkhead box transcription factor FOXQ1 has been shown to be upregulated in colorectal cancer (CRC) and metastatic breast cancer and involved in tumor development, epithelial-mesenchymal transition and chemoresistance. Yet, its transcriptional regulation is still unknown. Methods FOXQ1 mRNA and protein expression were analysed in a panel of CRC cell lines, and laser micro-dissected human biopsy samples by qRT-PCR, microarray GeneChip® U133 Plus 2.0 and western blots. FOXQ1 regulation was assayed by chromatin immunoprecipitation and luciferase reporter assays. Results FOXQ1 was robustly induced in CRC compared to other tumors, but had no predictive value with regards to grade, metastasis and survival in CRC. Prototype-based gene coexpression and gene set enrichment analysis showed a significant association between FOXQ1 and the Wnt pathway in tumors and cancer cell lines from different tissues. In vitro experiments confirmed, on a molecular level, FOXQ1 as a direct Wnt target. Analysis of known Wnt targets identified FOXQ1 as the most suitable marker for canonical Wnt activation across a wide panel of cell lines derived from different tissues. Conclusions Our data show that FOXQ1 is one of the most over-expressed genes in CRC and a direct target of the canonical Wnt pathway. It is a potential new marker for detection of early CRC and Wnt activation in tumors of different origins. PMID:23555880

Soluble human leukocyte antigen G5 polarizes differentiation of macrophages toward a decidual macrophage-like phenotype.

PubMed

Lee, Cheuk-Lun; Guo, YiFan; So, Kam-Hei; Vijayan, Madhavi; Guo, Yue; Wong, Vera H H; Yao, YuanQing; Lee, Kai-Fai; Chiu, Philip C N; Yeung, William S B

2015-10-01

What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized macrophages showed enhanced phagocytic activity. They also had higher expression and activity of indoleamine 2,3-dioxygenase 1, a phenotypic marker of decidual macrophages, which inhibited proliferation of autologous T-cells via induction of G0/G1 cell cycle arrest. In addition, sHLAG5-polarized macrophages had an increased secretion of interleukin-6 and C-X-C motif ligand 1, which inhibited interferon-γ production in T-cells and induction of trophoblast invasion, respectively. Most information on the phenotypes and biological activities of human decidual macrophages are based on past literatures. A direct comparison between sHLAG5-polarized macrophages and primary decidual macrophages is required to verify the present observations. This is the first study on the role of sHLAG5 in macrophage differentiation. Further study on the mechanism that regulates the differentiation process of macrophages would enhance our understanding on the physiology of early pregnancy. This work was supported in part by the Hong Kong Research Grant Council Grant HKU774212 and the University of Hong Kong Grant 201309176126. The authors have no competing interests to declare. Nil. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.

PubMed

Rehman, Jalees; Li, Jingling; Orschell, Christie M; March, Keith L

2003-03-04

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7+/-0.3%), Mac-1 (57.6+/-13.5%), and CD11c (90.8+/-4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2+/-0.7%) or stem/progenitor-cell markers AC133 (0.16+/-0.05%) and c-kit (1.3+/-0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture.

PubMed

Wang, Jun; Ti, Yunfan; Wang, Yicun; Guo, Guodong; Jiang, Hui; Chang, Menghan; Qian, Hongbo; Zhao, Jianning; Sun, Guojing

2018-04-19

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4 + T cells in patients with long bone fracture. We found that LAG-3 + cells represented approximately 13% of peripheral blood CD4 + T cells on average. Compared to LAG-3 - CD4 + T cells, LAG-3 + CD4 + T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3 + CD4 + T cells expressed significantly higher levels of IL-10 and TGF-β than LAG-3 - CD4 + T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3 + CD4 + T cells. The frequency of LAG-3 + CD4 + T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4 + T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.

Transcriptional expression levels and biochemical markers of oxidative stress in the earthworm Eisenia andrei after exposure to 2,4-dichlorophenoxyacetic acid (2,4-D).

PubMed

Hattab, Sabrine; Boughattas, Iteb; Boussetta, Hamadi; Viarengo, Aldo; Banni, Mohamed; Sforzini, Susanna

2015-12-01

This study investigated the stress response of earthworms (Eisenia andrei) to exposure to a commonly used herbicide, 2,4 dichloro-phenoxy-acetic acid (2,4-D). We evaluated both stress biomarkers and the transcriptional expression levels and activity of three enzymes involved in oxidative stress responses. Earthworms were exposed to three sublethal concentration of 2,4-D (3.5, 7, and 14 mg kg(-1)) for 7 and 14 days. Exposure to 7 and 14 mg kg(-1) 2,4-D significantly reduced both worm body weight and lysosomal membrane stability (LMS); the latter is a sensitive stress biomarker in coelomocytes. Exposure to 2,4-D caused a pronounced increase in the accumulation of malonedialdehyde (MDA), a marker of oxidative stress, and significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD),and glutathione-S-transferase (GST). Compared to expression in controls, the expression levels of the sod, cat, and gst genes increased in worms exposed to all three 2,4-D doses for 7 days. However, after 14 days of exposure, only the expression of the gst gene remained higher than controls. These data provide new insights into the cytotoxicity of 2,4-D in the earthworm E. andrei and should be carefully considered in view of the biological effects of herbicides in soils organisms. Copyright © 2015 Elsevier Inc. All rights reserved.

Ex-vivo characterization of regulatory T cells in pulmonary tuberculosis patients, latently infected persons, and healthy endemic controls.

PubMed

Zewdie, Martha; Howe, Rawleigh; Hoff, Søren T; Doherty, T Mark; Getachew, Nahom; Tarekegne, Azeb; Tessema, Bamlak; Yamuah, Lawrence; Aseffa, Abraham; Abebe, Markos

2016-09-01

Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ex-vivo characterization of regulatory T cells in pulmonary tuberculosis patients, latently infected persons, and healthy endemic controls

PubMed Central

Zewdie, Martha; Howe, Rawleigh; Hoff, Søren T.; Doherty, T. Mark; Getachew, Nahom; Tarekegne, Azeb; Tessema, Bamlak; Yamuah, Lawrence; Aseffa, Abraham; Abebe, Markos

2016-01-01

SUMMARY Background Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. Method We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. Results The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4+CD25+/hi T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4+CD25+FOXP3+CD127lo Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4+CD25+FOXP3+CD127loCD45RO+Ki-67+) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO+Ki-67−) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5–21.9) of Tregs to 9.3% (7.0–12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection. PMID:27553411

YB-1 expression promotes epithelial-to-mesenchymal transition in prostate cancer that is inhibited by a small molecule fisetin

PubMed Central

Khan, Mohammad Imran; Adhami, Vaqar Mustafa; Lall, Rahul Kumar; Sechi, Mario; Joshi, Dinesh C.; Haidar, Omar M.; Syed, Deeba Nadeem; Siddiqui, Imtiaz Ahmad; Chiu, Shing-Yan; Mukhtar, Hasan

2014-01-01

Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from β1 - β4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from −11.9845 to −9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 μM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation. PMID:24770864

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

Coxiella burnetii Induces Inflammatory Interferon-Like Signature in Plasmacytoid Dendritic Cells: A New Feature of Immune Response in Q Fever

PubMed Central

Ka, Mignane B.; Mezouar, Soraya; Ben Amara, Amira; Raoult, Didier; Ghigo, Eric; Olive, Daniel; Mege, Jean-Louis

2016-01-01

Plasmacytoid dendritic cells (pDCs) play a major role in antiviral immunity via the production of type I interferons (IFNs). There is some evidence that pDCs interact with bacteria but it is not yet clear whether they are protective or contribute to bacterial pathogenicity. We wished to investigate whether Coxiella burnetii, the agent of Q fever, interacts with pDCs. The stimulation of pDCs with C. burnetii increased the expression of activation and migratory markers (CD86 and CCR7) as determined by flow cytometry and modulated gene expression program as revealed by a microarray approach. Indeed, genes encoding for pro-inflammatory cytokines, chemokines, and type I INF were up-regulated. The up-regulation of type I IFN was correlated with an increase in IFN-α release by C. burnetii-stimulated pDCs. We also investigated pDCs in patients with Q fever endocarditis. Using flow cytometry and a specific gating strategy, we found that the number of circulating pDCs was significantly lower in patients with Q fever endocarditis as compared to healthy donors. In addition, the remaining circulating pDCs expressed activation and migratory markers. As a whole, our study identified non-previously reported activation of pDCs by C. burnetii and their modulation during Q fever. PMID:27446817

Intragraft expression of the IL-10 gene is up-regulated in renal protocol biopsies with early interstitial fibrosis, tubular atrophy, and subclinical rejection.

PubMed

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; O'Valle, Francisco; Pérez-Riba, Mercè; Del Moral, Raimundo García; Grinyó, Josep M; Serón, Daniel

2010-04-01

Grafts with subclinical rejection associated with interstitial fibrosis and tubular atrophy (SCR+IF/TA) show poorer survival than grafts with subclinical rejection without IF/TA (SCR). Aiming to detect differences among SCR+IF/TA and SCR, we immunophenotyped the inflammatory infiltrate (CD45, CD3, CD20, CD68) and used a low-density array to determine levels of T(H)1 (interleukin IL-2, IL-3, gamma-interferon, tumor necrosis factor-alpha, lymphotoxin-alpha, lymphotoxin-beta, granulocyte-macrophage colony-stimulating factor) and T(H)2 (IL-4, IL-5, IL-6, IL-10, and IL-13) transcripts as well as of IL-2R (as marker for T-cell activation) in 31 protocol biopsies of renal allografts. Here we show that grafts with early IF/TA and SCR can be distinguished from grafts with SCR on the basis of the activation of IL-10 gene expression and of an increased infiltration by B-lymphocytes in a cellular context in which the degree of T-cell activation is similar in both groups of biopsies, as demonstrated by equivalent levels of IL-2R mRNA. These results suggest that the up-regulation of the IL-10 gene expression, as well as an increased proportion of B-lymphocytes in the inflammatory infiltrates, might be useful as markers of early chronic lesions in grafts with SCR.

Intragraft Expression of the IL-10 Gene Is Up-Regulated in Renal Protocol Biopsies with Early Interstitial Fibrosis, Tubular Atrophy, and Subclinical Rejection

PubMed Central

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; O'Valle, Francisco; Pérez-Riba, Mercè; del Moral, Raimundo García; Grinyó, Josep M.; Serón, Daniel

2010-01-01

Grafts with subclinical rejection associated with interstitial fibrosis and tubular atrophy (SCR+IF/TA) show poorer survival than grafts with subclinical rejection without IF/TA (SCR). Aiming to detect differences among SCR+IF/TA and SCR, we immunophenotyped the inflammatory infiltrate (CD45, CD3, CD20, CD68) and used a low-density array to determine levels of TH1 (interleukin IL-2, IL-3, γ-interferon, tumor necrosis factor-α, lymphotoxin-α, lymphotoxin-β, granulocyte-macrophage colony-stimulating factor) and TH2 (IL-4, IL-5, IL-6, IL-10, and IL-13) transcripts as well as of IL-2R (as marker for T-cell activation) in 31 protocol biopsies of renal allografts. Here we show that grafts with early IF/TA and SCR can be distinguished from grafts with SCR on the basis of the activation of IL-10 gene expression and of an increased infiltration by B-lymphocytes in a cellular context in which the degree of T-cell activation is similar in both groups of biopsies, as demonstrated by equivalent levels of IL-2R mRNA. These results suggest that the up-regulation of the IL-10 gene expression, as well as an increased proportion of B-lymphocytes in the inflammatory infiltrates, might be useful as markers of early chronic lesions in grafts with SCR. PMID:20150436

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

PubMed

Bischoff, Florian C; Werner, Astrid; John, David; Boeckel, Jes-Niels; Melissari, Maria-Theodora; Grote, Phillip; Glaser, Simone F; Demolli, Shemsi; Uchida, Shizuka; Michalik, Katharina M; Meder, Benjamin; Katus, Hugo A; Haas, Jan; Chen, Wei; Pullamsetti, Soni S; Seeger, Werner; Zeiher, Andreas M; Dimmeler, Stefanie; Zehendner, Christoph M

2017-08-04

Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays ( P <0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α ( P <0.001), ATF6 ( P <0.01), and soluble BiP ( P <0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure ( P <0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease. © 2017 American Heart Association, Inc.

A forkhead Transcription Factor Is Wound-Induced at the Planarian Midline and Required for Anterior Pole Regeneration

PubMed Central

Scimone, M. Lucila; Lapan, Sylvain W.; Reddien, Peter W.

2014-01-01

Planarian regeneration requires positional information to specify the identity of tissues to be replaced as well as pluripotent neoblasts capable of differentiating into new cell types. We found that wounding elicits rapid expression of a gene encoding a Forkhead-family transcription factor, FoxD. Wound-induced FoxD expression is specific to the ventral midline, is regulated by Hedgehog signaling, and is neoblast-independent. FoxD is subsequently expressed within a medial subpopulation of neoblasts at wounds involving head regeneration. Ultimately, FoxD is co-expressed with multiple anterior markers at the anterior pole. Inhibition of FoxD with RNA interference (RNAi) results in the failure to specify neoblasts expressing anterior markers (notum and prep) and in anterior pole formation defects. FoxD(RNAi) animals fail to regenerate a new midline and to properly pattern the anterior blastema, consistent with a role for the anterior pole in organizing pattern of the regenerating head. Our results suggest that wound signaling activates a forkhead transcription factor at the midline and, if the head is absent, FoxD promotes specification of neoblasts at the prior midline for anterior pole regeneration. PMID:24415944

Expression of the autoantigen TRIM33/TIF1γ in skin and muscle of patients with dermatomyositis is upregulated, together with markers of cellular stress.

PubMed

Scholtissek, B; Ferring-Schmitt, S; Maier, J; Wenzel, J

2017-08-01

Dermatomyositis (DM) is an autoimmune disorder associated with a dysregulation of immune homeostasis of both the innate and adaptive immune system. Earlier data suggested that these two arms of the immune system interconnect in DM. In the current study, we analysed the association of autoantigen expression [adaptive system components: Mi2, transcriptional intermediary factor (TIF)1γ, small ubiquitin-like modifier 1 activating enzyme subunit (SAE)1, melanoma differentiation-associated protein (MDA)5] with markers of cellular stress (innate system components: MxA, p53) in skin and muscle (immunohistology and gene expression data, respectively). We found that distinctive self-antigens of DM were elevated in both skin and muscle tissue. In particular, TIF1γ expression was seen in autoimmune diseases including DM, but not in other inflammatory skin disorders. This upregulation was closely associated with p53 expression and type I interferon-regulated inflammation, suggesting that upregulation of autoantigens in the skin and muscle of patients with DM might be driven by cellular stress. Better understanding of these mechanisms could pave the way for new therapeutic concepts focusing on stress reduction. © 2017 British Association of Dermatologists.

Cigarette smoke-induced alveolar epithelial-mesenchymal transition is mediated by Rac1 activation.

PubMed

Shen, Hui-juan; Sun, Yan-hong; Zhang, Shui-juan; Jiang, Jun-xia; Dong, Xin-wei; Jia, Yong-liang; Shen, Jian; Guan, Yan; Zhang, Lin-hui; Li, Fen-fen; Lin, Xi-xi; Wu, Xi-mei; Xie, Qiang-min; Yan, Xiao-feng

2014-06-01

Epithelial-mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT. EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively. Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression. Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.