Increased expression of CD44 is associated with more aggressive behavior in clear cell renal cell carcinoma.

PubMed

Zanjani, Leili Saeednejad; Madjd, Zahra; Abolhasani, Maryam; Rasti, Arezoo; Fodstad, Oystein; Andersson, Yvonne; Asgari, Mojgan

2018-01-01

Although CD44 has been suggested as a prognostic marker in renal cell carcinoma (RCC), the prognostic significance of this marker in three main subtypes of RCC is still unclear. Thus, the present study was conducted to evaluate the expression and prognostic significance of CD44 as a cancer stem cell marker in different histological subtypes of RCC. Methodology & results: CD44 expression was evaluated in 206 well-defined renal tumor samples using immunohistochemistry on tissue microarrays. Higher CD44 expression was associated with more aggressive behavior, tumor progression and worse prognosis in clear cell RCC (ccRCC) but not in papillary and chromophobe RCC subtypes. Cancer stem cell marker CD44 may be a promising target for cancer treatment only in ccRCC.

Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters.

PubMed

Kappler, Matthias; Taubert, Helge; Holzhausen, Hans-Jürgen; Reddemann, Rolf; Rot, Swetlann; Becker, Axel; Kuhnt, Thomas; Dellas, Kathrin; Dunst, Jürgen; Vordermark, Dirk; Hänsgen, Gabriele; Bache, Matthias

2008-08-01

Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant Levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. Tumortissue sections of 34 patients with advanced head-and-neck cancertreated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1alpha and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. Bivariate analysis showed a significant correlation of HIF-1alpha expression with high P53 and high OPN expression, high serum VEGF Levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1alpha and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1alpha or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1alpha or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). Expression of HIF-1alpha and CAIX was correlated with different tumor parameters. Only combined HIF-1alpha and CAIX expression was significantly predictive of patients' overall survival.

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao.

PubMed

Lima, L S; Gramacho, K P; Carels, N; Novais, R; Gaiotto, F A; Lopes, U V; Gesteira, A S; Zaidan, H A; Cascardo, J C M; Pires, J L; Micheli, F

2009-07-14

In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.

Prognostic value of CD44 expression in penile squamous cell carcinoma: a pilot study.

PubMed

Minardi, Daniele; Lucarini, Guendalina; Filosa, Alessandra; Zizzi, Antonio; Simonetti, Oriana; Offidani, Anna Maria; d'Anzeo, Gianluca; Di Primio, Roberto; Montironi, Rodolfo; Muzzonigro, Giovanni

2012-10-01

Several studies have reported on the prognostic value of molecular markers for metastasis risk and survival in penile squamous cell carcinoma (SCC) patients. The usefulness of CD44 expression as such a marker has been studied in different tumors, but not in penile SCC. Our aim was to determine whether CD44 expression may serve as a prognostic marker for lymph node metastasis and survival in penile SCC patients. CD44 immunoistochemical expression was investigated in tissue specimens from 39 patients with penile SCC. CD44 cell positivity, staining intensity and distribution were analyzed and correlated with tumor stage, grade, lymph node status and disease-specific survival. CD44 expression was detected in epithelial cells of both intratumoral and normal tissues with different intensities and staining distributions. In normal tissues CD44 protein was mainly detected in cell membranes, whereas in the tumor compartments it was found in both the cell membranes and the cytoplasm. The intensities and percentages of CD44 expressing cells did not correlate with tumor stage and/or grade. Seventy-three percent of the patients with lymph node metastasis showed high intensities of CD44 staining, as compared to 44% of the patients without lymph node metastasis (P = 0.03). Lymph node-positive patients showed both cytoplasmic and membranous CD44 expression. High CD44 expression was found to be significantly correlated with a decreased 5 year overall survival (P = 0.01). CD44 levels and patterns of expression can be considered as markers for penile SCC aggressiveness and, in addition, may serve as predictive markers for lymph node metastasis, also in patients with clinically negative lymph nodes. CD44 expression may provide prognostic information for penile SCC patients, next to classical clinical-pathological factors.

CXCR6 identifies a putative population of retained human lung T cells characterised by co-expression of activation markers.

PubMed

Morgan, Angela J; Guillen, Cristina; Symon, Fiona A; Birring, Surinder S; Campbell, James J; Wardlaw, Andrew J

2008-01-01

Expressions of activation markers have been described on the surface of T cells in the blood and the lung in both health and disease. We have studied the distribution of activation markers on human lung T cells and have found that only certain populations exist. Importantly, the presence or absence of some markers appears to predict those of others, in particular cells which express CD103 also express CD49a and CD69, whereas cells which do not express CD69 also do not express CD49a or CD103. In view of the paucity of activation marker expression in the peripheral blood, we have hypothesised that these CD69+, CD49a+, and CD103+ (triple positive) cells are retained in the lung, possess effector function (IFNgamma secretion) and express particular chemokine receptors which allow them to be maintained in this environment. We have found that the ability of the triple negative cells to secrete IFNgamma is significantly less than the triple positive cells, suggesting that the expression of activation markers can highlight a highly specialised effector cell. We have studied the expression of 14 chemokine receptors and have found that the most striking difference between the triple negative cells and the triple positive cells is the expression of CXCR6 with 12.8+/-9.8% of triple negative cells expressing CXCR6 compared to 89.5+/-5.5% of triple positive cells. We propose therefore that CXCR6 may play an important role in the retention of T cells within the lung.

Developmental origin of lung macrophage diversity

PubMed Central

Tan, Serena Y. S.; Krasnow, Mark A.

2016-01-01

Macrophages are specialized phagocytic cells, present in all tissues, which engulf and digest pathogens, infected and dying cells, and debris, and can recruit and regulate other immune cells and the inflammatory response and aid in tissue repair. Macrophage subpopulations play distinct roles in these processes and in disease, and are typically recognized by differences in marker expression, immune function, or tissue of residency. Although macrophage subpopulations in the brain have been found to have distinct developmental origins, the extent to which development contributes to macrophage diversity between tissues and within tissues is not well understood. Here, we investigate the development and maintenance of mouse lung macrophages by marker expression patterns, genetic lineage tracing and parabiosis. We show that macrophages populate the lung in three developmental waves, each giving rise to a distinct lineage. These lineages express different markers, reside in different locations, renew in different ways, and show little or no interconversion. Thus, development contributes significantly to lung macrophage diversity and targets each lineage to a different anatomical domain. PMID:26952982

Transcriptome analysis of cattle muscle identifies potential markers for skeletal muscle growth rate and major cell types.

PubMed

Guo, Bing; Greenwood, Paul L; Cafe, Linda M; Zhou, Guanghong; Zhang, Wangang; Dalrymple, Brian P

2015-03-13

This study aimed to identify markers for muscle growth rate and the different cellular contributors to cattle muscle and to link the muscle growth rate markers to specific cell types. The expression of two groups of genes in the longissimus muscle (LM) of 48 Brahman steers of similar age, significantly enriched for "cell cycle" and "ECM (extracellular matrix) organization" Gene Ontology (GO) terms was correlated with average daily gain/kg liveweight (ADG/kg) of the animals. However, expression of the same genes was only partly related to growth rate across a time course of postnatal LM development in two cattle genotypes, Piedmontese x Hereford (high muscling) and Wagyu x Hereford (high marbling). The deposition of intramuscular fat (IMF) altered the relationship between the expression of these genes and growth rate. K-means clustering across the development time course with a large set of genes (5,596) with similar expression profiles to the ECM genes was undertaken. The locations in the clusters of published markers of different cell types in muscle were identified and used to link clusters of genes to the cell type most likely to be expressing them. Overall correspondence between published cell type expression of markers and predicted major cell types of expression in cattle LM was high. However, some exceptions were identified: expression of SOX8 previously attributed to muscle satellite cells was correlated with angiogenesis. Analysis of the clusters and cell types suggested that the "cell cycle" and "ECM" signals were from the fibro/adipogenic lineage. Significant contributions to these signals from the muscle satellite cells, angiogenic cells and adipocytes themselves were not as strongly supported. Based on the clusters and cell type markers, sets of five genes predicted to be representative of fibro/adipogenic precursors (FAPs) and endothelial cells, and/or ECM remodelling and angiogenesis were identified. Gene sets and gene markers for the analysis of many of the major processes/cell populations contributing to muscle composition and growth have been proposed, enabling a consistent interpretation of gene expression datasets from cattle LM. The same gene sets are likely to be applicable in other cattle muscles and in other species.

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.

Radiation Dose-effects on Cell Cycle, Apoptosis, and Marker Expression of Ataxia Telangiectasia-Heterozygous Human Breast Epithelial Cells

NASA Technical Reports Server (NTRS)

Cruz, A.; Bors, K.; Jansen, H.; Richmond, R.

2003-01-01

Ataxia-telangiectasia (A-T) is a radiation-sensitive genetic condition. AT-heterozygous human mammary epithelial cells (HMEC) were irradiated using a Cs137 source in order to compare cell cycle, apoptosis, and marker expression responses across 3 radiation doses. No differences in cell cycle and apoptosis were found with any of the radiation doses used (30, 60, and 90 rads) compared with the unirradiated control (0 rad). At the same doses, however, differences were found in marker expression, such as keratin 18 (kl8), keratin 14 (k14), insulin-like growth factor I receptor (IGF-IR), and connexin 43 (cx43). This may indicate that radiation sensitivity in the heterozygous state may be initiated through signal transduction responses.

Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration.

PubMed

Gayen, Srimonta; Mandal, Chandi Charan; Samanta, Milan Kumar; Dey, Avishek; Sen, Soumitra Kumar

2016-04-01

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

PB-1: The Relationship Between Anti Apoptotic Marker (BCL-2) and Biochemical Markers in Type 2 Diabetes Patients

PubMed Central

Damitri, TD; Faridah, AR; Imran, Y; Hasnan, J.

2006-01-01

Purpose : To investigate the expression of anti apoptotic marker (bcl-2) and the level of biochemical markers in type 2 diabetes patients. METHODS : A cross-sectional study was conducted from August 2003 to November 2005. Forty one type 2 diabetes patients and 36 non diabetes (control) subjects aged between 20 to 70 years were included in this study. Blood samples were collected for fasting plasma glucose (FPG), triglycerides (TG), Total cholesterol (TC), High density lipoprotein cholesterol (HDLC), Low density lipoprotein cholesterol (LDLC) and analyzed in the Chemical Pathology laboratory, while glycosylated hemoglobin A1c (A1C) was analyzed in the Endocrine laboratory. The skin biopsy tissue samples were stained with immunohistochemistry (IHC) stain for expression of bcl-2 in the Pathology laboratory. RESULTS : There was a significant difference (p<0.001) between both groups for mean FPG (diabetics=11.02±4.25, control=4.41±1.12 mmol/L), HDLC (diabetics=1.00±0.38, control=1.47±0.72 mmol/L) and A1C (diabetics=9.50±2.24%, control=5.00±0.67%). However, there was no significant difference for TG, TC, and LDLC between both groups. Interestingly, the difference of mean bcl-2 expression were very highly significant (p<0.001) when compared between both groups. Mean bcl-2 expression was dibetics= 1.88±0.33 and control= 1.47±0.51. Positive bcl-2 expression was found in only 5 (12.2%) diabetics while 36 (87.8%) diabetics showed negative expression. Positive bcl-2 expression was observed in 19 (52.8%) controls while 17 (47.2%) showed negative expression. CONCLUSION : The expression of anti apoptotic marker bcl-2 was increased in non diabetic subjects in order to prevent cell death. However, the reduced expression of bcl-2 in diabetic patients may be associated with programmed cell death. The detailed mechanism for the gene expression of bcl-2 may help us to understand how bcl-2 is involved in apoptosis in diabetic microvasculature complications.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Temporal Expression of Peripheral Blood Leukocyte Biomarkers in a Macaca fascicularis Infection Model of Tuberculosis; Comparison with Human Datasets and Analysis with Parametric/Non-parametric Tools for Improved Diagnostic Biomarker Identification

PubMed Central

Wareham, Alice; Lewandowski, Kuiama S.; Williams, Ann; Dennis, Michael J.; Sharpe, Sally; Vipond, Richard; Silman, Nigel; Ball, Graham

2016-01-01

A temporal study of gene expression in peripheral blood leukocytes (PBLs) from a Mycobacterium tuberculosis primary, pulmonary challenge model Macaca fascicularis has been conducted. PBL samples were taken prior to challenge and at one, two, four and six weeks post-challenge and labelled, purified RNAs hybridised to Operon Human Genome AROS V4.0 slides. Data analyses revealed a large number of differentially regulated gene entities, which exhibited temporal profiles of expression across the time course study. Further data refinements identified groups of key markers showing group-specific expression patterns, with a substantial reprogramming event evident at the four to six week interval. Selected statistically-significant gene entities from this study and other immune and apoptotic markers were validated using qPCR, which confirmed many of the results obtained using microarray hybridisation. These showed evidence of a step-change in gene expression from an ‘early’ FOS-associated response, to a ‘late’ predominantly type I interferon-driven response, with coincident reduction of expression of other markers. Loss of T-cell-associate marker expression was observed in responsive animals, with concordant elevation of markers which may be associated with a myeloid suppressor cell phenotype e.g. CD163. The animals in the study were of different lineages and these Chinese and Mauritian cynomolgous macaque lines showed clear evidence of differing susceptibilities to Tuberculosis challenge. We determined a number of key differences in response profiles between the groups, particularly in expression of T-cell and apoptotic makers, amongst others. These have provided interesting insights into innate susceptibility related to different host `phenotypes. Using a combination of parametric and non-parametric artificial neural network analyses we have identified key genes and regulatory pathways which may be important in early and adaptive responses to TB. Using comparisons between data outputs of each analytical pipeline and comparisons with previously published Human TB datasets, we have delineated a subset of gene entities which may be of use for biomarker diagnostic test development. PMID:27228113

Two-dimensional gel analysis of protein expression in ovarian tumors shows a low degree of intratumoral heterogeneity.

PubMed

Alaiya, A A; Franzén, B; Moberger, B; Silfverswärd, C; Linder, S; Auer, G

1999-01-01

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. In the present study, tumor cells were prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression was low. In contrast, large differences were observed when the protein profiles of different tumors were compared. The differences in gene expression between pairs of malignant carcinomas were slightly larger than the differences observed between pairs of benign tumors. We conclude that 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

Detergents with different chemical properties induce variable degree of cytotoxicity and mRNA expression of lipid-metabolizing enzymes and differentiation markers in cultured keratinocytes.

PubMed

Wei, Tianling; Geijer, Sophia; Lindberg, Magnus; Berne, Berit; Törmä, Hans

2006-12-01

The knowledge how detergents with different chemical properties influence epidermal keratinocytes is sparse. In the present study, the effects of five detergents were examined with respect to cell-toxicity and mRNA expression of key-enzymes in barrier lipid production and keratinocyte differentiation markers. First, the LD(50) for each detergent were determined. Secondly, keratinocytes were exposed to sub-toxic concentrations and the mRNA expression was analysed by real-time PCR after 24 h exposure to the detergents. SLS and CAPB induced a concentration-dependent increase in the expression of enzymes producing cholesterol and ceramides, while transcripts of enzymes producing fatty acids were unaffected. SLES and cocoglucoside increased the expression of certain enzymes involved in cholesterol and fatty acid synthesis while sodium cocoamphoacetate (SCAA) stimulated expression of transcripts involved in fatty acid synthesis. The expression of differentiation markers were increased by SLS, SLES and CAPB, while SCAA and cocoglucoside exhibited no effect. The present findings show that detergents have variable effects on lipid synthesis and keratinocyte differentiation, which could partly explain their barrier destruction potential in vivo.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

PubMed Central

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy.

PubMed

Tao, Wensi; Ayala-Haedo, Juan A; Field, Matthew G; Pelaez, Daniel; Wester, Sara T

2017-12-01

The purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls. Orbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell-specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR. Orbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO. Our results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways.

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy

PubMed Central

Tao, Wensi; Ayala-Haedo, Juan A.; Field, Matthew G.; Pelaez, Daniel; Wester, Sara T.

2017-01-01

Purpose The purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls. Methods Orbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell–specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR. Results Orbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO. Conclusion Our results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways. PMID:29214313

Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis

PubMed Central

Aljadi, Zenib; Mansouri, Ladan; Nopp, Anna; Paulsson, Josefin M; Winqvist, Ola; Russom, Aman; Ståhl, Mårten; Hylander, Britta; Jacobson, Stefan H; Lundahl, Joachim

2014-01-01

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood–membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis. PMID:24712758

Effects of space flight on surface marker expression

NASA Astrophysics Data System (ADS)

Sonnenfeld, G.

1999-01-01

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.

Temporal and spatial distribution of macrophage phenotype markers in the foreign body response to glutaraldehyde-crosslinked gelatin hydrogels.

PubMed

Yu, Tony; Wang, Wenbo; Nassiri, Sina; Kwan, Thomas; Dang, Chau; Liu, Wei; Spiller, Kara L

2016-01-01

Currently, it is not well understood how changes in biomaterial properties affect the foreign body response (FBR) or macrophage behavior. Because failed attempts at biomaterial degradation by macrophages have been linked to frustrated phagocytosis, a defining feature of the FBR, we hypothesized that increased hydrogel crosslinking density (and decreased degradability) would exacerbate the FBR. Gelatin hydrogels were crosslinked with glutaraldehyde (0.05, 0.1, and 0.3%) and implanted subcutaneously in C57BL/6 mice over the course of 3 weeks. Interestingly, changes in hydrogel crosslinking did not affect the thickness of the fibrous capsule surrounding the hydrogels, expression of the pan-macrophage marker F480, expression of three macrophage phenotype markers (iNOS, Arg1, CD163), or expression of the myofibroblast marker aSMA, determined using semi-quantitative immunohistochemical analysis. With respect to temporal changes, the level of expression of the M1 marker (iNOS) remained relatively constant throughout the study, while the M2 markers Arg1 and CD163 increased over time. Expression of these M2 markers was highly correlated with fibrous capsule thickness. Differences in spatial distribution of staining also were noted, with the strongest staining for iNOS at the hydrogel surface and increasing expression of the myofibroblast marker aSMA toward the outer edge of the fibrous capsule. These results confirm previous reports that macrophages in the FBR exhibit characteristics of both M1 and M2 phenotypes. Understanding the effects (or lack of effects) of biomaterial properties on the FBR and macrophage phenotype may aid in the rational design of biomaterials to integrate with surrounding tissue.

Stem Cell-Associated Marker Expression in Canine Hair Follicles

PubMed Central

Gerhards, Nora M.; Sayar, Beyza S.; Origgi, Francesco C.; Galichet, Arnaud; Müller, Eliane J.; Welle, Monika M.; Wiener, Dominique J.

2016-01-01

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients. PMID:26739040

Stem Cell-Associated Marker Expression in Canine Hair Follicles.

PubMed

Gerhards, Nora M; Sayar, Beyza S; Origgi, Francesco C; Galichet, Arnaud; Müller, Eliane J; Welle, Monika M; Wiener, Dominique J

2016-03-01

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients. © 2016 The Histochemical Society.

The not face: A grammaticalization of facial expressions of emotion.

PubMed

Benitez-Quiroz, C Fabian; Wilbur, Ronnie B; Martinez, Aleix M

2016-05-01

Facial expressions of emotion are thought to have evolved from the development of facial muscles used in sensory regulation and later adapted to express moral judgment. Negative moral judgment includes the expressions of anger, disgust and contempt. Here, we study the hypothesis that these facial expressions of negative moral judgment have further evolved into a facial expression of negation regularly used as a grammatical marker in human language. Specifically, we show that people from different cultures expressing negation use the same facial muscles as those employed to express negative moral judgment. We then show that this nonverbal signal is used as a co-articulator in speech and that, in American Sign Language, it has been grammaticalized as a non-manual marker. Furthermore, this facial expression of negation exhibits the theta oscillation (3-8 Hz) universally seen in syllable and mouthing production in speech and signing. These results provide evidence for the hypothesis that some components of human language have evolved from facial expressions of emotion, and suggest an evolutionary route for the emergence of grammatical markers. Copyright © 2016 Elsevier B.V. All rights reserved.

The Not Face: A grammaticalization of facial expressions of emotion

PubMed Central

Benitez-Quiroz, C. Fabian; Wilbur, Ronnie B.; Martinez, Aleix M.

2016-01-01

Facial expressions of emotion are thought to have evolved from the development of facial muscles used in sensory regulation and later adapted to express moral judgment. Negative moral judgment includes the expressions of anger, disgust and contempt. Here, we study the hypothesis that these facial expressions of negative moral judgment have further evolved into a facial expression of negation regularly used as a grammatical marker in human language. Specifically, we show that people from different cultures expressing negation use the same facial muscles as those employed to express negative moral judgment. We then show that this nonverbal signal is used as a co-articulator in speech and that, in American Sign Language, it has been grammaticalized as a non-manual marker. Furthermore, this facial expression of negation exhibits the theta oscillation (3–8 Hz) universally seen in syllable and mouthing production in speech and signing. These results provide evidence for the hypothesis that some components of human language have evolved from facial expressions of emotion, and suggest an evolutionary route for the emergence of grammatical markers. PMID:26872248

Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

NASA Technical Reports Server (NTRS)

Richmond, Robert C.

2001-01-01

Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

Expression of neuronal markers in the endometrium of women with and those without endometriosis.

PubMed

Newman, T A; Bailey, J L; Stocker, L J; Woo, Y L; Macklon, N S; Cheong, Y C

2013-09-01

How do the expression patterns of neuronal markers differ in the endometrium of women with and without endometriosis? The neuronal markers, PGP9.5, NGFp75 and VR1, are expressed in the endometrium at levels that do not differ between women with and without endometriosis. Aberrant neuronal growth within the uterus may contribute to abnormal fertility and uterine dysfunction. However, controversy still exists as to whether aberrant innervation in the endometrium is associated with gynaecological pathology such as endometriosis. This may reflect the use of subjective methods such as histology to assess the innervation of the endometrium. We, therefore, employed a quantitative method, western blotting, to study markers of endometrial innervation in the presence and absence of endometriosis. This study included 45 women undergoing laparoscopic examination for the diagnosis of endometriosis. Endometrial samples were analysed by western blot for the expression of neuronal and neurotrophic markers, PGP9.5, VR1 and NGFp75. Endometrial pipelle biopsies were obtained from patients with (n = 20, study group) and without (n = 25, control group) endometriosis. Tissue was analysed by immunohistochemistry and western blot analysis for the expression of pan-neuronal marker, PGP9.5, sensory nociceptive marker, TPVR1, and low-affinity neurotrophic growth factor receptor, NGFRp75. PGP9.5, NGFp75 and VR1 were expressed in the endometrium of women, independent of the presence of endometriosis. Furthermore, the expression level of PGP9.5, VR1 and NGFp75 did not alter between the two cohorts of women. Studies of this nature are subject to the heterogeneous nature of patient population and tissue samples despite attempts to standardize these parameters. Hence, further studies using similar methodology will be required to confirm our results. Our results highlight that sensory neuronal markers are present in women with and without endometriosis. Future work will assess what the targets of the endometrial nerves are and investigate their function, their impact on endometrial biology and, in particular, whether aberrant neuronal function, rather than the mere presence of neuronal function, could be the root cause of subfertility and/or pain affecting many endometriosis sufferers. Our results do not, however, confirm the previous paradigm of increased innervation in the endometrium of women with endometriosis, nor the use of nerve cell detection from pipelle biopsies to diagnose endometriosis.

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.

Cytoplasmic expression of CD133 stemness marker is associated with tumor aggressiveness in clear cell renal cell carcinoma.

PubMed

Saeednejad Zanjani, Leili; Madjd, Zahra; Abolhasani, Maryam; Andersson, Yvonne; Rasti, Arezoo; Shariftabrizi, Ahmad; Asgari, Mojgan

2017-10-01

Prominin-1 (CD133) is one of the most commonly used markers for cancer stem cells (CSCs), which are characterized by their ability for self-renewal and tumorigenicity. However, the clinical and prognostic significance of CSCs in renal cell carcinoma (RCC) remains unclear. The aim of this study was to investigate the expression patterns and prognostic significance of the cancer stem cell marker CD133 in different histological subtypes of RCC. CD133 expression was evaluated using immunohistochemistry in 193 well-defined renal tumor samples on tissue microarrays, including 136 (70.5%) clear cell renal cell carcinomas (CCRCCs), 26 (13.5%) papillary RCCs, and 31 (16.1%) chromophobe RCCs. The association between CD133 expression and clinicopathological features as well as the survival outcomes was determined. There was a statistically significant difference between CD133 expression among the different RCC subtypes. In CCRCC, higher cytoplasmic expression of CD133 was significantly associated with increase in grade, stage, microvascular invasion (MVI) and lymph node invasion (LNI), while no association was found with the membranous expression. Moreover, on multivariate analysis, TNM stage and nuclear grade were independent prognostic factors for overall survival (OS) in cytoplasmic expression. We showed that higher cytoplasmic CD133 expression was associated with more aggressive tumor behavior and more advanced disease in CCRCC but not in the other examined subtypes. Our results demonstrated that higher cytoplasmic CD133 expression is clinically significant in CCRCC and is associated with increased tumor aggressiveness and is useful for predicting cancer progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Claudin-18 overexpression in intestinal-type mucinous borderline tumour of the ovary.

PubMed

Halimi, Sultan Ahmad; Maeda, Daichi; Shinozaki-Ushiku, Aya; Koso, Takahiro; Matsusaka, Keisuke; Tanaka, Mariko; Arimoto, Takahide; Oda, Katsutoshi; Kawana, Kei; Yano, Tetsu; Fujii, Tomoyuki; Fukayama, Masashi

2013-10-01

Mucinous borderline tumours of the ovary are subclassified as intestinal-type (IMBT) and endocervical-like (EMBT), which differ in their clinicopathological features. In this study, we attempted to elucidate characteristics of the mucinous epithelium in each subtype. The expression of claudin-18, a marker of gastric differentiation, MUCs, CDX2, CK7, CK20, oestrogen receptor (ER), progesterone receptor (PgR), CA-125 and vimentin in IMBTs (n = 54), EMBTs (n = 25) and serous borderline tumours (SBTs) (n = 22) were compared by immunohistochemistry. Claudin-18 positivity was identified in 98% of the IMBTs, whereas only 4% of the EMBTs were claudin-18-positive. Expression of intestinal markers such as CDX2 and MUC2 was relatively infrequent in IMBTs (48% and 33%, respectively). Müllerian-lineage markers such as ER, PgR and vimentin were expressed rarely in IMBTs, while most EMBTs and SBTs were positive for these markers. Hierarchial clustering revealed a close association between EMBTs and SBTs, while IMBTs were clearly separate. Claudin-18 positivity is a specific phenotype that is characteristic of IMBTs. Frequent and diffuse expression of gastric markers, along with less frequent and usually focal expression of intestinal markers, suggests that IMBTs are essentially composed of gastrointestinal-type mucinous epithelium (gastric-type epithelium with a variable degree of intestinal differentiation). © 2013 John Wiley & Sons Ltd.

Androgen deprivation and stem cell markers in prostate cancers

PubMed Central

Tang, Yao; Hamburger, Anne W; Wang, Linbo; Khan, Mohammad Afnan; Hussain, Arif

2010-01-01

In our previous studies using human LNCaP xenografts and TRAMP (transgenic adenocarcinoma of mouse prostate) mice, androgen deprivation therapy (ADT) resulted in a temporary cessation of prostate cancer (PCa) growth, but then tumors grew faster with more malignant behaviour. To understand whether cancer stem cells might play a role in PCa progression in these animal models, we investigated the expressions of stem cell-related markers in tumors at different time points after ADT. In both animal models, enhanced expressions of stem cell markers were observed in tumors of castrated mice, as compared to non-castrated controls. This increased cell population that expressed stem cell markers is designated as stem-like cells (SLC) in this article. We also observed that the SLC peaked at relatively early time points after ADT, before tumors resumed their growth. These results suggest that the SLC population may play a role in tumor re-growth and disease progression, and that targeting the SLC at their peak-expression time point may prevent tumor recurrence following ADT. PMID:20126580

Mass spectrometric identification of diagnostic markers for chronic prostatitis in seminal plasma by analysis of seminal plasma protein clinical samples.

PubMed

Rokka, A; Mehik, A; Tonttila, P; Vaarala, M

2017-08-15

There are few specific diagnostic markers for chronic prostatitis. Therefore, we used mass spectrometry to evaluate differences in seminal plasma protein expression among patients with prostatitis and young and middle-aged healthy controls. We analysed pooled seminal plasma protein samples from four prostatitis patients (two pools), three young controls (one pool), and three middle-aged controls (one pool). The samples were analysed by liquid chromatography-tandem mass spectrometry. Of the 349 proteins identified, 16 were differentially expressed between the two control pools. Five proteins were up- or down-regulated in both of the prostatitis pools compared to middle-aged controls but not between young and middle-aged pools. Progestagen-associated endometrial protein (PAEP) was over-expressed in prostatitis samples compared to young and middle-aged controls. Our findings and those of previous studies indicate that PAEP is a potential seminal plasma marker for chronic prostatitis. In conclusion, we found age-related changes in seminal plasma protein expression. PAEP expression in seminal plasma should be investigated further to evaluate its potential as a diagnostic marker for chronic prostatitis.

[Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment].

PubMed

Hao, Tong; Li, Jun-Jie; Du, Zhi-Yan; Duan, Cui-Mi; Wang, Yan-Meng; Wang, Chang-Yong; Song, Jing-Ping; Wang, Lin-Jie; Li, Ying-Hui; Wang, Yan

2012-10-01

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.

Differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of pain in human rotator cuff tendinopathy.

PubMed

Dean, Benjamin John Floyd; Snelling, Sarah J B; Dakin, Stephanie G; Murphy, Richard J; Javaid, Muhammad Kassim; Carr, Andrew Jonathan

2015-07-10

The relationship between peripheral tissue characteristics and pain symptoms in soft tissue inflammation is poorly understood. The primary aim of this study was to determine immunohistochemical differences in tissue obtained from patients with persistent pain and patients who had become pain-free after surgical treatment for rotator cuff tendinopathy. The secondary aim was to investigate whether there would be differences in glutaminergic and inflammatory gene expression between disease-derived and healthy control cells in vitro. Supraspinatus tendon biopsies were obtained from nine patients with tendon pain before shoulder surgery and from nine further patients whose pain had resolved completely following shoulder surgery. Histological markers relating to the basic tendon characteristics, inflammation and glutaminergic signalling were quantified by immunohistochemical analysis. Gene expression of glutaminergic and inflammatory markers was determined in tenocyte explants derived from painful rotator cuff tendon tears in a separate cohort of patients and compared to that of explants from healthy control tendons. Dual labelling was performed to identify cell types expressing nociceptive neuromodulators. Tendon samples from patients with persistent pain demonstrated increased levels of metabotropic glutamate receptor 2 (mGluR2), kainate receptor 1 (KA1), protein gene product 9.5 (PGP9.5), CD206 (macrophage marker) and CD45 (pan-leucocyte marker) versus pain-free controls (p <0.05). NMDAR1 co-localised with CD206-positive cells, whereas PGP9.5 and glutamate were predominantly expressed by resident tendon cells. These results were validated by in vitro increases in the expression of mGluR2, N-methyl-D-aspartate receptor (NMDAR1), KA1, CD45, CD206 and tumour necrosis factor alpha (TNF-α) genes (p <0.05) in disease-derived versus control cells. We conclude that differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of shoulder pain in rotator cuff tendinopathy, and that disease-derived cells exhibit a distinctly different neuro-inflammatory gene expression profile to healthy control cells.

TNF Receptor 1/2 Predict Heart Failure Risk in Type 2 Diabetes Mellitus Patients.

PubMed

Ping, Zhang; Aiqun, Ma; Jiwu, Li; Liang, Shao

2017-04-06

Inflammation plays an important role in heart failure and diabetes mellitus. Traditional serum markers have limited predictive value in heart failure and diabetes. TNFR1 and TNFR2 (TNFR1/2) have been proven to be strongly associated with heart failure and diabetes complications. This study aimed to assess the association of sTNFR1 and sTNFR2 levels and incidental HF risk in diabetes patients.We detected the mRNA, protein, and serum expression of TNFR1/2, their downstream signaling pathway protein NF-kB, and JNK expression and some traditional serum inflammatory markers in a heart failure group without diabetes mellitus or abnormal glucose tolerance (n = 84), a diabetes mellitus group without heart failure (n = 86), and a heart failure with diabetes mellitus group (n = 86).TNFR1/2 were significantly higher in patients with heart failure and diabetes mellitus based on mRNA expression to protein expression and serum expression. However, there were no differences in mRNA, protein, and serum levels of TNFR1/2 between the HF group and DM group. Furthermore, there were no differences between the groups in some traditional serum inflammatory markers.This study demonstrated higher expressions of TNFR, NF-kB, and JNK in patients with heart failure and diabetes mellitus. Compared with traditional serum markers, TNFR1 and TNFR2 are associated with heart failure risk in type 2 diabetes mellitus patients.

Low meprin α expression differentiates primary ovarian mucinous carcinoma from gastrointestinal cancers that commonly metastasise to the ovaries

PubMed Central

Heinzelmann‐Schwarz, Viola A; Scolyer, Richard A; Scurry, James P; Smith, Alison N; Gardiner‐Garden, Margaret; Biankin, Andrew V; Baron‐Hay, Sally; Scott, Carolyn; Ward, Robyn L; Fink, Daniel; Hacker, Neville F; Sutherland, Robert L; O'Brien, Philippa M

2007-01-01

Background Currently, no specific immunohistochemical markers are available to differentiate primary mucinous epithelial ovarian cancer (MOC) from adenocarcinomas originating at other sites that have metastasised to the ovary, which may have an impact on patient management and prognosis. Aim To investigate the expression of two intestinal markers, galectin 4 and meprin α, in mucinous carcinomas of the ovary and gastrointestinal tract. Methods Using immunohistochemical analysis, the expression of galectin 4 and meprin α was investigated in 10 MOCs and in 38 mucinous adenocarcinomas of colon, pancreas, stomach and appendix, the most common sites of origin of ovarian metastases. Results Total cytoplasmic galectin 4 expression was relatively consistent between the different carcinomas. Membranous meprin α expression was significantly lower in MOCs compared with gastrointestinal carcinomas. Moreover, meprin α expression showed greater discrimination between the ovarian and gastrointestinal carcinomas than the cytokeratins CK7 and CK20, the current standard immunohistochemical markers used to determine the tissue origin of mucinous carcinomas involving the ovaries. Conclusions Meprin α is a useful additional marker in differentiating primary from secondary mucinous adenocarcinomas of the ovary. PMID:16822880

Different strains of Propionibacterium acnes modulate differently the cutaneous innate immunity.

PubMed

Jasson, Fiona; Nagy, Istvan; Knol, Anne Chantal; Zuliani, Thomas; Khammari, Amir; Dréno, Brigitte

2013-09-01

Acne is a chronic inflammatory illness of the pilosebaceous follicle where innate immunity plays a central role. In acne, the density of Propionibacterium acnes is increased in the pilosebaceous unit. We hypothesized that the severity of acne is not only dependent on the proliferation of P. acnes but also dependent on the pro-inflammatory potential of P. acnes strains and consequently constitutes potential triggering factor for acne scarring. We investigated pro-inflammatory potential of five different strains of P. acnes and P. avidum in skin explants and the preventive effect of zinc gluconate. The expression of immune markers was studied by immunohistochemistry, RT-qPCR and ELISA. P. acnes strains modulate differently the expression of immune markers both at gene and at protein levels. P. acnes type III had the highest pro-inflammatory potential by up-regulating the expression of PAR-2, TNF-alpha, MMP-13 and TIMP-2, whereas P. avidum had the weakest by up-regulating only MMP-13 and TIMP-2. Preincubation of zinc gluconate, which is a modulator of innate immunity, down-regulates the expression of most immune markers induced by P. acnes, PAR-2, TIMP-2, up-regulates MMP-1, TIMP-1. Our results demonstrate that different P. acnes strains have different inflammatory potential targeting markers of cutaneous innate immunity, and that inflammatory potential can be down-regulated by zinc gluconate. As such, the inflammatory potential of P. acnes strains on acne skin may influence the severity of inflammatory acne lesions and scars. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Failure of hepatocyte marker-expressing hematopoietic progenitor cells to efficiently convert into hepatocytes in vitro.

PubMed

Lian, Gewei; Wang, Chengyan; Teng, Chunbo; Zhang, Cong; Du, Liying; Zhong, Qian; Miao, Chenglin; Ding, Mingxiao; Deng, Hongkui

2006-03-01

Whether bone marrow (BM) hematopoietic stem/progenitor cells can directly differentiate into nonhematopoietic cells remains controversial. The aim of this study is to further investigate the potentiality of BM hematopoietic progenitor cells to convert into hepatocytes in vitro. Different subsets of BM cells from C57/BL6 mice were isolated using markers of hematopoietic stem cells by magnetic cell sorting and by flow cytometry. These cells were induced to transdifferentiate to hepatocytes in vitro in the presence of various cytokines or of hepatocytes (or tissue) from damaged liver, which have been reported to stimulate the conversion. Hepatic gene markers in freshly isolated or cultured BM cells were determined by reverse transcriptase polymerase chain reaction and immunofluorescence. Freshly isolated hematopoietic progenitor cells (HPC) expressed a low level of messenger RNAs of hepatic cell-specific markers including albumin and alpha-fetoprotein (AFP), but did not significantly upregulate expression of these markers, even in the presence of cytokines or cocultured hepatocytes (or tissue). HPCs induced in vitro did not express the message of alpha-anti-trypsin-a mature hepatocyte marker. At protein level, the specific staining of AFP was not detected in the HPCs, either freshly isolated or in vitro induced. Albumin protein was detected in freshly isolated albumin mRNA-positive and -negative BM cell subpopulations. Albumin-stained BM cells disappeared after being induced for 5 days, but restained if mouse serum was supplemented in medium for a 24-hour extended culture, suggesting that albumin was absorbed by BM cells instead of de novo expression. HPCs expressed mRNAs of hepatic cell markers, but could not efficiently convert into hepatocytes in vitro under our experimental conditions. Our observation raises a cautionary note in determining whether in vitro transdifferentiation of BM cells to hepatocytes can actually take place.

Discourse Markers in College English Listening Instruction: An Empirical Study of Chinese Learners

ERIC Educational Resources Information Center

Zhang, Jianfeng

2012-01-01

Discourse markers can clearly indicate the organization of discourse, remind the hearer of the preceding and the following contents, and express clearly the concrete speech acts. The aims of this study are to explore the relationship between the discourse-marker-based model and listening comprehension as well as the different effects of such a…

Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination

PubMed Central

2013-01-01

Background Antibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype. Results Allium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding completely marker free plants. Conclusions The present study establishes the efficient expression of the newly introduced insect resistant ASAL gene even after Cre/lox mediated recombination resulting in elimination of selectable marker gene. PMID:24144281

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis

PubMed Central

2012-01-01

Background Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult. PMID:23268714

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice

PubMed Central

Volynets, Valentina; Rings, Andreas; Bárdos, Gyöngyi; Ostaff, Maureen J.; Wehkamp, Jan; Bischoff, Stephan C.

2016-01-01

ABSTRACT The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest. PMID:27583194

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice.

PubMed

Volynets, Valentina; Rings, Andreas; Bárdos, Gyöngyi; Ostaff, Maureen J; Wehkamp, Jan; Bischoff, Stephan C

2016-01-01

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Cytoskeletal proteins and stem cell markers gene expression in human bone marrow mesenchymal stromal cells after different periods of simulated microgravity

NASA Astrophysics Data System (ADS)

Gershovich, P. M.; Gershovich, J. G.; Zhambalova, A. P.; Romanov, Yu. A.; Buravkova, L. B.

2012-01-01

Mesenchymal stem (stromal) cells (MSCs) are present in a variety of tissues during prenatal and postnatal human development. In adult organism, they are prevalent in bone marrow and supposed to be involved in space-flight induced osteopenia. We studied expression of various genes in human bone marrow MSCs after different terms of simulated microgravity (SMG) provided by Random Positioning Machine. Simulated microgravity induced transient changes in expression level of genes associated with actin cytoskeleton, especially after 48 h of SMG. However, after 120 h exposure in SMG partial restoration of gene expression levels (relative to the control) was found. Similar results were obtained with bmMSCs subjected to 24 h readaptation in static state after 24 h in SMG. Analysis of 84 genes related to identification, growth and differentiation of stem cells revealed that expression of nine genes was changed slightly after 48 h in SMG. More pronounced changes in gene expression of "stem cells markers" were observed after 120 h of simulated microgravity. Among 84 investigated genes, 30 were up-regulated and 24 were down-regulated. Finally, MSCs osteogenesis induced by long-term (10-20 days) simulation of microgravity was accompanied by down-regulation of gene expression of the main osteogenic differentiation markers ( ALPL, OMD) and master transcription osteogenic factor of MSCs ( Runx2). Thus, our study demonstrated that changes in expression level of some genes associated with actin cytoskeleton and stem cell markers are supposed to be one of the mechanisms, which contribute to precursor's cellular adaptation to the microgravity conditions. These results can clarify genomic mechanisms through which SMG reduces osteogenic differentiation of bmMSCs.

NANOG regulates epithelial-mesenchymal transition and chemoresistance in ovarian cancer.

PubMed

Qin, Shan; Li, Yanfang; Cao, Xuexia; Du, Jiexian; Huang, Xianghua

2017-02-28

A key transcription factor associated with poor prognosis and resistance to chemotherapy in ovarian cancer is NANOG. However, the mechanism by which NANOG functions remains undefined. It has been suggested that epithelial-to-mesenchymal transition (EMT) also contributes to development of drug resistance in different cancers. We thus determined whether NANOG expression was associated with EMT and chemoresistance in epithelial ovarian cancer cells. NANOG expression was increased in epithelial ovarian cancer cell lines compared with its expression in normal epithelial ovarian cell lines. NANOG expression in SKOV-3 or OV2008 cells directly correlated with high expression of mesenchymal cell markers and inversely with low expression of epithelial cell marker. RNAi-mediated silencing of NANOG in SKOV-3 reversed the expression of mesenchymal cell markers and restored expression of E-cadherin. Reversibly, stable overexpression of NANOG in Moody cells increased expression of N-cadherin whereas down-regulating expression of E-cadherin, cumulatively indicating that NANOG plays an important role in maintaining the mesenchymal cell markers. Modulating NANOG expression did not have any effect on proliferation or colony formation. Susceptibility to cisplatin increased in SKOV-3 cells on down-regulating NANOG and reversible results were obtained in Moody cells post-overexpression of NANOG. NANOG silencing in SKOV-3 and OV2008 robustly attenuated in vitro migration and invasion. NANOG expression exhibited a biphasic pattern in patients with ovarian cancer and expression was directly correlated to chemoresistance retrospectively. Cumulatively, our data demonstrate that NANOG expression modulates chemosensitivity and EMT resistance in ovarian cancer. © 2017 The Author(s).

Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

PubMed Central

Bernstein, Steven L; Guo, Yan; Peterson, Katherine; Wistow, Graeme

2009-01-01

Background The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. Results Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. Conclusion We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models. PMID:19778450

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.

Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis.

PubMed

Gomes Fernandes, Maria; Bialecka, Monika; Salvatori, Daniela C F; Chuva de Sousa Lopes, Susana M

2018-05-01

Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. Non-applicable. Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro. M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.

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.

Human-specific features of spatial gene expression and regulation in eight brain regions.

PubMed

Xu, Chuan; Li, Qian; Efimova, Olga; He, Liu; Tatsumoto, Shoji; Stepanova, Vita; Oishi, Takao; Udono, Toshifumi; Yamaguchi, Katsushi; Shigenobu, Shuji; Kakita, Akiyoshi; Nawa, Hiroyuki; Khaitovich, Philipp; Go, Yasuhiro

2018-06-13

Molecular maps of the human brain alone do not inform us of the features unique to humans. Yet, the identification of these features is important for understanding both the evolution and nature of human cognition. Here, we approached this question by analyzing gene expression and H3K27ac chromatin modification data collected in eight brain regions of humans, chimpanzees, gorillas, a gibbon and macaques. An analysis of spatial transcriptome trajectories across eight brain regions in four primate species revealed 1,851 genes showing human-specific transcriptome differences in one or multiple brain regions, in contrast to 240 chimpanzee-specific ones. More than half of these human-specific differences represented elevated expression of genes enriched in neuronal and astrocytic markers in the human hippocampus, while the rest were enriched in microglial markers and displayed human-specific expression in several frontal cortical regions and the cerebellum. An analysis of the predicted regulatory interactions driving these differences revealed the role of transcription factors in species-specific transcriptome changes, while epigenetic modifications were linked to spatial expression differences conserved across species. Published by Cold Spring Harbor Laboratory Press.

Relationships between immunophenotype, Ki-67 index, microvascular density, Ep-CAM/P-cadherin, and MMP-2 expression in early-stage invasive ductal breast cancer.

PubMed

Niemiec, Joanna A; Adamczyk, Agnieszka; Małecki, Krzysztof; Majchrzyk, Kaja; Ryś, Janusz

2012-12-01

There is still a lack of complete consensus on immunohistochemical surrogate markers for luminal A (LA) and luminal B (LB), HER2, and basal-like subtypes of breast carcinomas and their correlation with cancer cell adhesion and invasion-promoting factors. Therefore, early-stage invasive ductal breast cancer patients (N=209) were recruited to the study and divided into 4 subtypes, on the basis of the expression of the estrogen/progesterone receptor and HER2 (LA: 74.4% of cases; LB: 7.8%; HER2: 5.6%; and triple-negative phenotype: 12.2%). Regardless of the above-mentioned classification, we divided all carcinomas into 2 groups: carcinomas expressing at least 1 basal marker [cytokeratine (CK)5/6, CK5, vimentin, epidermal growth factor receptor, or aberrant CK8/18 expression-membranous or in <10% of cells] versus carcinomas negative for basal markers. Then we studied the relationships between the above subtypes (2 classifications) and (i) the expression of adhesion molecules (Ep-CAM, P-cadherin), (ii) matrix metalloproteinases (MMP)-2, (iii) the proliferation index (MIB-1 LI), and (iv) the microvascular density. We confirmed that triple-negative phenotypes are characterized by basal marker expression, a high tumor grade, and high MIB-1 LI. In this subtype, we found MMP-2 expression in stromal leukocytes less frequently. Both LA carcinomas and carcinomas negative for basal markers were more often negative for epithelial cell adhesion molecule (Ep-CAM) and P-cadherin. Moreover, we noted a higher mean value of microvascular density in CK5/6 and Ep-CAM-immunopositive tumors, carcinomas with aberrant CK8/18 expression, and carcinomas with no or strong expression of MMP-2 in stromal fibroblast-like cells. These results might suggest that mechanisms of stroma remodeling and carcinogenesis (Ep-CAM is the suggested marker of breast progenitors) may differ between breast cancer subtypes.

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.

On the evolution of harming and recognition in finite panmictic and infinite structured populations

PubMed Central

Lehmann, Laurent; Feldman, Marcus W.; Rousset, François

2010-01-01

Natural selection may favor two very different types of social behaviors that have costs in vital rates (fecundity and/or survival) to the actor: helping behaviors, which increase the vital rates of recipients, and harming behaviors, which reduce the vital rates of recipients. While social evolutionary theory has mainly dealt with helping behaviors, competition for limited resources creates ecological conditions where an actor may benefit from expressing behaviors that reduce the vital rates of neighbours. This may occur if the reduction in vital rates decreases the intensity of competition experienced by the actor or that experienced by its offspring. Here, we explore the joint evolution of neutral recognition markers and marker-based costly conditional harming whereby actors express harming, conditional on actor and recipient bearing different conspicuous markers. We do so for two complementary demographic scenarios: finite panmictic and infinite structured populations. We find that marker-based conditional harming can evolve under a large range of recombination rates and group sizes under both finite panmictic and infinite structured populations. Direct comparison with results for the evolution of marker-based conditional helping reveals that, if everything else is equal, marker-based conditional harming is often more likely to evolve than marker-based conditional. PMID:19624725

α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

PubMed

Cattavarayane, Sandhanakrishnan; Palovuori, Riitta; Tanjore Ramanathan, Jayendrakishore; Manninen, Aki

2015-02-27

The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.

Signatures of tumour immunity distinguish Asian and non-Asian gastric adenocarcinomas

PubMed Central

Lin, Suling J; Gagnon-Bartsch, Johann A; Tan, Iain Beehuat; Earle, Sophie; Ruff, Louise; Pettinger, Katherine; Ylstra, Bauke; van Grieken, Nicole; Rha, Sun Young; Chung, Hyun Cheol; Lee, Ju-Seog; Cheong, Jae Ho; Noh, Sung Hoon; Aoyama, Toru; Miyagi, Yohei; Tsuburaya, Akira; Yoshikawa, Takaki; Ajani, Jaffer A; Boussioutas, Alex; Yeoh, Khay Guan; Yong, Wei Peng; So, Jimmy; Lee, Jeeyun; Kang, Won Ki; Kim, Sung; Kameda, Yoichi; Arai, Tomio; zur Hausen, Axel; Speed, Terence P; Grabsch, Heike I; Tan, Patrick

2015-01-01

Objective Differences in gastric cancer (GC) clinical outcomes between patients in Asian and non-Asian countries has been historically attributed to variability in clinical management. However, recent international Phase III trials suggest that even with standardised treatments, GC outcomes differ by geography. Here, we investigated gene expression differences between Asian and non-Asian GCs, and if these molecular differences might influence clinical outcome. Design We compared gene expression profiles of 1016 GCs from six Asian and three non-Asian GC cohorts, using a two-stage meta-analysis design and a novel biostatistical method (RUV-4) to adjust for technical variation between cohorts. We further validated our findings by computerised immunohistochemical analysis on two independent tissue microarray (TMA) cohorts from Asian and non-Asian localities (n=665). Results Gene signatures differentially expressed between Asians and non-Asian GCs were related to immune function and inflammation. Non-Asian GCs were significantly enriched in signatures related to T-cell biology, including CTLA-4 signalling. Similarly, in the TMA cohorts, non-Asian GCs showed significantly higher expression of T-cell markers (CD3, CD45R0, CD8) and lower expression of the immunosuppressive T-regulatory cell marker FOXP3 compared to Asian GCs (p<0.05). Inflammatory cell markers CD66b and CD68 also exhibited significant cohort differences (p<0.05). Exploratory analyses revealed a significant relationship between tumour immunity factors, geographic locality-specific prognosis, and postchemotherapy outcomes. Conclusions Analyses of >1600 GCs suggest that Asian and non-Asian GCs exhibit distinct tumour immunity signatures related to T-cell function. These differences may influence geographical differences in clinical outcome, and the design of future trials particularly in immuno-oncology. PMID:25385008

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.

The influence of TSA and VPA on the in vitro differentiation of bone marrow mesenchymal stem cells into neuronal lineage cells: Gene expression studies.

PubMed

Fila-Danilow, Anna; Borkowska, Paulina; Paul-Samojedny, Monika; Kowalczyk, Malgorzata; Kowalski, Jan

2017-03-27

Epigenetic mechanisms regulate the transcription of genes, which can affect the differentiation of MSCs. The aim of the current work is to determine how the histone deacetylase inhibitors TSA and VPA affect the expression of neuronal lineage genes in a culture of rat MSCs (rMSCs). We analyzed the expression of early neuron marker gene (Tubb3), mature neuron markers genes (Vacht, Th, Htr2a) and the oligodendrocyte progenitor marker gene (GalC). Moreover, changes in the gene expression after three different periods of exposure to TSA and VPA were investigated for the first time. After six days of exposition to TSA and VPA, the expression of Tubb3 and GalC decreased, while the expression of Th increased. The highest increase of VAChT expression was observed after three days of TSA and VPA treatment. A decrease in Htr2a gene expression was observed after TSA treatment and an increase was observed after VPA treatment. We also observed that TSA and VPA inhibited cell proliferation and the formation of neurospheres in the rMSCs culture. The central findings of our study are that TSA and VPA affect the expression of neuronal lineage genes in an rMSCs culture. After exposure to TSA or VPA, the expression of early neuronal gene decreases but equally the expression of mature neuron genes increases. After TSA and VPA treatment ER of the oligodendrocyte progenitor marker decreased. TSA and VPA inhibit cell proliferation and the formation of neurospheres in rMSCs culture.

Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

PubMed Central

Fu, Jia; Wei, Chengguo; Lee, Kyung; Zhang, Weijia; He, Wu; Chuang, Peter

2016-01-01

Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate genes involved in the pathogenesis of diabetic nephropathy. To determine if the podocyte-specific gene information contained in mRNA profiles of the whole glomerulus of the diabetic kidney accurately reflects gene expression in the isolated podocytes, we crossed Nos3−/− IRG mice with podocin-rtTA and TetON-Cre mice for enhanced green fluorescent protein labeling of podocytes before diabetic injury. Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice were examined 10 weeks later. Expression of podocyte-specific markers in glomeruli was downregulated in diabetic mice compared with controls. However, expression of these markers was not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli were corrected for podocyte number per glomerulus, the differences in podocyte marker expression disappeared. Analysis of the differentially expressed genes in diabetic mice also revealed distinct upregulated pathways in the glomeruli (mitochondrial function, oxidative stress) and in podocytes (actin organization). In conclusion, our data suggest reduced expression of podocyte markers in glomeruli is a secondary effect of reduced podocyte number, thus podocyte-specific gene expression detected in the whole glomerulus may not represent that in podocytes in the diabetic kidney. PMID:26264855

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.

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

Evaluation of the collaborative network of highly correlating skin proteins and its change following treatment with glucocorticoids

PubMed Central

2010-01-01

Background Glucocorticoids (GC) represent the core treatment modality for many inflammatory diseases. Its mode of action is difficult to grasp, not least because it includes direct modulation of many components of the extracellular matrix as well as complex anti-inflammatory effects. Protein expression profile of skin proteins is being changed with topical application of GC, however, the knowledge about singular markers in this regard is only patchy and collaboration is ill defined. Material/Methods Scar formation was observed under different doses of GC, which were locally applied on the back skin of mice (1 to 3 weeks). After euthanasia we analyzed protein expression of collagen I and III (picrosirius) in scar tissue together with 16 additional protein markers, which are involved in wound healing, with immunhistochemistry. For assessing GC's effect on co-expression we compared our results with a model of random figures to estimate how many significant correlations should be expected by chance. Results GC altered collagen and protein expression with distinct results in different areas of investigation. Most often we observed a reduced expression after application of low dose GC. In the scar infiltrate a multivariate analysis confirmed the significant impact of both GC concentrations. Calculation of Spearman's correlation coefficient similarly resulted in a significant impact of GC, and furthermore, offered the possibility to grasp the entire interactive profile in between all variables studied. The biological markers, which were connected by significant correlations could be arranged in a highly cross-linked network that involved most of the markers measured. A marker highly cross-linked with more than 3 significant correlations was indicated by a higher variation of all its correlations to the other variables, resulting in a standard deviation of > 0.2. Conclusion In addition to immunohistochemical analysis of single protein markers multivariate analysis of co-expressions by use of correlation coefficients reveals the complexity of biological relationships and identifies complex biological effects of GC on skin scarring. Depiction of collaborative clusters will help to understand functional pathways. The functional importance of highly cross-linked proteins will have to be proven in subsequent studies. PMID:20509951

Enrichment and isolation of neurons from adult mouse brain for ex vivo analysis.

PubMed

Berl, Sabina; Karram, Khalad; Scheller, Anja; Jungblut, Melanie; Kirchhoff, Frank; Waisman, Ari

2017-05-01

Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS. Copyright © 2017 Elsevier B.V. All rights reserved.

Abnormalities in early markers of muscle involvement support a delay in myogenesis in spinal muscular atrophy.

PubMed

Martínez-Hernández, Rebeca; Bernal, Sara; Alias, Laura; Tizzano, Eduardo F

2014-06-01

Spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the spinal cord that results in muscle denervation and profound weakness in affected patients. We sought evidence for primary muscle involvement in the disease during human development by analyzing the expression of several muscle cytoskeletal components (i.e. slow, fast, and developmental myosin, desmin, and vimentin) in fetal or postnatal skeletal muscle samples from 5 SMA cases and 6 controls. At 14 weeks' gestation, SMA samples had higher percentages of myotubes expressing fast myosin and lower percentages of myotubes expressing slow myosin versus control samples. Desmin and vimentin were highly expressed at prenatal stages without notable differences between control and SMA samples, although both proteins showed persistent immunostaining in atrophic fibers in postnatal SMA samples. We also studied the expression of Pax7-positive nuclei as a marker of satellite cells and found no differences between control and SMA prenatal samples. There was, however, a significant increase in satellite cells in postnatal atrophic SMA fibers, suggesting an abnormal myogenic process. Together, these results support the hypothesis of a delay in muscle maturation as one of the primary pathologic components of SMA. Furthermore, myosins and Pax7 may be useful research markers of muscle involvement in this disease.

Significantly greater expression of ER, PR, and ECAD in advanced-stage low-grade ovarian serous carcinoma as revealed by immunohistochemical analysis.

PubMed

Wong, Kwong-Kwok; Lu, Karen H; Malpica, Anais; Bodurka, Diane C; Shvartsman, Hyun S; Schmandt, Rosemarie E; Thornton, Angela D; Deavers, Michael T; Silva, Elvio G; Gershenson, David M

2007-10-01

A 2-tier system that classifies ovarian serous carcinoma (OSC) as low grade or high grade is gaining acceptance. Women with low-grade OSC generally have higher 5-year survival rates than do women with high-grade OSC. We examined the expression of various markers to further understand the molecular differences between low-grade and high-grade OSCs: the potential therapeutic targets or prognostic markers Her-2/neu, estrogen receptor, and progesterone receptor (PR); the metastasis-associated markers cyclin D1 (BCL1), E-cadherin, matrix metalloproteinase (MMP) 2, and MMP-9; and the cell proliferation-associated markers BCL1, Ki-67 antigen (Ki-67), and p53. For this immunohistochemical analysis, we used paraffin-embedded specimens from 47 patients with advanced-stage low-grade OSC and from 49 patients with advanced-stage high-grade OSC. Our results showed that low-grade tumors expressed significantly higher levels of estrogen receptor, PR, and E-cadherin than did high-grade tumors, suggesting the involvement of gonadal steroid hormones, especially in the pathogenesis of low-grade OSC; the PR positivity was also observed in the stromal component of these low-grade tumors. On the other hand, high-grade tumors trended toward increased expression of MMP-9, BCL1, p53, and Ki-67, and robust MMP-9 positivity was observed in the stromal component of these high-grade tumors. These differences may lead to the development of different therapeutic strategies for women with either the low-grade or the high-grade form of OSC.

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

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

The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

PubMed Central

Bay-Jensen, Anne-Christine; Tabassi, Nadine CB; Sondergaard, Lene V; Andersen, Thomas L; Dagnaes-Hansen, Frederik; Garnero, Patrick; Kassem, Moustapha; Delaissé, Jean-Marie

2009-01-01

Introduction The urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue. Methods The type II collagen degradation markers CTX-II and Helix-II were measured in the body fluids of premenopausal and postmenopausal women and in those of ovariectomised rats receiving oestrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analysed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression. Results As expected, urinary levels of CTX-II are significantly increased in postmenopausal women and also in oestrogen-deprived rats, although only transiently. However, in neither case were these elevations paralleled by a significant increase of Helix-II levels and PIIANP levels did not change at any time. CTX-II immunoreactivity and collagen expression were detected in different cartilage areas. The upper zone is the area where CTX-II immunoreactivity and collagen expression best reflected the differences in urinary levels of CTX-II measured in response to oestrogen. However, correlations between urinary levels of CTX-II and tissue immunostainings in individual rats were not statistically significant. Conclusions We found only a small effect of oestrogen deprivation on cartilage. It was detected by CTX-II, but not by other type II collagen turnover markers typically affected in osteoarthritis. PMID:20527083

Earth Mover's Distance (EMD): A True Metric for Comparing Biomarker Expression Levels in Cell Populations.

PubMed

Orlova, Darya Y; Zimmerman, Noah; Meehan, Stephen; Meehan, Connor; Waters, Jeffrey; Ghosn, Eliver E B; Filatenkov, Alexander; Kolyagin, Gleb A; Gernez, Yael; Tsuda, Shanel; Moore, Wayne; Moss, Richard B; Herzenberg, Leonore A; Walther, Guenther

2016-01-01

Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational "gating" tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover's Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains.

Aberrant Expression of Calretinin, D2-40 and Mesothelin in Mucinous and Non-Mucinous Colorectal Carcinomas and Relation to Clinicopathological Features and Prognosis.

PubMed

Foda, Abd AlRahman Mohammad; El-Hawary, Amira Kamal; Hamed, Hazem

2016-10-01

CRC is a heterogeneous disease in terms of morphology, invasive behavior, metastatic capacity, and clinical outcome. Recently, many so-called mesothelial markers, including calretinin, D2-40, WT1, thrombomodulin, mesothelin, and others, have been certified. The aim of this study was to assess the immunohistochemical expression of calretinin and other mesothelial markers (D2-40 and mesothelin) in colorectal mucinous adenocarcinoma (MA) and non mucinous adenocarcinoma (NMA) specimens and relation to clinicopathological features and prognosis using manual tissue microarray technique. We studied tumor tissue specimens from 150 patients with colorectal MA and NMA who underwent radical surgery from January 2007 to January 2012. High-density manual tissue microarrays were constructed using a modified mechanical pencil tip technique, and paraffin sections were submitted for immunohistochemistry using Calretinin, D2-40 and mesothelin expressions. We found that NMA showed significantly more calretinin and D2-40 expression than MA In contrast, no statistically significant difference between NMA and MA was detected in mesothelin expression. There were no statistically significant relations between any of the clinicopathological or histological parameters and any of the three markers. In a univariate analysis, neither calretinin nor D2-40 expressions showed any significant relations to DFS or OS. However, mesothelin luminal expression was significantly associated with worse DFS. Multivariate Cox regression analysis proved that luminal mesothelin expression was an independent negative prognostic factor in NMA. In conclusion, Calretinin, D2-40 and mesothelin are aberrantly expressed in a proportion of CRC cases with more expression in NMA than MA. Aberrant expression of these mesothelial markers was not associated with clinicopathological or histological features of CRCs. Only mesothelin expression appears to be a strong predictor of adverse prognosis.

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.

Stem Cell Markers (Cytokeratin 17 and Cytokeratin 19) in Scarring and Nonscarring Alopecia

PubMed Central

El Sakka, Dalia; Gaber, Mohamed Abdel Wahed; Abdou, Asmaa Gaber; Wahed, Moshira Abdel; Saleh, Ahmed Abdel-Wahab; Shehata, Walla

2016-01-01

Background: Alopecia is one of the most important hair follicle (HF) disorders, which is divided into scarring (cicatricial) and nonscarring (noncicatricial) types. Objective: The aim of this study is to investigate the expression of stem cell (SC) markers such as cytokeratin (CK) 17 and CK19 in scarring and nonscarring alopecia. Materials and Methods: Thirty patients with scalp alopecia (15 with scarring alopecia and 15 without) together with ten healthy volunteers were included in this study. Biopsies were taken from all participants and stained for CK17 and CK19 using immunohistochemistry. Results: There was a statistically significant difference between the nonscarring group and the control group with regard to CK17 expression in the outer layers of the HFs (P = 0.00) and CK19 staining of the inner layers of the HFs (P = 0.008). There was a statistically significant difference between the scarring and the control groups regarding CK17 expression in the outer (P = 0.00) and the inner layers (P = 0.00) of the HFs and CK19 expression in the inner layers of the HFs (P = 0.00). CK17 expression in the outer layers (P = 0.02) and the inner layers of the HFs (P = 0.00) together with CK19 expression in the inner layers of the HFs (P = 0.00) showed statistically significant differences between scarring and nonscarring alopecia groups. Conclusions: The presence of SC markers (CK17 and CK19) in the HFs was affected in both scarring and nonscarring alopecia, but the defect in scarring alopecia is more evident than that of nonscarring alopecia. The persistence of SC markers in some types of scarring alopecia could give a hope for the recovery of these lesions. Further studies are recommended to clarify the benefit from using HF SCs in the treatment of alopecia. PMID:27761086

Altered Expression of Retinal Molecular Markers in the Canine RPE65 Model of Leber Congenital Amaurosis

PubMed Central

Hernández, Maria; Pearce-Kelling, Susan E.; Rodriguez, F. David; Aguirre, Gustavo D.; Vecino, Elena

2010-01-01

Purpose. Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed. Methods. Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies. Results. Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas. Conclusions. The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans. PMID:20671290

Bcl-2 and BLIMP-1 expression predict worse prognosis in gastric diffuse large B cell lymphoma (DLCBL) while other markers for nodal DLBCL are not useful.

PubMed

Martin-Arruti, Maialen; Vaquero, Manuel; Díaz de Otazu, Ramón; Zabalza, Iñaki; Ballesteros, Javier; Roncador, Giovanna; García-Orad, Africa

2012-04-01

Previous studies have identified clinicopathological and immunohistochemical differences among diffuse large B cell lymphomas (DLBCL) as a function of disease location. Nevertheless, there is a continuing tendency to generalize the prognostic value of various identified markers without taking into account tumour site. Accordingly, we analysed the prognostic value of several of the immunohistochemical markers that have been proposed for nodal DLBCL in a group of patients with gastric DLBCL. Using histochemical methods, CD10, Bcl-6, Gcet1, MUM-1, Bcl-2 and BLIMP-1 expression was investigated in 43 cases of gastric DBLCL. As in nodal DLBCLs, expression of BLIMP-1, and of Bcl-2 in non-germinal centre B cell-like (non-GCB) patients, was associated with a worse prognosis. However, unlike nodal DBLCL, there was no significant association of prognosis with expression of CD10, Bcl-6, Gcet1 or MUM-1, or with categorization according to Hans or Muris algorithms. Although most markers of prognosis in nodal DLBCL are not useful indicators for gastric DLBCL, Bcl-2 or BLIMP-1 expression does correlate with worse prognosis. These data support the notion that clinicopathological features in DLBCL vary according to the disease location. © 2012 Blackwell Publishing Ltd.

Expression Pattern of Smad4/GATA3 as a Predictor of Survival in Invasive Ductal Carcinoma of the Breast.

PubMed

Min, Kyueng-Whan; Kim, Dong-Hoon; Do, Sung-Im; Chae, Seoung Wan; Kim, Kyungeun; Sohn, Jin Hee; Lee, Hyun Joo; Do, In-Gu; Pyo, Jung-Soo; Kim, Yuil; Kim, Dong Hyun; Yang, Jung-Ho; Lee, Sang-Jo; Oh, Young Ha; Oh, Sukjoong; Choi, Seon Hyeong; Park, Yong Lai; Park, Chan Heun; Kim, Eun-Kyung; Kwon, Mi Jung; Seo, Jinwon

2017-01-01

Smad4 and GATA3 proteins are known prognostic markers in various cancers. Smad4 is a mediator linked to both tumour suppression and progression. GATA3 is a regulator of development and morphogenesis of the mammary gland. We assessed and compared the predictive performance of Smad4 and GATA3 for clinical outcomes in patients with breast cancer. The combined expression pattern based on Smad4+/- and GATA3+/- was evaluated by immunostaining using breast cancer tissue microarray, and the relationships between protein expression and clinicopathological variables were analysed. Smad4 expression was only associated with an ill-defined tumour border, whereas GATA3 was associated with several good prognostic factors. On analysis of combined markers, there was a significant difference in the expression of fascin (an important factor for cancer invasiveness) between the Smad4+/GATA3- and Smad4-/GATA3+ groups. Smad4+/GATA3- was correlated with worse clinicopathological parameters, relapse-free survival (RFS), and overall survival (OS), compared to Smad4-/GATA3+. Combined markers of Smad4/GATA3 showed a superior performance compared to single markers for predicting RFS and OS in patients with breast cancer. © 2017 S. Karger AG, Basel.

Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma.

PubMed

Saeednejad Zanjani, Leili; Madjd, Zahra; Abolhasani, Maryam; Shariftabrizi, Ahmad; Rasti, Arezoo; Asgari, Mojgan

2018-01-01

CD105 is recently described as a cancer stem cell (CSC) marker. The present study was aimed to investigate the expression and prognostic significance of the CSC marker CD105 in different histological subtypes of renal cell carcinoma (RCC). Expression of CD105 was evaluated using immunohistochemistry in RCC samples on tissue microarrays including clear cell RCCs (ccRCCs), papillary, and chromophobe RCCs. The association between CD105 expression and clinicopathological features as well as survival outcomes was determined. In ccRCC, increased tumoral cytoplasmic and endothelial expression of CD105 were significantly associated with advanced stage, renal vein invasion, and microvascular invasion (MVI). In addition, MVI was associated with a worse overall survival (OS). Moreover, in multivariate analysis tumor stage and nuclear grade were independent prognostic factors for OS both in case of tumoral cytoplasmic and endothelial CD105 expression. Additionally, CD105 expression was found to be a predictor of worse OS in univariate analysis. However, in papillary and chromophobe RCC, no significant association was found between CD105 expression and clinicopathological parameters or prognosis. We showed that CD105 expression was associated with more aggressive tumor behavior, more advanced disease, and worse prognosis in ccRCC but not in the other RCC subtypes.

Similarity of markers identified from cancer gene expression studies: observations from GEO.

PubMed

Shi, Xingjie; Shen, Shihao; Liu, Jin; Huang, Jian; Zhou, Yong; Ma, Shuangge

2014-09-01

Gene expression profiling has been extensively conducted in cancer research. The analysis of multiple independent cancer gene expression datasets may provide additional information and complement single-dataset analysis. In this study, we conduct multi-dataset analysis and are interested in evaluating the similarity of cancer-associated genes identified from different datasets. The first objective of this study is to briefly review some statistical methods that can be used for such evaluation. Both marginal analysis and joint analysis methods are reviewed. The second objective is to apply those methods to 26 Gene Expression Omnibus (GEO) datasets on five types of cancers. Our analysis suggests that for the same cancer, the marker identification results may vary significantly across datasets, and different datasets share few common genes. In addition, datasets on different cancers share few common genes. The shared genetic basis of datasets on the same or different cancers, which has been suggested in the literature, is not observed in the analysis of GEO data. © The Author 2013. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

LRRK2 modulates vulnerability to mitochondrial dysfunction in C. elegans

PubMed Central

Saha, Shamol; Guillily, Maria; Ferree, Andrew; Lanceta, Joel; Chan, Diane; Ghosh, Joy; Hsu, Cindy H.; Segal, Lilach; Raghavan, Kesav; Matsumoto, Kunihiro; Hisamoto, Naoki; Kuwahara, Tomoki; Iwatsubo, Takeshi; Moore, Landon; Goldstein, Lee; Cookson, Mark; Wolozin, Benjamin

2009-01-01

Summary Mutations in leucine rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease. We generated lines of C. elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 expression increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C or kinase dead LRRK2 was less than protection by wild type LRRK2. Knockdown of lrk-1, the endogenous orthologue of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT∷GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the WT line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing WT LRRK2; however loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different than that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition, and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson’s disease. PMID:19625511

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

PubMed

Amatya, Vishwa Jeet; Kushitani, Kei; Mawas, Amany Sayed; Miyata, Yoshihiro; Okada, Morihito; Kishimoto, Takumi; Inai, Kouki; Takeshima, Yukio

2017-05-01

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer

PubMed Central

WANG, HAIYING; MOLINA, JULIAN; JIANG, JOHN; FERBER, MATTHEW; PRUTHI, SANDHYA; JATKOE, TIMOTHY; DERECHO, CARLO; RAJPUROHIT, YASHODA; ZHENG, JIAN; WANG, YIXIN

2013-01-01

Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and response to treatment. PMID:24649289

Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer.

PubMed

Wang, Haiying; Molina, Julian; Jiang, John; Ferber, Matthew; Pruthi, Sandhya; Jatkoe, Timothy; Derecho, Carlo; Rajpurohit, Yashoda; Zheng, Jian; Wang, Yixin

2013-11-01

Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and response to treatment.

Multilayered epithelium in a rat model and human Barrett's esophagus: Similar expression patterns of transcription factors and differentiation markers

PubMed Central

Chen, Xiaoxin; Qin, Rong; Liu, Ba; Ma, Yan; Su, Yinghao; Yang, Chung S; Glickman, Jonathan N; Odze, Robert D; Shaheen, Nicholas J

2008-01-01

Background In rats, esophagogastroduodenal anastomosis (EGDA) without concomitant chemical carcinogen treatment leads to gastroesophageal reflux disease, multilayered epithelium (MLE, a presumed precursor in intestinal metaplasia), columnar-lined esophagus, dysplasia, and esophageal adenocarcinoma. Previously we have shown that columnar-lined esophagus in EGDA rats resembled human Barrett's esophagus (BE) in its morphology, mucin features and expression of differentiation markers (Lab. Invest. 2004;84:753–765). The purpose of this study was to compare the phenotype of rat MLE with human MLE, in order to gain insight into the nature of MLE and its potential role in the development of BE. Methods Serial sectioning was performed on tissue samples from 32 EGDA rats and 13 patients with established BE. Tissue sections were immunohistochemically stained for a variety of transcription factors and differentiation markers of esophageal squamous epithelium and intestinal columnar epithelium. Results We detected MLE in 56.3% (18/32) of EGDA rats, and in all human samples. As expected, both rat and human squamous epithelium, but not intestinal metaplasia, expressed squamous transcription factors and differentiation markers (p63, Sox2, CK14 and CK4) in all cases. Both rat and human intestinal metaplasia, but not squamous epithelium, expressed intestinal transcription factors and differentiation markers (Cdx2, GATA4, HNF1α, villin and Muc2) in all cases. Rat MLE shared expression patterns of Sox2, CK4, Cdx2, GATA4, villin and Muc2 with human MLE. However, p63 and CK14 were expressed in a higher proportion of rat MLE compared to humans. Conclusion These data indicate that rat MLE shares similar properties to human MLE in its expression pattern of these markers, not withstanding small differences, and support the concept that MLE may be a transitional stage in the metaplastic conversion of squamous to columnar epithelium in BE. PMID:18190713

Multilayered epithelium in a rat model and human Barrett's esophagus: similar expression patterns of transcription factors and differentiation markers.

PubMed

Chen, Xiaoxin; Qin, Rong; Liu, Ba; Ma, Yan; Su, Yinghao; Yang, Chung S; Glickman, Jonathan N; Odze, Robert D; Shaheen, Nicholas J

2008-01-11

In rats, esophagogastroduodenal anastomosis (EGDA) without concomitant chemical carcinogen treatment leads to gastroesophageal reflux disease, multilayered epithelium (MLE, a presumed precursor in intestinal metaplasia), columnar-lined esophagus, dysplasia, and esophageal adenocarcinoma. Previously we have shown that columnar-lined esophagus in EGDA rats resembled human Barrett's esophagus (BE) in its morphology, mucin features and expression of differentiation markers (Lab. Invest. 2004;84:753-765). The purpose of this study was to compare the phenotype of rat MLE with human MLE, in order to gain insight into the nature of MLE and its potential role in the development of BE. Serial sectioning was performed on tissue samples from 32 EGDA rats and 13 patients with established BE. Tissue sections were immunohistochemically stained for a variety of transcription factors and differentiation markers of esophageal squamous epithelium and intestinal columnar epithelium. We detected MLE in 56.3% (18/32) of EGDA rats, and in all human samples. As expected, both rat and human squamous epithelium, but not intestinal metaplasia, expressed squamous transcription factors and differentiation markers (p63, Sox2, CK14 and CK4) in all cases. Both rat and human intestinal metaplasia, but not squamous epithelium, expressed intestinal transcription factors and differentiation markers (Cdx2, GATA4, HNF1alpha, villin and Muc2) in all cases. Rat MLE shared expression patterns of Sox2, CK4, Cdx2, GATA4, villin and Muc2 with human MLE. However, p63 and CK14 were expressed in a higher proportion of rat MLE compared to humans. These data indicate that rat MLE shares similar properties to human MLE in its expression pattern of these markers, not withstanding small differences, and support the concept that MLE may be a transitional stage in the metaplastic conversion of squamous to columnar epithelium in BE.

Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs. PMID:24376422

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.

Expression of Epithelial Mesenchymal Transition and Cancer Stem Cell Markers in Circulating Tumor Cells.

PubMed

Werner, Stefan; Stenzl, Arnulf; Pantel, Klaus; Todenhöfer, Tilman

2017-01-01

The characterization of circulating tumor cells (CTC) has the potential not only to provide important insights into molecular alterations of advanced tumor disease but also to facilitate risk prediction. Epithelial mesenchymal transition (EMT) has been discovered as important process for the development of metastases and the dissemination of tumor cells into the blood stream. In different tumor types, CTC with a mesenchymal phenotype have been reported that have presumably underwent EMT. Moreover, CTC with stem-cell like characteristics have been postulated as important drivers of tumor progression. Different platforms have been introduced to allow CTC enrichment independent of expression of epithelial antigens, as these may be downregulated in EMT- or stem-cell-like CTC. Both for CTCs with EMT- or stem-cell features different markers have been proposed. However, there is still a lack of evidence on the association of these markers with functional features and characteristics for stem cells and cells undergoing EMT.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

PubMed

Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

2017-02-01

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

PubMed

Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

Referential Choices in a Collaborative Storytelling Task: Discourse Stages and Referential Complexity Matter.

PubMed

Fossard, Marion; Achim, Amélie M; Rousier-Vercruyssen, Lucie; Gonzalez, Sylvia; Bureau, Alexandre; Champagne-Lavau, Maud

2018-01-01

During a narrative discourse, accessibility of the referents is rarely fixed once and for all. Rather, each referent varies in accessibility as the discourse unfolds, depending on the presence and prominence of the other referents. This leads the speaker to use various referential expressions to refer to the main protagonists of the story at different moments in the narrative. This study relies on a new, collaborative storytelling in sequence task designed to assess how speakers adjust their referential choices when they refer to different characters at specific discourse stages corresponding to the introduction, maintaining, or shift of the character in focus, in increasingly complex referential contexts. Referential complexity of the stories was manipulated through variations in the number of characters (1 vs. 2) and, for stories in which there were two characters, in their ambiguity in gender (different vs. same gender). Data were coded for the type of reference markers as well as the type of reference content (i.e., the extent of the information provided in the referential expression). Results showed that, beyond the expected effects of discourse stages on reference markers (more indefinite markers at the introduction stage, more pronouns at the maintaining stage, and more definite markers at the shift stage), the number of characters and their ambiguity in gender also modulated speakers' referential choices at specific discourse stages, For the maintaining stage, an effect of the number of characters was observed for the use of pronouns and of definite markers, with more pronouns when there was a single character, sometimes replaced by definite expressions when two characters were present in the story. For the shift stage, an effect of gender ambiguity was specifically noted for the reference content with more specific information provided in the referential expression when there was referential ambiguity. Reference content is an aspect of referential marking that is rarely addressed in a narrative context, yet it revealed a quite flexible referential behavior by the speakers.

Referential Choices in a Collaborative Storytelling Task: Discourse Stages and Referential Complexity Matter

PubMed Central

Fossard, Marion; Achim, Amélie M.; Rousier-Vercruyssen, Lucie; Gonzalez, Sylvia; Bureau, Alexandre; Champagne-Lavau, Maud

2018-01-01

During a narrative discourse, accessibility of the referents is rarely fixed once and for all. Rather, each referent varies in accessibility as the discourse unfolds, depending on the presence and prominence of the other referents. This leads the speaker to use various referential expressions to refer to the main protagonists of the story at different moments in the narrative. This study relies on a new, collaborative storytelling in sequence task designed to assess how speakers adjust their referential choices when they refer to different characters at specific discourse stages corresponding to the introduction, maintaining, or shift of the character in focus, in increasingly complex referential contexts. Referential complexity of the stories was manipulated through variations in the number of characters (1 vs. 2) and, for stories in which there were two characters, in their ambiguity in gender (different vs. same gender). Data were coded for the type of reference markers as well as the type of reference content (i.e., the extent of the information provided in the referential expression). Results showed that, beyond the expected effects of discourse stages on reference markers (more indefinite markers at the introduction stage, more pronouns at the maintaining stage, and more definite markers at the shift stage), the number of characters and their ambiguity in gender also modulated speakers' referential choices at specific discourse stages, For the maintaining stage, an effect of the number of characters was observed for the use of pronouns and of definite markers, with more pronouns when there was a single character, sometimes replaced by definite expressions when two characters were present in the story. For the shift stage, an effect of gender ambiguity was specifically noted for the reference content with more specific information provided in the referential expression when there was referential ambiguity. Reference content is an aspect of referential marking that is rarely addressed in a narrative context, yet it revealed a quite flexible referential behavior by the speakers. PMID:29515493

Age and sex differences in human skeletal muscle fibrosis markers and transforming growth factor-β signaling.

PubMed

Parker, Lewan; Caldow, Marissa K; Watts, Rani; Levinger, Pazit; Cameron-Smith, David; Levinger, Itamar

2017-07-01

The aim of the study was to determine whether higher fibrosis markers in skeletal muscle of older adults are accompanied by increased expression of components of the canonical TGF-β signal transduction pathway. Fourteen healthy young (21-35 years; 9 males and 5 females) and seventeen older (55-75 years; 9 males and 8 females) participants underwent vastus lateralis biopsies to determine intramuscular mRNA and protein expression of fibrogenic markers and TGF-β signaling molecules related to TGF-β1 and myostatin. Expression of mRNA encoding the pro-fibrotic factors; axin 2, collagen III, β-catenin and fibronectin, were all significantly higher (all p < 0.05) in the older participants (350, 170, 298, and 641%, respectively). Furthermore, axin 2 and β-catenin mRNA were significantly higher in older females than older males (p < 0.05). Gene expression of ActRIIB, myostatin, and TGF-β1 were higher in older adults compared to younger adults (all p < 0.05). There was, however, no difference in the total protein content of myostatin, myoD or myogenin (all p > 0.05), whereas Smad3 protein phosphorylation was 48% lower (p < 0.05) in muscle from older adults. Increased abundance of mRNA of fibrotic markers was observed in muscle from older adults and was partly accompanied by altered abundance of pro-fibrotic ligands in a sex specific manner.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

PubMed

Camilleri, Emily T; Gustafson, Michael P; Dudakovic, Amel; Riester, Scott M; Garces, Catalina Galeano; Paradise, Christopher R; Takai, Hideki; Karperien, Marcel; Cool, Simon; Sampen, Hee-Jeong Im; Larson, A Noelle; Qu, Wenchun; Smith, Jay; Dietz, Allan B; van Wijnen, Andre J

2016-08-11

Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

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.

Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality.

PubMed

Patel, Ronak; Page, Shyanne; Al-Ahmad, Abraham Jacob

2017-07-01

The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes, and neurons. Recently, isogenic in vitro models of the BBB based on human pluripotent stem cells have been documented, yet the impact of inter-individual variability on the yield and phenotype of such models remains to be documented. In this study, we investigated the impact of inter-individual variability on the yield and phenotype of isogenic models of the BBB, using patient-derived induced pluripotent stem cells (iPSCs). Astrocytes, BMECs, and neurons were differentiated from four asymptomatic patient-derived iPSCs (two males, two females). We differentiated such cells using existing differentiation protocols and quantified expression of cell lineage markers, as well as BBB phenotype, barrier induction, and formation of neurite processes. iPSC-derived BMECs showed barrier properties better than hCMEC/D3 monolayers; however, we noted differences in the expression and activity among iPSC lines. In addition, we noted differences in the differentiation efficiency of these cells into neural stem cells and progenitor cells (as noted by differences in expression of cell lineage markers). Such differences were reflected later in the terminal differentiation, as seen as ability to induce barrier function and to form neurite processes. Although we demonstrated our ability to obtain an isogenic model of the BBB with different patients' iPSCs, we also noted subtle differences in the expression of cell lineage markers and cell maturation processes, suggesting the presence of inter-individual polymorphisms. © 2017 International Society for Neurochemistry.

"Ahora" (Now) and "Ahorita" (Right Now) as Deictic Markers.

ERIC Educational Resources Information Center

Matus-Mendoza, Mariadelaluz

This study explores the different meanings of "ahora" (now) and "ahorita" (right now) in Spanish usage by a migrant community of mushroom pickers in Mexico and the United States. Both terms are used as temporal expressions in Mexican Spanish spoken on both sides of the border. They are discourse markers. After studying groups…

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

PubMed

Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

1998-01-01

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

PubMed Central

Maguire, Colin T.; Demarest, Bradley L.; Hill, Jonathon T.; Palmer, James D.; Brothman, Arthur R.; Yost, H. Joseph; Condic, Maureen L.

2013-01-01

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage. PMID:23326421

Isolation and characterization of human CXCR4-positive pancreatic cells.

PubMed

Koblas, T; Zacharovová, K; Berková, Z; Mindlová, M; Girman, P; Dovolilová, E; Karasová, L; Saudek, F

2007-01-01

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues. Here we report that CXCR4-positive pancreatic cells express markers of pancreatic endocrine progenitors (neurogenin-3, nestin) and markers of pluripotent stem cells (Oct-4, Nanog, ABCG2, CD133, CD117). Upon in vitro differentiation, these cells form ILCC and produce key islet hormones including insulin. Based on our results, we assume that CXCR4 marks pancreatic endocrine progenitors and in combination with other cell surface markers may be used in the attempt to identify and isolate PSC.

Expression of CD markers' in immune thrombocytopenic purpura: prognostic approaches.

PubMed

Behzad, Masumeh Maleki; Asnafi, Ali Amin; Jaseb, Kaveh; Jalali Far, Mohammad Ali; Saki, Najmaldin

2017-12-01

Immune Thrombocytopenic Purpura (ITP) is a common autoimmune bleeding disorder characterized by a reduction in peripheral blood platelet counts. In this disease, autoantibodies (Auto-Abs) are produced against platelet GPIIb/GPIIIa by B cells, which require interaction with T cells. In this review, the importance of B and T lymphocytes in ITP prognosis has been studied. Relevant literature was identified by a PubMed search (1990-2016) of English-language papers using the terms B and T lymphocyte, platelet, CD markers and immune thrombocytopenic purpura. T and B lymphocytes are the main immune cells in the body. Defective function causes disrupted balance of different subgroups of lymphocytes, and abnormal expression of surface markers of these cells results in self-tolerance dysfunction, as well as induction of Auto-Abs against platelet glycoproteins (PG). Given the role of B and T cells in production of autoantibodies against PG, it can be stated that the detection of changes in CD markers' expression in these cells can be a good approach for assessing prognosis in ITP patients. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

PubMed

Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew

2002-08-01

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies suggest that within CNS and non-CNS tissues there exist subpopulations of macrophages that are SIV-infected and express PCNA.

Signatures of tumour immunity distinguish Asian and non-Asian gastric adenocarcinomas.

PubMed

Lin, Suling J; Gagnon-Bartsch, Johann A; Tan, Iain Beehuat; Earle, Sophie; Ruff, Louise; Pettinger, Katherine; Ylstra, Bauke; van Grieken, Nicole; Rha, Sun Young; Chung, Hyun Cheol; Lee, Ju-Seog; Cheong, Jae Ho; Noh, Sung Hoon; Aoyama, Toru; Miyagi, Yohei; Tsuburaya, Akira; Yoshikawa, Takaki; Ajani, Jaffer A; Boussioutas, Alex; Yeoh, Khay Guan; Yong, Wei Peng; So, Jimmy; Lee, Jeeyun; Kang, Won Ki; Kim, Sung; Kameda, Yoichi; Arai, Tomio; Zur Hausen, Axel; Speed, Terence P; Grabsch, Heike I; Tan, Patrick

2015-11-01

Differences in gastric cancer (GC) clinical outcomes between patients in Asian and non-Asian countries has been historically attributed to variability in clinical management. However, recent international Phase III trials suggest that even with standardised treatments, GC outcomes differ by geography. Here, we investigated gene expression differences between Asian and non-Asian GCs, and if these molecular differences might influence clinical outcome. We compared gene expression profiles of 1016 GCs from six Asian and three non-Asian GC cohorts, using a two-stage meta-analysis design and a novel biostatistical method (RUV-4) to adjust for technical variation between cohorts. We further validated our findings by computerised immunohistochemical analysis on two independent tissue microarray (TMA) cohorts from Asian and non-Asian localities (n=665). Gene signatures differentially expressed between Asians and non-Asian GCs were related to immune function and inflammation. Non-Asian GCs were significantly enriched in signatures related to T-cell biology, including CTLA-4 signalling. Similarly, in the TMA cohorts, non-Asian GCs showed significantly higher expression of T-cell markers (CD3, CD45R0, CD8) and lower expression of the immunosuppressive T-regulatory cell marker FOXP3 compared to Asian GCs (p<0.05). Inflammatory cell markers CD66b and CD68 also exhibited significant cohort differences (p<0.05). Exploratory analyses revealed a significant relationship between tumour immunity factors, geographic locality-specific prognosis, and postchemotherapy outcomes. Analyses of >1600 GCs suggest that Asian and non-Asian GCs exhibit distinct tumour immunity signatures related to T-cell function. These differences may influence geographical differences in clinical outcome, and the design of future trials particularly in immuno-oncology. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

PubMed

Jespersen, David; Belanger, Faith C; Huang, Bingru

2017-01-01

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass

PubMed Central

Jespersen, David; Belanger, Faith C.; Huang, Bingru

2017-01-01

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection. PMID:28187136

A Brassica rapa Linkage Map of EST-based SNP Markers for Identification of Candidate Genes Controlling Flowering Time and Leaf Morphological Traits

PubMed Central

Li, Feng; Kitashiba, Hiroyasu; Inaba, Kiyofumi; Nishio, Takeshi

2009-01-01

For identification of genes responsible for varietal differences in flowering time and leaf morphological traits, we constructed a linkage map of Brassica rapa DNA markers including 170 EST-based markers, 12 SSR markers, and 59 BAC sequence-based markers, of which 151 are single nucleotide polymorphism (SNP) markers. By BLASTN, 223 markers were shown to have homologous regions in Arabidopsis thaliana, and these homologous loci covered nearly the whole genome of A. thaliana. Synteny analysis between B. rapa and A. thaliana revealed 33 large syntenic regions. Three quantitative trait loci (QTLs) for flowering time were detected. BrFLC1 and BrFLC2 were linked to the QTLs for bolting time, budding time, and flowering time. Three SNPs in the promoter, which may be the cause of low expression of BrFLC2 in the early-flowering parental line, were identified. For leaf lobe depth and leaf hairiness, one major QTL corresponding to a syntenic region containing GIBBERELLIN 20 OXIDASE 3 and one major QTL containing BrGL1, respectively, were detected. Analysis of nucleotide sequences and expression of these genes suggested possible involvement of these genes in leaf morphological traits. PMID:19884167

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.

Identification of immunohistochemical markers for distinguishing lung adenocarcinoma from squamous cell carcinoma

PubMed Central

Zhan, Cheng; Yan, Li; Wang, Lin; Sun, Yang; Wang, Xingxing; Lin, Zongwu; Zhang, Yongxing; Wang, Qun

2015-01-01

Background Immunohistochemical staining has been widely used in distinguishing lung adenocarcinoma (LUAD) from lung squamous cell carcinoma (LUSC), which is of vital importance for the diagnosis and treatment of lung cancer. Due to the lack of a comprehensive analysis of different lung cancer subtypes, there may still be undiscovered markers with higher diagnostic accuracy. Methods Herein first, we systematically analyzed high-throughput data obtained from The Cancer Genome Atlas (TCGA) database. Combining differently expressed gene screening and receiver operating characteristic (ROC) curve analysis, we attempted to identify the genes which might be suitable as immunohistochemical markers in distinguishing LUAD from LUSC. Then we detected the expression of six of these genes (MLPH, TMC5, SFTA3, DSG3, DSC3 and CALML3) in lung cancer sections using immunohistochemical staining. Results A number of genes were identified as candidate immunohistochemical markers with high sensitivity and specificity in distinguishing LUAD from LUSC. Then the staining results confirmed the potentials of the six genes (MLPH, TMC5, SFTA3, DSG3, DSC3 and CALML3) in distinguishing LUAD from LUSC, and their sensitivity and specificity were not less than many commonly used markers. Conclusions The results revealed that the six genes (MLPH, TMC5, SFTA3, DSG3, DSC3 and CALML3) might be suitable markers in distinguishing LUAD from LUSC, and also validated the feasibility of our methods for identification of candidate markers from high-throughput data. PMID:26380766

High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: a Cancer and Leukemia Group B (CALGB) study

PubMed Central

Langer, Christian; Radmacher, Michael D.; Ruppert, Amy S.; Whitman, Susan P.; Paschka, Peter; Mrózek, Krzysztof; Baldus, Claudia D.; Vukosavljevic, Tamara; Liu, Chang-Gong; Ross, Mary E.; Powell, Bayard L.; de la Chapelle, Albert; Kolitz, Jonathan E.; Larson, Richard A.; Marcucci, Guido

2008-01-01

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile. PMID:18378853

Histopathology of aortic complications in bicuspid aortic valve versus Marfan syndrome: relevance for therapy?

PubMed

Grewal, Nimrat; Franken, Romy; Mulder, Barbara J M; Goumans, Marie-José; Lindeman, Johannes H N; Jongbloed, Monique R M; DeRuiter, Marco C; Klautz, Robert J M; Bogers, Ad J J C; Poelmann, Robert E; Groot, Adriana C Gittenberger-de

2016-05-01

Patients with bicuspid aortic valve (BAV) and patients with Marfan syndrome (MFS) are more prone to develop aortic dilation and dissection compared to persons with a tricuspid aortic valve (TAV). To elucidate potential common and distinct pathways of clinical relevance, we compared the histopathological substrates of aortopathy. Ascending aortic wall biopsies were divided in five groups: BAV (n = 36) and TAV (n = 23) without and with dilation and non-dilated MFS (n = 8). General histologic features, apoptosis, the expression of markers for vascular smooth muscle cell (VSMC) maturation, markers predictive for ascending aortic dilation in BAV, and expression of fibrillin-1 were investigated. Both MFS and BAV showed an altered distribution and decreased fibrillin-1 expression in the aorta and a significantly lower level of differentiated VSMC markers. Interestingly, markers predictive for aortic dilation in BAV were not expressed in the MFS aorta. The aorta in MFS was similar to the aorta in dilated TAV with regard to the presence of medial degeneration and apoptosis, while other markers for degeneration and aging like inflammation and progerin expression were low in MFS, comparable to BAV. Both MFS and BAV aortas have immature VSMCs, while MFS and TAV patients have a similar increased rate of medial degeneration. However, the mechanism leading to apoptosis is expected to be different, being fibrillin-1 mutation induced increased angiotensin-receptor-pathway signaling in MFS and cardiovascular aging and increased progerin in TAV. Our findings could explain why angiotensin inhibition is successful in MFS and less effective in TAV and BAV patients.

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.

Gene Expression Analysis Reveals New Possible Mechanisms of Vancomycin-Induced Nephrotoxicity and Identifies Gene Markers Candidates

PubMed Central

Dieterich, Christine; Puey, Angela; Lyn, Sylvia; Swezey, Robert; Furimsky, Anna; Fairchild, David; Mirsalis, Jon C.; Ng, Hanna H.

2009-01-01

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription–polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury. PMID:18930951

Gene expression analysis reveals new possible mechanisms of vancomycin-induced nephrotoxicity and identifies gene markers candidates.

PubMed

Dieterich, Christine; Puey, Angela; Lin, Sylvia; Lyn, Sylvia; Swezey, Robert; Furimsky, Anna; Fairchild, David; Mirsalis, Jon C; Ng, Hanna H

2009-01-01

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription-polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.

MGDB: crossing the marker genes of a user microarray with a database of public-microarrays marker genes.

PubMed

Huerta, Mario; Munyi, Marc; Expósito, David; Querol, Enric; Cedano, Juan

2014-06-15

The microarrays performed by scientific teams grow exponentially. These microarray data could be useful for researchers around the world, but unfortunately they are underused. To fully exploit these data, it is necessary (i) to extract these data from a repository of the high-throughput gene expression data like Gene Expression Omnibus (GEO) and (ii) to make the data from different microarrays comparable with tools easy to use for scientists. We have developed these two solutions in our server, implementing a database of microarray marker genes (Marker Genes Data Base). This database contains the marker genes of all GEO microarray datasets and it is updated monthly with the new microarrays from GEO. Thus, researchers can see whether the marker genes of their microarray are marker genes in other microarrays in the database, expanding the analysis of their microarray to the rest of the public microarrays. This solution helps not only to corroborate the conclusions regarding a researcher's microarray but also to identify the phenotype of different subsets of individuals under investigation, to frame the results with microarray experiments from other species, pathologies or tissues, to search for drugs that promote the transition between the studied phenotypes, to detect undesirable side effects of the treatment applied, etc. Thus, the researcher can quickly add relevant information to his/her studies from all of the previous analyses performed in other studies as long as they have been deposited in public repositories. Marker-gene database tool: http://ibb.uab.es/mgdb © The Author 2014. Published by Oxford University Press.

[Study on transformation of P-dissolving Penicillium oxalicum P8 with double-marker vector expressing green fluorescent protein and hygromycin B resistance].

PubMed

Zhang, Lei; Fan, Bing-Quan; Huang, Wei-Yi

2005-12-01

P-dissolving Penicillium oxalicum P8 was isolated previously in this lab which has a considerable ability to dissolve many kinds of inorganic phosphorus and improve crop growth. In order to study rhizosphere colonization of plants by Penicillium oxalicum P8, protoplasts were transformed with a double-marker expression vector of green fluorescent protein and hygromycin B resistance. Some transformants were selected which expressed both the GFP and hygromycin B phosphotransferase and did not show significant morphological or physiological differences as compared to wild-type strain. Southern blot analysis confirmed the heterogeneous genomic integration of the vector DNA into the transformants.

Nestin expression in neuroepithelial tumors.

PubMed

Schiffer, Davide; Manazza, Andrea; Tamagno, Ilaria

2006-05-29

Nestin is a marker of early stages of neurocytogenesis. It has been studied in 50 neuroepithelial tumors, mostly gliomas of different malignancy grades, by immunohistochemistry, immunofluorescence, immunoblotting, and confocal microscopy and compared with GFAP and Vimentin. As an early marker of differentiation, Nestin is almost not expressed in diffuse astrocytomas, variably expressed in anaplastic astrocytomas and strongly and irregularly expressed in glioblastomas. Negative in oligodendrogliomas, it stains ependymomas and shows a gradient of expression in pilocytic astrocytomas. In glioblastomas, Nestin distribution does not completely correspond to that of GFAP and Vimentin with which its expression varies in tumor cells in a complementary way, as confirmed by confocal microscopy. Tumor cells can thus either derive from or differentiate toward the neurocytogenetic stages. Hypothetically, they could be put in relation with radial glia where during embriogenesis the three antigens are successively expressed. Completely negative cells of invasive or recurrent glioblastomas may represent malignant selected clones after accumulation of mutations or early stem cells not expressing antigens.

A Comparison of Clinicopathological Features and Molecular Markers in British and Nigerian Women with Breast Cancer

PubMed Central

Gukas, Isaac D.; Girling, Anne C.; Mandong, Barnabas. M.; Prime, Wendy; Jennings, Barbara A.; Leinster, Samuel J.

2008-01-01

Background Some studies have suggested that breast cancer in black women is more aggressive than in white women. This study’s aim was to look for evidence of differences in tumour biology between the two cohorts. Methods This study compared the stage, grade and pathological expression of five immunohistochemical markers (oestrogen receptor [ER], progesterone receptor [PR], ERBB2, P53 and cyclin D1 [CCND1]) in tumour biopsies from age-matched cohorts of patients from Nigeria and England. Sixty-eight suitable samples from Nigerian (n = 34) and British (n = 34) breast cancer patients were retrieved from histology tissue banks. Results There were significant differences between the two cohorts in the expression of ER and CCND1; and stark differences in the clinical stage at presentation. But no significant differences were observed for tumour grade. Conclusion There was a significantly, low ER expression in the Nigerian cases which also predicts a poor response to hormonal therapy as well as a poorer prognosis. Differences in clinical stage at presentation will most likely influence prognosis between Nigerian and British women with breast cancer. PMID:21892296

EFEMP1 as a novel DNA methylation marker for prostate cancer: array-based DNA methylation and expression profiling.

PubMed

Kim, Yong-June; Yoon, Hyung-Yoon; Kim, Seon-Kyu; Kim, Young-Won; Kim, Eun-Jung; Kim, Isaac Yi; Kim, Wun-Jae

2011-07-01

Abnormal DNA methylation is associated with many human cancers. The aim of the present study was to identify novel methylation markers in prostate cancer (PCa) by microarray analysis and to test whether these markers could discriminate normal and PCa cells. Microarray-based DNA methylation and gene expression profiling was carried out using a panel of PCa cell lines and a control normal prostate cell line. The methylation status of candidate genes in prostate cell lines was confirmed by real-time reverse transcriptase-PCR, bisulfite sequencing analysis, and treatment with a demethylation agent. DNA methylation and gene expression analysis in 203 human prostate specimens, including 106 PCa and 97 benign prostate hyperplasia (BPH), were carried out. Further validation using microarray gene expression data from the Gene Expression Omnibus (GEO) was carried out. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was identified as a lead candidate methylation marker for PCa. The gene expression level of EFEMP1 was significantly higher in tissue samples from patients with BPH than in those with PCa (P < 0.001). The sensitivity and specificity of EFEMP1 methylation status in discriminating between PCa and BPH reached 95.3% (101 of 106) and 86.6% (84 of 97), respectively. From the GEO data set, we confirmed that the expression level of EFEMP1 was significantly different between PCa and BPH. Genome-wide characterization of DNA methylation profiles enabled the identification of EFEMP1 aberrant methylation patterns in PCa. EFEMP1 might be a useful indicator for the detection of PCa.

Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations.

PubMed

Bahadorani, M; Hosseini, S M; Abedi, P; Hajian, M; Hosseini, S E; Vahdati, A; Baharvand, H; Nasr-Esfahani, Mohammad H

2012-01-01

To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs). Crude testicular cells were plated over Datura-Stramonium Agglutinin (DSA) for 1 h, and non-adhering cells were cultured in the presence of different serum concentrations (1, 5, 10, and 15%) for 7 days in a highly enriched medium initially developed in mice. Colonies developed in each group were used for the assessment of morphology, immunocytochemistry, and gene expression. Brief incubation of testicular cells with DSA resulted in a significant increase in the number of cells that expressed the germ cell marker (VASA). The expression of THY1, a specific marker of undifferentiated spermatogonia, was significantly higher in colonies developed in the presence of 1% rather than 5, 10 and 15% serum. Goat SSCs could proliferate and maintain in SSC culture media for 1 week at serum concentrations as low as 1%, while higher concentrations had detrimental effects on SSC culture/expansion.

Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer.

PubMed

Hyun, Kyung-A; Koo, Gi-Bang; Han, Hyunju; Sohn, Joohyuk; Choi, Wonshik; Kim, Seung-Il; Jung, Hyo-Il; Kim, You-Sun

2016-04-26

The dissemination of circulating tumor cells (CTCs) requires the Epithelial-to-Mesenchymal transition (EMT), in which cells lose their epithelial characteristics and acquire more mesenchymal-like phenotypes. Current isolation of CTCs relies on affinity-based approaches reliant on the expression of Epithelial Cell Adhesion Molecule (EpCAM). Here we show EMT-induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy; however, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label-free microfluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and potentially miss critical subpopulations. Further characterization of CTCs, including low-EpCAM populations, using this technology may improve detection techniques and cancer diagnosis, ultimately improving cancer treatment.

Preclinical validation of 111In-girentuximab-F(ab')2 as a tracer to image hypoxia related marker CAIX expression in head and neck cancer xenografts.

PubMed

Huizing, Fokko J; Hoeben, Bianca A W; Franssen, Gerben; Lok, Jasper; Heskamp, Sandra; Oosterwijk, Egbert; Boerman, Otto C; Bussink, Johan

2017-09-01

Hypoxia is a major cause of radio- and chemoresistance. Carbonic anhydrase IX (CAIX) is an endogenous hypoxia-related marker and an important prognostic marker. Assessment of CAIX expression may allow patient selection for hypoxia or CAIX-targeted treatment. The radioactive tracer 111 In-girentuximab-F(ab') 2 targets CAIX and can be used for SPECT imaging. Aim of this study was to validate and optimize 111 In-girentuximab-F(ab') 2 for imaging of CAIX expression in head and neck tumor xenografts. Affinity and internalization kinetics of 111 In-girentuximab-F(ab') 2 were determined in vitro using CAIX-expressing SK-RC-52 cells. Tumor targeting characteristics were determined in athymic mice with six different head and neck squamous cell carcinoma (SCCNij) xenografts. Tracer uptake was measured by ex vivo radioactivity counting. Intratumoral distribution of tracer uptake was measured using autoradiography and CAIX expression was determined immunohistochemically. 26% of the tracer was internalized into the SK-RC-52 cells within 24h. The half maximal inhibitory concentration (IC 50 ) was 0.69±0.08nM. In biodistribution studies SCCNij153 tumors showed the highest tracer uptake: 4.1±0.8ID/g at 24h p.i. Immunohistochemical and autoradiographic analyses of the xenografts showed a distinct spatial correlation between localization of the tracer and CAIX expression. 111 In-girentuximab-F(ab') 2 has a high affinity for CAIX. In vivo tumor uptake correlated strongly with CAIX expression in different head and neck xenografts. These results suggest that 111 In-girentuximab-F(ab') 2 is a promising tracer for imaging of hypoxia-related CAIX expression. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Cytogenetic and molecular markers for detecting Aegilops uniaristata chromosomes in a wheat background.

PubMed

Gong, Wenping; Li, Guangrong; Zhou, Jianping; Li, Genying; Liu, Cheng; Huang, Chengyan; Zhao, Zhendong; Yang, Zujun

2014-09-01

Aegilops uniaristata has many agronomically useful traits that can be used for wheat breeding. So far, a Triticum turgidum - Ae. uniaristata amphiploid and one set of Chinese Spring (CS) - Ae. uniaristata addition lines have been produced. To guide Ae. uniaristata chromatin transformation from these lines into cultivated wheat through chromosome engineering, reliable cytogenetic and molecular markers specific for Ae. uniaristata chromosomes need to be developed. Standard C-banding shows that C-bands mainly exist in the centromeric regions of Ae. uniaristata but rarely at the distal ends. Fluorescence in situ hybridization (FISH) using (GAA)8 as a probe showed that the hybridization signal of chromosomes 1N-7N are different, thus (GAA)8 can be used to identify all Ae. uniaristata chromosomes in wheat background simultaneously. Moreover, a total of 42 molecular markers specific for Ae. uniaristata chromosomes were developed by screening expressed sequence tag - sequence tagged site (EST-STS), expressed sequence tag - simple sequence repeat (EST-SSR), and PCR-based landmark unique gene (PLUG) primers. The markers were subsequently localized using the CS - Ae. uniaristata addition lines and different wheat cultivars as controls. The cytogenetic and molecular markers developed herein will be helpful for screening and identifying wheat - Ae. uniaristata progeny.

Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.

PubMed

Boesmans, Werend; Lasrado, Reena; Vanden Berghe, Pieter; Pachnis, Vassilis

2015-02-01

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca(2+) imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co-express glial fibrillary acidic protein (GFAP), S100β, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease. © 2014 Wiley Periodicals, Inc.

Differences of immunophenotypic markers and signaling molecules between adenocarcinomas of gastric cardia and distal stomach.

PubMed

Xue, Liying; Zhang, Xianghong; Li, Yuehong; Yang, Haiyan; Li, Xuemin; Mi, Jianmin; Wang, Hengshu; Wang, Junling; Yan, Xia

2011-04-01

During the past decades, the subsites of gastric carcinoma underwent significant changes. The incidence of the adenocarcinoma at distal stomach has been decreased, whereas cardiac adenocarcinoma remained increasing in many countries. The aim of this study was to investigate the differences between gastric cardiac and distal adenocarcinomas. We detected expressions of cytokeratins (cytokeratins 7, 14, 19, and 20) and mucins (mucins 1, 2, and 5AC) by immunohistochemistry and signaling molecules (p38, mitogen-activated protein kinase-interacting kinase 1 (MNK1), extracellular signal-regulated kinase, Jun N-terminal kinase, and phosphoinositide 3 kinase) by reverse transcription-polymerase chain reaction in both groups. The incidence of mucin 2 expression was lower in total (50.0%) and advanced-stage cases (52.0%) with cardiac adenocarcinomas than those in distal cases with total (70.2%) and advanced stage (71.4%), respectively. However, the staining for cytokeratin 14 was also significantly higher in total or advanced-stage tumors from the cardia. Our data showed no significant difference of cytokeratin 7/cytokeratin 20 pattern between 2 groups, but cytokeratin 20 expression was significantly higher in advanced-stage carcinomas of the cardia (58.7%) than in distal ones with advanced stage (38.3%). A multivariate analysis demonstrated different relationships between immunophenotypic markers and pathologic parameters in adenocarcinomas of the cardia and distal stomach. Moreover, significantly lower expressions of MNK1 and p38 in cardiac tumors were also detected. In summary, we found significant differences in patterns of immunophenotypic markers and expressions of signaling molecules between the 2 groups. It is indicated that adenocarcinoma of the cardia was different in histotype and histologic origin from distal adenocarcinoma. The cardiac adenocarcinoma might be a special subtype or an independent entity of gastric carcinoma in China. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

PubMed

Oliveira, C S; de Souza, M M; Saraiva, N Z; Tetzner, T A D; Lima, M R; Lopes, F L; Garcia, J M

2012-06-01

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. © 2011 Blackwell Verlag GmbH.

Database of cattle candidate genes and genetic markers for milk production and mastitis

PubMed Central

Ogorevc, J; Kunej, T; Razpet, A; Dovc, P

2009-01-01

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3′UTRs of candidate genes. PMID:19508288

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.

Variation of musculoskeletal stress markers in the medieval population from Cedynia (Poland)--proposal of standardized scoring method application.

PubMed

Myszka, Anna; Piontek, Janusz

2012-09-01

The objective of this paper is: (a) to present a rating scale for the evaluation of the musculoskeletal stress markers; (b) to analyze the medieval population from Cedynia in terms of the degree of expression and frequency of the musculoskeletal stress markers. The presented rating scale was developed based on the variability of the morphology of muscle attachment sites, observed in the skeletal material from Cedynia (102 males and 99 females). The scale encompasses 10 musculoskeletal stress markers located on the scapula, humerus, radius, femur and tibia. The system reflects three degrees (1, 2, 3) of complexity of the muscle attachment sites morphology. The analysis of asymmetry and sexual dimorphism of the musculoskeletal stress markers was made based on the chi2 (Pearson) statistics or chi2 statistics for 2 x 2 tables. Moderate degree (2) of muscle attachment site complexity is the most frequent degree of musculoskeletal stress markers development in the population from Cedynia. Low (1) and high (3) complexity of muscle attachment site are the most seldom observed categories. No statistically significant differences between the frequencies of the musculoskeletal stress markers on the bones of the right and left side of the skeleton were noted in females. Also in males the differences found were not statistically significant. Only in the case of deltoid tuberosity (H2) p = 0.052 oscillating around the threshold value may suggest existence of a statistically significant difference in the degree of expression of this stress marker on the bone of the right and left side of the skeleton. On the bones of the right side of the skeleton dimorphic differences were observed in the glenoid tuberosity (S2), bicipital groove (H1), pronator teres origin (R2), tibial tuberosity (T1), soleal crest (T2) and linea aspera (F2). On the bones of the left side of the skeleton dimorphic differences were noted for the bicipital groove (H1), pronator teres origin (R2) and glenoid tuberosity (S2).

Establishment of embryonic neuroepithelial cell lines exhibiting an epiplastic expression pattern of region specific markers.

PubMed

Nardelli, Jeannette; Catala, Martin; Charnay, Patrick

2003-09-15

Neuroepithelial b2T cells were derived from the hindbrain and the spinal cord of mouse transgenic embryos, which expressed SV40 T antigen under the control of a Hoxb2 enhancer. Strikingly, b2T cell lines of either origin exhibit a very similar gene expression pattern, including markers of the hindbrain and the spinal cord, such as Hox genes, but not of more anterior cephalic regions. In addition, the broad expression pattern of b2T cells, probably linked to culture conditions, appeared to be appropriately modulated when the cells were reimplanted at different longitudinal levels into chick host embryos, suggesting that these cells are responsive to exogenous signalling mechanisms. Further support for these allegations was obtained by culturing b2T cells in defined medium and by assessing the expression of Krox20, an odd-numbered rhombomere marker, which appeared to be modulated by a complex interplay between FGF, retinoic acid (RA), and noggin. With respect to these as yet unique properties, b2T cells constitute an original alternative tool to in vivo models for the analysis of molecular pathways involved in the patterning of the neural tube. Copyright 2003 Wiley-Liss, Inc.

Mesencephalic basolateral domain specification is dependent on Sonic Hedgehog

PubMed Central

Martinez-Lopez, Jesus E.; Moreno-Bravo, Juan A.; Madrigal, M. Pilar; Martinez, Salvador; Puelles, Eduardo

2015-01-01

In the study of central nervous system morphogenesis, the identification of new molecular markers allows us to identify domains along the antero-posterior and dorso-ventral (DV) axes. In the past years, the alar and basal plates of the midbrain have been divided into different domains. The precise location of the alar-basal boundary is still under discussion. We have identified Barhl1, Nhlh1 and Six3 as appropriate molecular markers to the adjacent domains of this transition. The description of their expression patterns and the contribution to the different mesencephalic populations corroborated their role in the specification of these domains. We studied the influence of Sonic Hedgehog on these markers and therefore on the specification of these territories. The lack of this morphogen produced severe alterations in the expression pattern of Barhl1 and Nhlh1 with consequent misspecification of the basolateral (BL) domain. Six3 expression was apparently unaffected, however its distribution changed leading to altered basal domains. In this study we confirmed the localization of the alar-basal boundary dorsal to the BL domain and demonstrated that the development of the BL domain highly depends on Shh. PMID:25741244

Novel Nomogram That Predicts Aggressive Disease and Treatment Failure Among African-American Men with Prostate Cancer

DTIC Science & Technology

2015-12-01

analyzed including TMPRSS2-ERG, AMACR, PSMA , RB, c- Myc, and AR. We observed statistically significant differences in biomarker expression between EA...biomarkers have been analyzed so far including TMPRSS2-ERG, AMACR, PSMA , RB, c-Myc, and AR. In this cohort we observed statistically significant...markers or markers that give relative intensity - p53, p63, Ki67, PSMA , AR - Other 12 markers will be read as present/absent/indeterminate etc

Computer-analyzed facial expression as a surrogate marker for autism spectrum social core symptoms.

PubMed

Owada, Keiho; Kojima, Masaki; Yassin, Walid; Kuroda, Miho; Kawakubo, Yuki; Kuwabara, Hitoshi; Kano, Yukiko; Yamasue, Hidenori

2018-01-01

To develop novel interventions for autism spectrum disorder (ASD) core symptoms, valid, reliable, and sensitive longitudinal outcome measures are required for detecting symptom change over time. Here, we tested whether a computerized analysis of quantitative facial expression measures could act as a marker for core ASD social symptoms. Facial expression intensity values during a semi-structured socially interactive situation extracted from the Autism Diagnostic Observation Schedule (ADOS) were quantified by dedicated software in 18 high-functioning adult males with ASD. Controls were 17 age-, gender-, parental socioeconomic background-, and intellectual level-matched typically developing (TD) individuals. Statistical analyses determined whether values representing the strength and variability of each facial expression element differed significantly between the ASD and TD groups and whether they correlated with ADOS reciprocal social interaction scores. Compared with the TD controls, facial expressions in the ASD group appeared more "Neutral" (d = 1.02, P = 0.005, PFDR < 0.05) with less variation in Neutral expression (d = 1.08, P = 0.003, PFDR < 0.05). Their expressions were also less "Happy" (d = -0.78, P = 0.038, PFDR > 0.05) with lower variability in Happy expression (d = 1.10, P = 0.003, PFDR < 0.05). Moreover, the stronger Neutral facial expressions in the ASD participants were positively correlated with poorer ADOS reciprocal social interaction scores (ρ = 0.48, P = 0.042). These findings indicate that our method for quantitatively measuring reduced facial expressivity during social interactions can be a promising marker for core ASD social symptoms.

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

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque

PubMed Central

Soares, David; Goldrick, Isabelle; Lemon, Roger N.; Kraskov, Alexander; Greensmith, Linda

2017-01-01

Abstract There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration “thin” spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin‐positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32‐postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. PMID:28213922

Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells

PubMed Central

Jabbari, Esmaiel; Sarvestani, Samaneh K.; Daneshian, Leily; Moeinzadeh, Seyedsina

2015-01-01

Introduction The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells’ tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment. Methods Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers. Results The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm. Conclusion The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells’ tissue origin. PMID:26168187

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.

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/.

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors.

PubMed

Gareau, Tia; Lara, Giovanna G; Shepherd, Robert D; Krawetz, Roman; Rancourt, Derrick E; Rinker, Kristina D; Kallos, Michael S

2014-04-01

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue-engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm(2) ), 60 rpm (3 dyne/cm(2) )] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct-4, Nanog, Sox-2 and Rex-1 were assessed using gene expression profiles and flow-cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright © 2012 John Wiley & Sons, Ltd.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

PubMed

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

PubMed

Kersten, Bas; Valkering, Matthijs; Wouters, Rolf; van Amerongen, Rosa; Hanekamp, Diana; Kwidama, Zinia; Valk, Peter; Ossenkoppele, Gert; Zeijlemaker, Wendelien; Kaspers, Gertjan; Cloos, Jacqueline; Schuurhuis, Gerrit J

2016-04-01

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment. © 2016 John Wiley & Sons Ltd.

Manufacturing porcine islets: culture at 22°C has no advantage above culture at 37°C

PubMed Central

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

Background The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37°C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Methods Porcine islets were isolated from three donors and cultured at 37°C for 1 day, and then under three different conditions: 37°C for 6 days (condition A); 22°C for 6 days (condition B); or 22°C for 5 days followed by 37°C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Results Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129–159 nmol/min.mgDNA before culture, and 259–291, 204–212, and 207–228 nmol/min•mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular most of lymphocyte markers showed a >10-fold reduction and also 6 markers in the lymphokine and chemokine series: these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 hours at 37°C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22°C to those in islets cultured at 37°C. Results were consistent among the preparations from the three donors. Conclusions Culture of porcine islets at 37°C provides benefits over culture at 22°C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. PMID:23941232

Soy consumption and histopathologic markers in breast tissue using tissue microarrays.

PubMed

Maskarinec, Gertraud; Erber, Eva; Verheus, Martijn; Hernandez, Brenda Y; Killeen, Jeffrey; Cashin, Suzanne; Cline, J Mark

2009-01-01

This study examined the relation of soy intake with hormonal and proliferation markers in benign and malignant breast tissue using tissue microarrays (TMAs). TMAs with up to 4 malignant and 4 benign tissue samples for 268 breast cancer cases were constructed. Soy intake in early life and in adulthood was assessed by questionnaire. The TMAs were stained for estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), proliferating cell nuclear antigen (PCNA), and Ki-67 using standard immunohistochemical methods. Logistic regression was applied for statistical analysis. A higher percentage of women showed positive marker expression in malignant than in benign tissue. With one exception, HER2/neu, no significant associations between soy intake and pathologic markers were observed. Early life soy intake was associated with lower HER2/neu and PCNA staining of malignant tissue. In benign tissue, early life soy intake showed higher ER and PR expression, but no difference in proliferation markers. The results of this investigation provide some assurance that soy intake does not adversely affect markers of proliferation. TMAs were shown to be a useful tool for epidemiologic research.

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.

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

PubMed Central

Nakamura, Teppei; Otsuka, Saori; Ichii, Osamu; Sakata, Yuko; Nagasaki, Ken-Ichi; Hashimoto, Yoshiharu; Kon, Yasuhiro

2013-01-01

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown. PMID:24124609

Individuality in harpsichord performance: disentangling performer- and piece-specific influences on interpretive choices

PubMed Central

Gingras, Bruno; Asselin, Pierre-Yves; McAdams, Stephen

2013-01-01

Although a growing body of research has examined issues related to individuality in music performance, few studies have attempted to quantify markers of individuality that transcend pieces and musical styles. This study aims to identify such meta-markers by discriminating between influences linked to specific pieces or interpretive goals and performer-specific playing styles, using two complementary statistical approaches: linear mixed models (LMMs) to estimate fixed (piece and interpretation) and random (performer) effects, and similarity analyses to compare expressive profiles on a note-by-note basis across pieces and expressive parameters. Twelve professional harpsichordists recorded three pieces representative of the Baroque harpsichord repertoire, including three interpretations of one of these pieces, each emphasizing a different melodic line, on an instrument equipped with a MIDI console. Four expressive parameters were analyzed: articulation, note onset asynchrony, timing, and velocity. LMMs showed that piece-specific influences were much larger for articulation than for other parameters, for which performer-specific effects were predominant, and that piece-specific influences were generally larger than effects associated with interpretive goals. Some performers consistently deviated from the mean values for articulation and velocity across pieces and interpretations, suggesting that global measures of expressivity may in some cases constitute valid markers of artistic individuality. Similarity analyses detected significant associations among the magnitudes of the correlations between the expressive profiles of different performers. These associations were found both when comparing across parameters and within the same piece or interpretation, or on the same parameter and across pieces or interpretations. These findings suggest the existence of expressive meta-strategies that can manifest themselves across pieces, interpretive goals, or expressive devices. PMID:24348446

Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

PubMed

Di Giancamillo, Alessia; Deponti, Daniela; Modina, Silvia; Tessaro, Irene; Domeneghini, Cinzia; Peretti, Giuseppe Maria

2017-11-01

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Cryptochrome-1 expression: a new prognostic marker in B-cell chronic lymphocytic leukemia.

PubMed

Lewintre, Eloisa Jantus; Martín, Cristina Reinoso; Ballesteros, Carlos García; Montaner, David; Rivera, Rosa Farrás; Mayans, José Ramón; García-Conde, Javier

2009-02-01

Chronic lymphocytic leukemia is an adult-onset leukemia with a heterogeneous clinical behavior. When chronic lymphocytic leukemia cases were divided on the basis of IgV(H) mutational status, widely differing clinical courses were revealed. Since IgV(H) sequencing is difficult to perform in a routine diagnostic laboratory, finding a surrogate for IgV(H) mutational status seems an important priority. In the present study, we proposed the use of Cryptochrome-1 as a new prognostic marker in early-stage chronic lymphocytic leukemia. Seventy patients (Binet stage A, without treatment) were included in the study. We correlated Cryptochrome-1 mRNA with well established prognostic markers such as IgV(H) mutations, ZAP70, LPL or CD38 expression and chromosomal abnormalities. High Cryptochrome-1 expression correlated with IgV(H) unmutated samples. In addition, Cryptochrome-1 was a valuable predictor of disease progression in early-stage chronic lymphocytic leukemia, therefore it can be introduced in clinical practice with the advantage of a simplified method of quantification.

Characterization of Putative Iron Responsive Genes as Species-Specific Indicators of Iron Stress in Thalassiosiroid Diatoms

PubMed Central

Whitney, LeAnn P.; Lins, Jeremy J.; Hughes, Margaret P.; Wells, Mark L.; Chappell, P. Dreux; Jenkins, Bethany D.

2011-01-01

Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations. PMID:22275908

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

PubMed

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Immunohistochemical Analysis of P63 Expression in Odontogenic Lesions

PubMed Central

Atarbashi Moghadam, Saede; Atarbashi Moghadam, Fazele; Eini, Ebrahim

2013-01-01

P63 may have a role in tumorigenesis and cytodifferentiation of odontogenic lesions. We investigated the immunohistochemical expression of P63 in a total of 30 cases of odontogenic cysts and tumors. The percentage of positive cells was calculated in the lining of odontogenic cysts and islands of ameloblastoma. P63 expression was evident in all types of odontogenic lesions. P63 was expressed throughout the lining epithelium of odontogenic keratocyst except surface parakeratinized layer. In addition, calcifying odontogenic cyst showed P63 expression in all layers. In almost all radicular and dentigerous cysts, the basal and parabasal layers were immunoreactive. Peripheral cells of ameloblastoma expressed P63; however, stellate reticulum had weaker immunostaining. No significant difference in P63 expression was observed between studied lesions (P = 0.86). Expression of P63 in odontogenic lesions suggests that this protein is important in differentiation and proliferation of odontogenic epithelial cells. However, it seems that it could not be a useful marker to differentiate between aggressive and nonaggressive lesions. P63 also represents a progenitor or basal cell marker, and it is not expressed in mature differentiated cells. PMID:24350278

Manufacturing porcine islets: culture at 22 °C has no advantage above culture at 37 °C: a gene expression evaluation.

PubMed

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors. Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. © 2013 John Wiley & Sons A/S.

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.

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.

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

McLetchie, D.N.; Tuskan, G.A.

Gender, the expression of maleness or femaleness, in dioecious plants has been associated with changes in morphology, physiology, ecological position, and commercial importance of several species, including members of the Salicaceae family. Various mechanisms have been proposed to explain the expression of gender in Salicaceae, including sex chromosomes, simple Mendelian genes, quantitative genes, environment, and genotype-by-environment interactions. Published reports would favor a genetic basis for gender. The objective of this study was to identify molecular markers associated with gender in a segregating family of hybrid poplars. Bulked segregant analysis and chi-squared analysis were used to test for the occurrence ofmore » sex chromosomes, individual loci, and chromosome ratios (i.e., ploidy levels) as the mechanisms for gender determination. Examination of 2488 PCR based RAPD markers from 1219 primers revealed nine polymorphic bands between male and female bulked samples. However, linkage analysis indicated that none of these markers were significantly associated with gender. Chisquared results for difference in male-to-female ratios between diploid and triploid genotypes also revealed no significant differences. These findings suggest gender is not controlled via sex chromosomes, simple Mendelian loci or ratios of autosome to gender-determining loci. It is possible that gender is determined genetically by regions of the genome not sampled by the tested markers or by a complex of loci operating in an additive threshold manner or in an epistatic manner. It is also possible that gender is determined environmentally at an early zygote stage, canalizing gender expression.« less

Biomarkers in systemic juvenile idiopathic arthritis: a comparison with biomarkers in cryopyrin-associated periodic syndromes.

PubMed

Nirmala, Nanguneri; Grom, Alexei; Gram, Hermann

2014-09-01

This review summarizes biomarkers in systemic juvenile idiopathic arthritis (sJIA). Broadly, the markers are classified under protein, cellular, gene expression and genetic markers. We also compare the biomarkers in sJIA to biomarkers in cryopyrin-associated periodic syndrome (CAPS). Recent publications showing the similarity of clinical response of sJIA and CAPS to anti-interleukin 1 therapies prompted a comparison at the biomarker level. sJIA traditionally is classified under the umbrella of juvenile idiopathic arthritis. At the clinical phenotypic level, sJIA has several features that are more similar to those seen in CAPS. In this review, we summarize biomarkers in sJIA and CAPS and draw upon the various similarities and differences between the two families of diseases. The main differences between sJIA and CAPS biomarkers are genetic markers, with CAPS being a family of monogenic diseases with mutations in NLRP3. There have been a small number of publications describing cellular biomarkers in sJIA with no such studies described for CAPS. Many of the protein marker's characteristics of sJIA are also seen to characterize CAPS. The gene expression data in both sJIA and CAPS show a strong upregulation of innate immunity pathways. In addition, we describe a strong similarity between sJIA and CAPS at the gene expression level in which several genes that form a part of the erythropoiesis signature are upregulated in both sJIA and CAPS.

Biomarkers in Systemic Juvenile Idiopathic Arthritis: A comparison with biomarkers in Cryopyrin Associated Periodic Syndromes

PubMed Central

Nirmala, Nanguneri; Grom, Alexei; Gram, Hermann

2015-01-01

Purpose of review This review summarizes biomarkers in Systemic Juvenile Idiopathic Arthritis (sJIA). Broadly, the markers are classified under protein, cellular, gene expression and genetic markers. We also compare the biomarkers in sJIA to biomarkers in cryopyrin associated periodic syndromes (CAPS). Recent findings Recent publications showing the similarity of clinical response of sJIA and CAPS to anti IL1 therapies prompted a comparison at the biomarker level. Summary sJIA traditionally is classified under the umbrella of juvenile idiopathic arthritis. At the clinical phenotypic level, sJIA has several features that are more similar to those seen in Cryopyrin Associated Periodic Syndromes (CAPS). In this review, we summarize biomarkers in sJIA and CAPS and draw upon the various similarities and differences between the two families of diseases. The main difference between sJIA and CAPS biomarkers are genetic markers with CAPS being a family of monogenic diseases with mutations in NLRP3. There have been a small number of publications describing cellular biomarkers in sJIA with no such studies described for CAPS. Many of the protein markers characteristic of sJIA are also seen to characterize CAPS. The gene expression data in both sJIA and CAPS show a strong upregulation of innate immunity pathways. In addition, we describe a strong similarity between sJIA and CAPS at the gene expression level where several genes that form a part of the erythropoiesis signature are upregulated in both sJIA and CAPS. PMID:25050926

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.

Accuracy of microRNAs as markers for the detection of neck lymph node metastases in patients with head and neck squamous cell carcinoma.

PubMed

de Carvalho, Ana Carolina; Scapulatempo-Neto, Cristovam; Maia, Danielle Calheiros Campelo; Evangelista, Adriane Feijó; Morini, Mariana Andozia; Carvalho, André Lopes; Vettore, André Luiz

2015-05-09

The presence of metastatic disease in cervical lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients is a very important determinant in therapy choice and prognosis, with great impact in overall survival. Frequently, routine lymph node staging cannot detect occult metastases and the post-surgical histologic evaluation of resected lymph nodes is not sensitive in detecting small metastatic deposits. Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers. Herein, we evaluated the feasibility of using the expression of microRNAs to detect metastatic cells in formalin-fixed paraffin-embedded (FFPE) lymph nodes and in fine-needle aspiration (FNA) biopsies of HNSCC patients. An initial screening compared the expression of 667 microRNAs in a discovery set comprised by metastatic and non-metastatic lymph nodes from HNSCC patients. The most differentially expressed microRNAs were validated by qRT-PCR in two independent cohorts: i) 48 FFPE lymph node samples, and ii) 113 FNA lymph node biopsies. The accuracy of the markers in identifying metastatic samples was assessed through the analysis of sensitivity, specificity, accuracy, negative predictive value, positive predictive value, and area under the curve values. Seven microRNAs highly expressed in metastatic lymph nodes from the discovery set were validated in FFPE lymph node samples. MiR-203 and miR-205 identified all metastatic samples, regardless of the size of the metastatic deposit. Additionally, these markers also showed high accuracy when FNA samples were examined. The high accuracy of miR-203 and miR-205 warrant these microRNAs as diagnostic markers of neck metastases in HNSCC. These can be evaluated in entire lymph nodes and in FNA biopsies collected at different time-points such as pre-treatment samples, intraoperative sentinel node biopsy, and during patient follow-up. These markers can be useful in a clinical setting in the management of HNSCC patients from initial disease staging and therapy planning to patient surveillance.

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.

Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

PubMed

Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

2014-04-01

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

Identification of innovative potential quality markers in rocket and melon fresh-cut produce.

PubMed

Cavaiuolo, Marina; Cocetta, Giacomo; Bulgari, Roberta; Spinardi, Anna; Ferrante, Antonio

2015-12-01

Ready-to-eat fresh cut produce are exposed to pre- and postharvest abiotic stresses during the production chain. Our work aimed to identify stress responsive genes as new molecular markers of quality that can be widely applied to leaves and fruits and easily determined at any stage of the production chain. Stress responsive genes associated with quality losses were isolated in rocket and melon fresh-cut produce and their expression levels analyzed by quantitative real time PCR (qRT-PCR) at different time points after harvest at 20 °C and 4 °C. qRT-PCR results were supported by correlation analysis with physiological and biochemical determinations evaluated at the same conditions such as chlorophyll a fluorescence indices, total, reducing sugars, sucrose, ethylene, ascorbic acid, lipid peroxidation and reactive oxygen species. In both species the putative molecular markers increased their expression soon after harvest suggesting a possible use as novel and objective quality markers of fresh-cut produces. Copyright © 2015 Elsevier Ltd. All rights reserved.

[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.

Use of CD25 as an immunohistochemical marker for acquired ocular toxoplasmosis.

PubMed

Miyamoto, Cristina; Mattos Neto, Rubens Belfort; Cesare, Sebastian Di; Belfort Junior, Rubens; Burnier, Miguel N

2010-01-01

Toxoplasmosis is the most common cause of posterior infectious uveitis worldwide. It is often impossible to determine its congenital or acquired nature. Interleukin-2 (IL-2) in peripheral blood has been described as a possible marker for acquired toxoplasmosis. The purpose of this study is to evaluate the histopathological characteristics of ocular toxoplasmosis cases using CD25 as a marker for the expression of interleukin-2. Ten formalin-fixed, paraffin-embedded enucleated globes from ten immunocompetent patients with clinical diagnosis of toxoplasmosis were evaluated. Four patients had the acquired form of ocular toxoplasmosis (positive IgM) while six were IgM negative and IgG positive for toxoplasmosis. Histopathological slides were reviewed for the extension of the retinal necrosis, number of toxo cysts, the granulomatous inflammatory reaction, the presence of T and B cells within the choroid and the IL-2 expression. Immunohistochemistry using monoclonal antibodies was performed to observe the expression of CD4, CD8, CD20, CD25, and CD68. The histopathological evaluation disclosed no differences between acquired and the other ocular toxoplasmosis cases regarding the characteristics studied. However, CD25 showed a higher expression of IL-2 on the 4 acquired cases of ocular toxoplasmosis compared to the remainders. To the best of our knowledge, this is the first report showing that the use of CD25 as a marker for interleukin-2 could differentiate acquired ocular toxoplasmosis.

Markers of epithelial-to-mesenchymal transition reflect tumor biology according to patient age and Gleason score in prostate cancer

PubMed Central

Jędroszka, Dorota; Hamouz, Raneem; Górniak, Karolina; Bednarek, Andrzej K.

2017-01-01

Introduction Prostate carcinoma (PRAD) is one of the most frequently diagnosed malignancies amongst men worldwide. It is well-known that androgen receptor (AR) plays a pivotal role in a vast majority of prostate tumors. However, recent evidence emerged stating that estrogen receptors (ERs) may also contribute to prostate tumor development. Moreover, progression and aggressiveness of prostate cancer may be associated with differential expression genes of epithelial-to-mesenchymal transition (EMT). Therefore we aimed to assess the significance of receptors status as well as EMT marker genes expression among PRAD patients in accordance to their age and Gleason score. Materials and methods We analyzed TCGA gene expression profiles of 497 prostate tumor samples according to 43 genes involved in EMT and 3 hormone receptor genes (AR, ESR1, ESR2) as well as clinical characteristic of cancer patients. Then patients were divided into four groups according to their age and 5 groups according to Gleason score. Next, we evaluated PRAD samples according to relationship between the set of variables in different combinations and compared differential expression in subsequent groups of patients. The analysis was applied using R packages: FactoMineR, gplots, RColorBrewer and NMF. Results MFA analysis resulted in distinct grouping of PRAD patients into four age categories according to expression level of AR, ESR1 and ESR2 with the most distinct group of age less than 50 years old. Further investigations indicated opposite expression profiles of EMT markers between different age groups as well as strong association of EMT gene expression with Gleason score. We found that depending on age of prostate cancer patients and Gleason score EMT genes with distinctly altered expression are: KRT18, KRT19, MUC1 and COL4A1, CTNNB1, SNAI2, ZEB1 and MMP3. Conclusions Our major observation is that prostate cancer from patients under 50 years old compared to older ones has entirely different EMT gene expression profiles showing potentially more aggressive invasive phenotype, despite Gleason score classification. PMID:29206234

Markers of epithelial-to-mesenchymal transition reflect tumor biology according to patient age and Gleason score in prostate cancer.

PubMed

Jędroszka, Dorota; Orzechowska, Magdalena; Hamouz, Raneem; Górniak, Karolina; Bednarek, Andrzej K

2017-01-01

Prostate carcinoma (PRAD) is one of the most frequently diagnosed malignancies amongst men worldwide. It is well-known that androgen receptor (AR) plays a pivotal role in a vast majority of prostate tumors. However, recent evidence emerged stating that estrogen receptors (ERs) may also contribute to prostate tumor development. Moreover, progression and aggressiveness of prostate cancer may be associated with differential expression genes of epithelial-to-mesenchymal transition (EMT). Therefore we aimed to assess the significance of receptors status as well as EMT marker genes expression among PRAD patients in accordance to their age and Gleason score. We analyzed TCGA gene expression profiles of 497 prostate tumor samples according to 43 genes involved in EMT and 3 hormone receptor genes (AR, ESR1, ESR2) as well as clinical characteristic of cancer patients. Then patients were divided into four groups according to their age and 5 groups according to Gleason score. Next, we evaluated PRAD samples according to relationship between the set of variables in different combinations and compared differential expression in subsequent groups of patients. The analysis was applied using R packages: FactoMineR, gplots, RColorBrewer and NMF. MFA analysis resulted in distinct grouping of PRAD patients into four age categories according to expression level of AR, ESR1 and ESR2 with the most distinct group of age less than 50 years old. Further investigations indicated opposite expression profiles of EMT markers between different age groups as well as strong association of EMT gene expression with Gleason score. We found that depending on age of prostate cancer patients and Gleason score EMT genes with distinctly altered expression are: KRT18, KRT19, MUC1 and COL4A1, CTNNB1, SNAI2, ZEB1 and MMP3. Our major observation is that prostate cancer from patients under 50 years old compared to older ones has entirely different EMT gene expression profiles showing potentially more aggressive invasive phenotype, despite Gleason score classification.

Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.

PubMed

Kumar, S; Arul, L; Talwar, D

2010-01-01

We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

PubMed Central

Guallar, D.; Pérez-Palacios, R.; Climent, M.; Martínez-Abadía, I.; Larraga, A.; Fernández-Juan, M.; Vallejo, C.; Muniesa, P.; Schoorlemmer, J.

2012-01-01

Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription. PMID:22844087

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.

IGF-IEc expression is increased in secondary compared to primary foci in neuroendocrine neoplasms.

PubMed

Alexandraki, Krystallenia I; Philippou, Anastassios; Boutzios, Georgios; Theohari, Irini; Koutsilieris, Michael; Delladetsima, Ioanna Kassiani; Kaltsas, Gregory A

2017-10-03

Different Insulin-like growth factor-I (IGF-I) mRNA transcripts are produced by alternative splicing and particularly the IGF-IEc isoform has been implicated in the development and/or progression of various types of cancer. In the present study, we examined the potential role of IGF-IEc expression as a new immunohistochemical marker of aggressiveness in neuroendocrine neoplasms (NENs). We utilized immunohistochemical analysis in tissue specimens of 47 patients with NENs, to evaluate the expression of IGF-IEc (%) and Ki-67 proliferation index (%). Specimens from patients with tumors of different tissue origin, of either primary or metastatic lesions and of different grade were examined. Cytoplasmic IGF-IEc staining was found in 23 specimens of NENs or NECs: 10 pancreatic, 4 small bowel, 3 gastric, 1 lung, 1 uterine and 4 poorly differentiated of unknown primary origin. Ki-67 and IGF-IEc expression was positively correlated in all the samples studied (r=0.31, p=0.03). IGF-1Ec expression was more prevalent in specimens originating from metastatic foci with high Ki-67 compared to primary sites with low Ki-67 expression (p=0.036). These findings suggest a possible role of IGF-IEc in NEN tumorigenesis and progression to metastases that could be used as an additional new marker of a more aggressive behavior and a potential drugable target.

[Regulation of moxibustion for expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer].

PubMed

Yang, Zong-Bao; Wang, Chen-Guang; Gong, An; Xie, Yu-feng; Liu, Qiong; Yang, Qing

2013-11-01

To explore relevant material basis of moxibustion for recovering gastric mucosal lesion. METHODL Forty-five SD rats were randomly divided into a normal goup, a model group, an acupoint group and a control group, 15 rats in the model group and 10 rats in the rest three groups. Except the normal group, binding and cold stress method were used to establish gastric mucosa injury model. The suspended moxibustion was applied in the acupoint group and control group at acupoints of the stomach meridian ("Liangmen" (ST 21) and "Zusanli" (ST36) and control acupoints (Laterally 1cm next to the "Liangmen" (ST 21) and Zusanli" (ST36), once a day, consectutively for 12 days. After 12 days, morphology of gastric mucosal was observed under optical microscope; protein fingerprints of gastric mucosa cell in rats were detected by protein fingerprint technology, weak cation chip and weak anion chip. Also mass to charge ratio of differential proteins in groups were compared and analyzed. Compared with the model group, index of gastric mucosal lesion in the acupoint group was reduced and its morphology was obviously improved (P<0.05). Campared with control group, index and morphology of gastric mucosal lesion were significantly improved in the acupoint group (P<0.05). According to test of weak cation chip, there was four marker proteins that had expression differences, indicating moxibustion at acupoints of stomach meridian could inrease expression of three marker protein whose molecular weight was 1354Da, 5692Da and 8432Da (all P<0.05) while reduce expression of marker protein with molecular weight of 3287Da (_<0.05). According to test of weak anion chip, moxibustion at acupoints of stomach meridian could increase expression of three marker proteins whose molecular weight was 2412 Da, 3026Da and 6475 Da (allP<0.05). Moxibustion at acupoints of the stomach meridian could regulate differential expression of gastric mucosa cell-related marker protein in rats with acute gastric ulcer and recover gastric mucosal lesion, it's effect is better than that of the points of laterally 1 cm next to acupoint.

Annexin A1: A new immunohistological marker of cholangiocarcinoma

PubMed Central

Hongsrichan, Nuttanan; Rucksaken, Rucksak; Chamgramol, Yaovalux; Pinlaor, Porntip; Techasen, Anchalee; Yongvanit, Puangrat; Khuntikeo, Narong; Pairojkul, Chawalit; Pinlaor, Somchai

2013-01-01

AIM: To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). METHODS: Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N-nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA. RESULTS: In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-κB gene expression. CONCLUSION: ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis. PMID:23674846

Testin (TES) as a candidate tumour suppressor and prognostic marker in human astrocytoma.

PubMed

Steponaitis, Giedrius; Kazlauskas, Arunas; Skiriute, Daina; Valiulyte, Indre; Skauminas, Kestutis; Tamasauskas, Arimantas; Vaitkiene, Paulina

2016-11-01

Astrocytomas are one of the most common brain tumours; however, the current methods used to characterize these tumours are inadequate. The establishment of molecular markers may identify variables required to improve tumour characterization and subtyping, and may aid to specify targets for improved treatment with essential prognostic value for patient survival. One such candidate is testin (TES), which was reported to have prognostic value for glioblastoma patients. However, the role of TES protein in gliomagenesis is currently unknown. In the present study, the methylation status of the TES promoter was investigated in post-operative astrocytoma tumours of different malignancy grade, and its association with the survival of astrocytoma patients was evaluated. In addition, the expression of TES protein was investigated in the same set of astrocytoma tumours tissue, and the association of protein expression with glioma patients survival was evaluated. The methylation status of TES was assessed by methylation-specific polymerase chain reaction in 138 different grade astrocytoma samples. Western blot analysis was used to characterize the expression pattern of TES in 86 different grade astrocytoma specimens: 13 of pathological grade I, 31 of pathological grade II, 17 of pathological grade III and 25 of pathological grade IV (glioblastoma). Statistical analyses were conducted to investigate the association between tumour molecular pattern, patient clinical variables and overall survival. The methylation analysis of the TES promoter exhibited a distinct profile between astrocytomas of different malignancy grade (P<0.001). Furthermore, gene promoter methylation was significantly associated with patients' age, survival and pathological grade (P<0.001). The protein expression level of TES was significantly lower in glioblastoma (grade IV astrocytoma) than in lower grade (II-III) astrocytoma tissue (P=0.028 and P=0.04, respectively). Additionally, short overall survival of patients was markedly associated with low TES protein expression (P=0.007). However, no association between TES methylation and TES protein expression was noticed. The present study demonstrated that decreased expression of TES may be important in tumour progression and prognosis in human astrocytomas. TES may be a useful marker for predicting the clinical outcome of astrocytoma patients.

Testin (TES) as a candidate tumour suppressor and prognostic marker in human astrocytoma

PubMed Central

Steponaitis, Giedrius; Kazlauskas, Arunas; Skiriute, Daina; Valiulyte, Indre; Skauminas, Kestutis; Tamasauskas, Arimantas; Vaitkiene, Paulina

2016-01-01

Astrocytomas are one of the most common brain tumours; however, the current methods used to characterize these tumours are inadequate. The establishment of molecular markers may identify variables required to improve tumour characterization and subtyping, and may aid to specify targets for improved treatment with essential prognostic value for patient survival. One such candidate is testin (TES), which was reported to have prognostic value for glioblastoma patients. However, the role of TES protein in gliomagenesis is currently unknown. In the present study, the methylation status of the TES promoter was investigated in post-operative astrocytoma tumours of different malignancy grade, and its association with the survival of astrocytoma patients was evaluated. In addition, the expression of TES protein was investigated in the same set of astrocytoma tumours tissue, and the association of protein expression with glioma patients survival was evaluated. The methylation status of TES was assessed by methylation-specific polymerase chain reaction in 138 different grade astrocytoma samples. Western blot analysis was used to characterize the expression pattern of TES in 86 different grade astrocytoma specimens: 13 of pathological grade I, 31 of pathological grade II, 17 of pathological grade III and 25 of pathological grade IV (glioblastoma). Statistical analyses were conducted to investigate the association between tumour molecular pattern, patient clinical variables and overall survival. The methylation analysis of the TES promoter exhibited a distinct profile between astrocytomas of different malignancy grade (P<0.001). Furthermore, gene promoter methylation was significantly associated with patients' age, survival and pathological grade (P<0.001). The protein expression level of TES was significantly lower in glioblastoma (grade IV astrocytoma) than in lower grade (II–III) astrocytoma tissue (P=0.028 and P=0.04, respectively). Additionally, short overall survival of patients was markedly associated with low TES protein expression (P=0.007). However, no association between TES methylation and TES protein expression was noticed. The present study demonstrated that decreased expression of TES may be important in tumour progression and prognosis in human astrocytomas. TES may be a useful marker for predicting the clinical outcome of astrocytoma patients. PMID:27899997

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

Analysis of chronic lymphotic leukemia transcriptomic profile: differences between molecular subgroups.

PubMed

Jantus Lewintre, Eloisa; Reinoso Martín, Cristina; Montaner, David; Marín, Miguel; José Terol, María; Farrás, Rosa; Benet, Isabel; Calvete, Juan J; Dopazo, Joaquín; García-Conde, Javier

2009-01-01

B cell chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder with a variable clinical course. Patients with unmutated IgV(H) gene show a shorter progression-free and overall survival than patients with immunoglobulin heavy chain variable regions (IgV(H)) gene mutated. In addition, BCL6 mutations identify a subgroup of patients with high risk of progression. Gene expression was analysed in 36 early-stage patients using high-density microarrays. Around 150 genes differentially expressed were found according to IgV(H) mutations, whereas no difference was found according to BCL6 mutations. Functional profiling methods allowed us to distinguish KEGG and gene ontology terms showing coordinated gene expression changes across subgroups of CLL. We validated a set of differentially expressed genes according to IgV(H) status, scoring them as putative prognostic markers in CLL. Among them, CRY1, LPL, CD82 and DUSP22 are the ones with at least equal or superior performance to ZAP70 which is actually the most used surrogate marker of IgV(H) status.

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

PubMed

De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

2013-01-01

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

Diverse effects of lead nitrate on the proliferation, differentiation, and gene expression of stem cells isolated from a dental origin.

PubMed

Abdullah, Mariam; Rahman, Fazliny Abd; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Abu Kasim, Noor Hayaty; Musa, Sabri

2014-01-01

Lead (Pb(2+)) exposure continues to be a significant public health problem. Therefore, it is vital to have a continuous epidemiological dataset for a better understanding of Pb(2+) toxicity. In the present study, we have exposed stem cells isolated from deciduous and permanent teeth, periodontal ligament, and bone marrow to five different types of Pb(2+) concentrations (160, 80, 40, 20, and 10 µM) for 24 hours to identify the adverse effects of Pb(2+) on the proliferation, differentiation, and gene expression on these cell lines. We found that Pb(2+) treatment altered the morphology and adhesion of the cells in a dose-dependent manner. There were no significant changes in terms of cell surface phenotypes. Cells exposed to Pb(2+) continued to differentiate into chondrogenesis and adipogenesis, and a severe downregulation was observed in osteogenesis. Gene expression studies revealed a constant expression of key markers associated with stemness (Oct 4, Rex 1) and DNA repair enzyme markers, but downregulation occurred with some ectoderm and endoderm markers, demonstrating an irregular and untimely differentiation trail. Our study revealed for the first time that Pb(2+) exposure not only affects the phenotypic characteristics but also induces significant alteration in the differentiation and gene expression in the cells.

Protein markers for identification of Yersinia pestis and their variation related to culture

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

Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.

2013-12-11

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y.more » pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.« less

Regulation of EMMPRIN (CD147) on monocyte subsets in patients with symptomatic coronary artery disease.

PubMed

Sturhan, Henrik; Ungern-Sternberg, Saskia N I v; Langer, Harald; Gawaz, Meinrad; Geisler, Tobias; May, Andreas E; Seizer, Peter

2015-06-01

The role of individual monocyte subsets in inflammatory cardiovascular diseases is insufficiently understood. Although the Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) regulates important processes for inflammation such as MMP-release, its expression and regulation on monocyte subsets has not been characterized. In this clinical study, blood was obtained from 80 patients with stable coronary artery disease (CAD), 49 with acute myocardial infarction (AMI) and 34 healthy controls. Monocytes were divided into 3 subsets: CD14(++)CD16(-) (low), CD14(++)CD16(+) (intermediate), CD14(+)CD16(++) (high) according to phenotypic markers analyzed by flow cytometry. Surface expression of EMMPRIN was evaluated and compared with CD36 and CD47 expression. In all patients, EMMPRIN expression was significantly different among monocyte subsets with the highest expression on "classical" CD14(++)CD16(-) monocytes. EMMPRIN was upregulated on all monocyte subsets in patients with AMI as compared to patients with stable CAD. Notably, neither CD47 nor CD36 revealed a significant difference in patients with AMI compared to patients with stable CAD. EMMPRIN could serve as a marker for classical monocytes, which is upregulated in patients with acute myocardial infarction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Training Performed Above Lactate Threshold Decreases p53 and Shelterin Expression in Mice.

PubMed

de Carvalho Cunha, Verusca Najara; Dos Santos Rosa, Thiago; Sales, Marcelo Magalhães; Sousa, Caio Victor; da Silva Aguiar, Samuel; Deus, Lysleine Alves; Simoes, Herbert Gustavo; de Andrade, Rosangela Vieira

2018-06-26

Telomere shortening is associated to sarcopenia leading to functional impairment during aging. There are mechanisms associated with telomere attrition, as well to its protection and repair. Physical training is a factor that attenuates telomere shortening, but little is known about the effects of different exercise intensities on telomere biology. Thus, we evaluated the effects of exercise intensity (moderate vs. high-intensity domain) on gene expression of senescence markers Checkpoint kinase 2 and tumor suppressor ( Chk2 and p53 , respectively), shelterin telomere repeat binding 1 and 2 ( Trf1 / Trf2 ), DNA repair ( Xrcc5 ), telomerase reverse transcriptase ( mTERT ) and telomere length in middle aged mice. Three groups were studied: a control group (CTL) and two groups submitted to swimming at intensities below the lactate threshold (LI group) and above the lactate threshold (HI group) for 40 and 20 min respectively, for 12 weeks. After training, the HI group showed reduction in p53 expression in the muscle, and decreased shelterin complex expression when compared to LI group. No differences were observed between groups for mTERT expression and telomere length. Thus, exercise training in high-intensity domain was more effective on reducing markers of senescence and apoptosis. The higher intensity exercise training also diminished shelterin expression, with no differences in telomere length and mTERT expression. Such results possibly indicate a more effective DNA protection for the higher-intensity exercise training. © Georg Thieme Verlag KG Stuttgart · New York.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

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

Kaseb, Hatem O.; Department of Clinical Pathology, National Cancer Institute; Fohrer-Ting, Helene

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumormore » cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. - Highlights: • Spheroid enrichment selects cancer stem cells (CSC) from head & neck tumors (HNSCC). • Compared to normal epithelial cells, isolated CSC express increased SC/CSC markers. • Isolated CSC display enhanced radioresistance, clonogenicity and tumorigenicity. • HNSCC CSC express chromosomal instability. • CD44+/CD66− is a promising, consistent biomarker for HNSCC CSC.« less

Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays.

PubMed Central

Baker, Valerie A; Harries, Helen M; Waring, Jeff F; Duggan, Colette M; Ni, Hong A; Jolly, Robert A; Yoon, Lawrence W; De Souza, Angus T; Schmid, Judith E; Brown, Roger H; Ulrich, Roger G; Rockett, John C

2004-01-01

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. PMID:15033592

Temporal relationship of serum markers and tissue damage during acute intestinal ischemia/reperfusion

PubMed Central

la Garza, Francisco Javier Guzmán-de; Ibarra-Hernández, Juan Manuel; Cordero-Pérez, Paula; Villegas-Quintero, Pablo; Villarreal-Ovalle, Claudia Ivette; Torres-González, Liliana; Oliva-Sosa, Norma Edith; Alarcón-Galván, Gabriela; Fernández-Garza, Nancy Esthela; Muñoz-Espinosa, Linda Elsa; Cámara-Lemarroy, Carlos Rodrigo; Carrillo-Arriaga, José Gerardo

2013-01-01

OBJECTIVE: It is essential to identify a serological marker of injury in order to study the pathophysiology of intestinal ischemia reperfusion. In this work, we studied the evolution of several serological markers after intestinal ischemia reperfusion injury in rats. The markers of non-specific cell damage were aspartate aminotransferase, alanine aminotransaminase, and lactic dehydrogenase, the markers of inflammation were tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta, and the markers of intestinal mucosal damage were intestinal fatty acid binding protein and D-lactate. We used Chiús classification to grade the histopathological damage. METHODS: We studied 35 Wistar rats divided into groups according to reperfusion time. The superior mesenteric artery was clamped for 30 minutes, and blood and biopsies were collected at 1, 3, 6, 12, 24, and 48 hours after reperfusion. We plotted the mean ± standard deviation and compared the baseline and maximum values for each marker using Student's t-test. RESULTS: The maximum values of interleukin-1 beta and lactic dehydrogenase were present before the maximal histopathological damage. The maximum tumor necrosis factor alpha and D-lactate expressions coincided with histopathological damage. Alanine aminotransaminase and aspartate aminotransferase had a maximum expression level that increased following the histopathological damage. The maximum expressions of interluken-6 and intestinal fatty acid binding protein were not significantly different from the Sham treated group. CONCLUSION: For the evaluation of injury secondary to acute intestinal ischemia reperfusion with a 30 minute ischemia period, we recommend performing histopathological grading, quantification of D-lactate, which is synthesized by intestinal bacteria and is considered an indicator of mucosal injury, and quantification of tumor necrosis factor alpha as indicators of acute inflammation three hours after reperfusion. PMID:23917671

The expression ratio of Map7/B2M is prognostic for survival in patients with stage II colon cancer.

PubMed

Blum, Craig; Graham, Amanda; Yousefzadeh, Matt; Shrout, Jessica; Benjamin, Katie; Krishna, Murli; Hoda, Raza; Hoda, Rana; Cole, David J; Garrett-Mayer, Elizabeth; Reed, Carolyn; Wallace, Michael; Mitas, Michael

2008-09-01

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative real-time RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making.

The expression ratio of Map7/B2M is prognostic for survival in patients with stage II colon cancer

PubMed Central

BLUM, CRAIG; GRAHAM, AMANDA; YOUSEFZADEH, MATT; SHROUT, JESSICA; BENJAMIN, KATIE; KRISHNA, MURLI; HODA, RAZA; HODA, RANA; COLE, DAVID J.; GARRETT-MAYER, ELIZABETH; REED, CAROLYN; WALLACE, MICHAEL; MITAS, MICHAEL

2012-01-01

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative real-time RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making. PMID:18695889

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

Patterns of expression of position-dependent integrated transgenes in mouse embryo.

PubMed Central

Bonnerot, C; Grimber, G; Briand, P; Nicolas, J F

1990-01-01

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution. Images PMID:1696727

Primate Short-Wavelength Cones Share Molecular Markers with Rods

PubMed Central

Craft, Cheryl M.; Huang, Jing; Possin, Daniel E.; Hendrickson, Anita

2015-01-01

Macaca, Callithrix jacchus marmoset monkey, Pan troglodytes chim- panzee and human retinas were examined to define if short wavelength (S) cones share molecular markers with L&M cone or rod photoreceptors. S cones showed consistent differences in their immunohistochemical staining and expression levels compared to L&M cones for “rod” Arrestin1 (S-Antigen), “cone” Arrestin4, cone alpha transducin, and Calbindin. Our data verify a similar pattern of expression in these primate retinas and provide clues to the structural divergence of rods and S cones versus L&M cones, suggesting S cone retinal function is “intermediate” between them. PMID:24664680

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

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

PubMed

Kurucz, Eva; Váczi, B; Márkus, R; Laurinyecz, Barbara; Vilmos, P; Zsámboki, J; Csorba, Kinga; Gateff, Elisabeth; Hultmark, D; Andó, I

2007-01-01

We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance

PubMed Central

Corona-Meraz, Fernanda-Isadora; Navarro-Hernández, Rosa-Elena; Ruíz-Quezada, Sandra-Luz; Madrigal-Ruíz, Perla-Monserrat; Castro-Albarrán, Jorge; Chavarría-Ávila, Efraín; Guzmán-Ornelas, Milton-Omar; Gómez-Bañuelos, Eduardo; Petri, Marcelo-Herón; Ramírez-Cedano, Joel-Isidro; Aguilar-Aldrete, María-Elena; Ríos-Ibarra, Clara; Vázquez-Del Mercado, Mónica

2016-01-01

Background. In obesity there is a subclinical chronic low-grade inflammatory response where insulin resistance (IR) may develop. Chemerin is secreted in white adipose tissue and promotes low-grade inflammatory process, where it expressed CMKLR1 receptor. The role of chemerin and CMKLR1 in inflammatory process secondary to obesity is not defined yet. Methods. Cross-sectional study with 134 individuals classified as with and without obesity by body mass index (BMI) and IR. Body fat storage measurements and metabolic and inflammatory markers were measured by routine methods. Soluble chemerin and basal levels of insulin by ELISA and relative expression of CMKLR1 were evaluated with qPCR and 2−ΔΔCT method. Results. Differences (P < 0.05) were observed between obesity and lean individuals in body fat storage measurements and metabolic-inflammatory markers. Both CMKLR1 expression and chemerin levels were increased in obesity without IR. Soluble chemerin levels correlate with adiposity and metabolic markers (r = 8.8% to 38.5%), P < 0.05. Conclusion. The increment of CMKLR1 expression was associated with insulin production. Increased serum levels of chemerin in obesity were observed, favoring a dysmetabolic response. The results observed in this study suggest that both chemerin and CMKLR1 have opposite expression in the context of low-grade inflammatory response manifested in the development of IR. PMID:27239101

Comparison of the value of PCNA and Ki-67 as markers of cell proliferation in ameloblastic tumor

PubMed Central

Mosqueda-Taylor, Adalberto; Molina-Frechero, Nelly; Mori-Estevez, Ana D.; Sánchez-Acuña, Guillermo

2013-01-01

Objectives: The aim of this study was to compare among PCNAand Ki-67 as the most reliable immunohistochemical marker for evaluating cell proliferation in ameloblastic tumors. Study Design: Observational, retrospective, and descriptive study of a large series of ameloblastic tumors, composed of 161 ameloblastomas and four ameloblastic carcinomas, to determine and compare PCNA and Ki-67 expression using immunohistochemistry techniques. Results: When analyzing Ki-67 positivity, the desmoplastic ameloblastoma demonstrated a significantly lower proliferation rate (1.9%) compared with the solid/multicystic and unicystic ameloblastomas and ameloblastic carcinomas (p<0.05), whereas the ameloblastic carcinomas displayed a significantly higher rate compared with all of the other ameloblastomas (48.7%) (p<0.05). When analyzing cell proliferation with PCNA, we found significant differences only between the ameloblastic carcinomas (93.3%) and the desmoplastic ameloblastomas (p<0.05). When differences between the immunopositivity for PCNA and Ki-67 were compared, the percentages were higher for PCNA in all types of ameloblastomas and ameloblastic carcinomas. In all cases, the percentages were greater than 80%, whereas the immunopositivity for Ki-67 was significantly lower; for example, the ameloblastic carcinoma expressed the highest positivity and only reached 48.7%, compared to 93.3% when we used PCNA. Conclusions: In the present study, when we used the proliferation cell marker Ki-67, the percentages of positivity were more specific and varied among the different types of ameloblastomas, suggesting that Ki-67 is a more specific marker for the proliferation of ameloblastic tumor cells. Key words:Ameloblastomas, ameloblastic carcinoma, PCNA, Ki-67, cell proliferation markers. PMID:23229269

Similarities and Differences between Porcine Mandibular and Limb Bone Marrow Mesenchymal Stem Cells

PubMed Central

Lloyd, Brandon; Tee, Boon Ching; Headley, Colwyn; Emam, Hany; Mallery, Susan; Sun, Zongyang

2017-01-01

Objective Research has shown promise of using bone marrow mesenchymal stem cells (BMSCs) for craniofacial bone regeneration; yet little is known about the differences of BMSCs from limb and craniofacial bones. This study compared pig mandibular and tibia BMSCs for their in vitro proliferation, osteogenic differentiation properties and gene expression. Design Bone marrow was aspirated from the tibia and mandible of 3–4 month-old pigs (n=4), followed by BMSC isolation, culture-expansion and characterization by flow cytometry. Proliferation rates were assessed using population doubling times. Osteogenic differentiation was evaluated by alkaline phosphatase activity. Affymetrix porcine microarray was used to compare gene expressions of tibial and mandibular BMSCs, followed by real-time RT-PCR evaluation of certain genes. Results Our results showed that BMSCs from both locations expressed MSC markers but not hematopoietic markers. The proliferation and osteogenic differentiation potential of mandibular BMSCs were significantly stronger than those of tibial BMSCs. Microarray analysis identified 404 highly abundant genes, out of which 334 genes were matched between the two locations and annotated into the same functional groups including osteogenesis and angiogenesis, while 70 genes were mismatched and annotated into different functional groups. In addition, 48 genes were differentially expressed by at least 1.5-fold difference between the two locations, including higher expression of cranial neural crest-related gene BMP-4 in mandibular BMSCs, which was confirmed by real-time RT-PCR. Conclusions Altogether, these data indicate that despite strong similarities in gene expression between mandibular and tibial BMSCs, mandibular BMSCs express some genes differently than tibial BMSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration. PMID:28135571

Expression of the carbohydrate tumour marker Sialyl Lewis A, Sialyl Lewis X, Lewis Y and Thomsen-Friedenreich antigen in normal squamous epithelium of the uterine cervix, cervical dysplasia and cervical cancer.

PubMed

Engelstaedter, V; Fluegel, B; Kunze, S; Mayr, D; Friese, K; Jeschke, U; Bergauer, F

2012-04-01

The carbohydrate molecules Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA), Lewis Y (LeY) and Thomsen-Friedenreich antigen (TF) are known to mediate the adhesion between tumor cells and endothelium. They are used as serum markers in diagnosis and treatment in a broad spectrum of human carcinomas, but their expression profile and role in the development of cervical cancer remains unclear. The aim of this study was to investigate the expression of SLeX, SLeA, LeY and TF in normal cervical squamous epithelium, cervical dysplasia and cervical cancer. Slides of paraffin-embedded tissue were fixed and incubated with monoclonal antibodies against SLeX, SLeA, LeY and TF. Immunohistochemical staining was evaluated by using a semi-quantitative score (IRS Score). We found a significant difference of SLeA expression in invasive cervical cancer compared to normal epithelium (p=0.006) and all grades of dysplasia (p=0.002). The expression of SLeX in normal epithelium was less intense than in carcinoma in situ (p=0.036). Staining for LeY showed the weakest results of the investigated markers. Significant differences were found when normal epithelium was compared to CIN I (p=0.011), to CIN II (p=0.013) and to invasive cervical cancer (p=0.005). For TF, significant differences were found in normal epithelium compared to CIN I (p=0.011), CIN II (p=0.013) and compared to invasive cervical cancer (p=0.005). This is the first study on the expression of SLeA, SLeX, LeY and TF in normal cervical endothelium, cervical dysplasia, carcinoma in situ and invasive cervical cancer. Further studies and higher numbers are desirable.

Comparison of culture media indicates a role for autologous serum in enhancing phenotypic preservation of rabbit limbal stem cells in explant culture.

PubMed

Gürdal, Mehmet; Barut Selver, Özlem; Baysal, Kemal; Durak, İsmet

2018-04-01

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.).

PubMed

Wang, Jiping; Li, Runzhi; Mao, Xinguo; Jing, Ruilian

2017-01-01

Calreticulin (CRT), an endoplasmic reticulum (ER)-localized Ca 2+ -binding/buffering protein, is highly conserved and extensively expressed in animal and plant cells. To understand the function of CRTs in wheat ( Triticum aestivum L.), particularly their roles in stress tolerance, we cloned the full-length genomic sequence of the TaCRT-D isoform from D genome of common hexaploid wheat, and characterized its function by transgenic Arabidopsis system. TaCRT-D exhibited different expression patterns in wheat seedling under different abiotic stresses. Transgenic Arabidopsis plants overexpressing ORF of TaCRT-D displayed more tolerance to drought, cold, salt, mannitol, and other abiotic stresses at both seed germination and seedling stages, compared with the wild-type controls. Furthermore, DNA polymorphism analysis and gene mapping were employed to develop the functional markers of this gene for marker-assistant selection in wheat breeding program. One SNP, S440 (T→C) was detected at the TaCRT-D locus by genotyping a wheat recombinant inbred line (RIL) population (114 lines) developed from Opata 85 × W7984. The TaCRT-D was then fine mapped between markers Xgwm645 and Xgwm664 on chromosome 3DL, corresponding to genetic distances of 3.5 and 4.4 cM, respectively, using the RIL population and Chinese Spring nulli-tetrasomic lines. Finally, the genome-specific and allele-specific markers were developed for the TaCRT-D gene. These findings indicate that TaCRT-D function importantly in plant stress responses, providing a gene target for genetic engineering to increase plant stress tolerance and the functional markers of TaCRT-D for marker-assistant selection in wheat breeding.

Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.)

PubMed Central

Wang, Jiping; Li, Runzhi; Mao, Xinguo; Jing, Ruilian

2017-01-01

Calreticulin (CRT), an endoplasmic reticulum (ER)-localized Ca2+-binding/buffering protein, is highly conserved and extensively expressed in animal and plant cells. To understand the function of CRTs in wheat (Triticum aestivum L.), particularly their roles in stress tolerance, we cloned the full-length genomic sequence of the TaCRT-D isoform from D genome of common hexaploid wheat, and characterized its function by transgenic Arabidopsis system. TaCRT-D exhibited different expression patterns in wheat seedling under different abiotic stresses. Transgenic Arabidopsis plants overexpressing ORF of TaCRT-D displayed more tolerance to drought, cold, salt, mannitol, and other abiotic stresses at both seed germination and seedling stages, compared with the wild-type controls. Furthermore, DNA polymorphism analysis and gene mapping were employed to develop the functional markers of this gene for marker-assistant selection in wheat breeding program. One SNP, S440 (T→C) was detected at the TaCRT-D locus by genotyping a wheat recombinant inbred line (RIL) population (114 lines) developed from Opata 85 × W7984. The TaCRT-D was then fine mapped between markers Xgwm645 and Xgwm664 on chromosome 3DL, corresponding to genetic distances of 3.5 and 4.4 cM, respectively, using the RIL population and Chinese Spring nulli-tetrasomic lines. Finally, the genome-specific and allele-specific markers were developed for the TaCRT-D gene. These findings indicate that TaCRT-D function importantly in plant stress responses, providing a gene target for genetic engineering to increase plant stress tolerance and the functional markers of TaCRT-D for marker-assistant selection in wheat breeding. PMID:28955354

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

Increased Expression of ALDH1A1 in Prostate Cancer is Correlated With Tumor Aggressiveness: A Tissue Microarray Study of Iranian Patients.

PubMed

Kalantari, Elham; Saadi, Faezeh H; Asgari, Mojgan; Shariftabrizi, Ahmad; Roudi, Raheleh; Madjd, Zahra

2017-09-01

Subpopulations of prostate cancer (PCa) cells expressing putative stem cell markers possess the ability to promote tumor growth, maintenance, and progression. This study aimed to evaluate the expression patterns and clinical significance of putative stem cell marker aldehyde dehydrogenase 1 A1 (ALDH1A1) in prostate tumor tissues. ALDH1A1 expression was examined in a well-defined series of prostate tissues, including 105 (68%) samples of PCa, 21 (13%) samples of high-grade prostatic intraepithelial neoplasia, and 31 (19%) samples of benign prostate hyperplasia, which were embedded in tissue microarray blocks. The correlation of ALDH1A1 expression with clinicopathologic parameters was also assessed. There was a significant difference between the expression level of ALDH1A1 in PCa compared with the high-grade prostatic intraepithelial neoplasia and benign prostate hyperplasia samples (P<0.001). PCa cells expressing ALDH1A1 were more often seen in samples with advanced Gleason score (P=0.05) and high serum prostate specific antigen level (P=0.02). In addition, a positive correlation was found between ALDH1A1 expression and primary tumor stage and regional lymph node involvement (P=0.04 and 0.03, respectively). The significant association between ALDH1A1 expressions with Gleason score indicates the potential role of this protein in PCa tumorigenesis and aggressive behavior; therefore, this cancer stem cell marker can be used as a promising candidate for targeted therapy of PCa, especially those with high Gleason score.

Vcsa1 Acts as a Marker of Erectile Function Recovery After Gene Therapeutic and Pharmacological Interventions

PubMed Central

Calenda, Giulia; Tong, Yuehong; Tar, Moses; Lowe, Daniel; Siragusa, Joseph; Melman, Arnold; Davies, Kelvin P.

2010-01-01

Purpose We identified molecular markers of erectile function, particularly those responding to erectile dysfunction treatment. Materials and Methods Sprague-Dawley retired breeder rats were intracorporeally injected with pVAX-hSlo, pSMAA-hSlo or the control plasmid pVAX. One week later the intracorporeal pressure-to-blood pressure ratio and gene expression were determined by microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. Rat corporeal cells were transfected in vitro with pVAX-hSlo, pSMAA-hSlo or pVAX and the change in gene expression was determined. We also determined whether Vcsa1 expression was changed after pharmacotherapy using tadalafil. Results Animals treated with vectors expressing hSlo had significantly improved erectile function compared to that in controls, accompanied by changed expression of a subset of genes. Vcsa1 was one of the genes that was most changed in expression (the third of approximately 31,000 with greater than 10-fold up-regulation). Changes in gene expression were different than those observed in corporeal cells transfected in vitro, distinguishing gene expression changes that were a direct effect of hSlo over expression. When tadalafil was administered in retired breeder rats, the Vcsa1 transcript increased 4-fold in corporeal tissue compared to that in untreated controls. Conclusions Our study identifies a set of genes that are changed in response to improved erectile function, rather than as a direct effect of treatment. We noted Vcsa1 may act as marker of the restoration of erectile function after gene transfer and pharmacotherapy. PMID:19375734

Vcsa1 acts as a marker of erectile function recovery after gene therapeutic and pharmacological interventions.

PubMed

Calenda, Giulia; Tong, Yuehong; Tar, Moses; Lowe, Daniel; Siragusa, Joseph; Melman, Arnold; Davies, Kelvin P

2009-06-01

We identified molecular markers of erectile function, particularly those responding to erectile dysfunction treatment. Sprague-Dawley retired breeder rats were intracorporeally injected with pVAX-hSlo, pSMAA-hSlo or the control plasmid pVAX. One week later the intracorporeal pressure-to-blood pressure ratio and gene expression were determined by microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. Rat corporeal cells were transfected in vitro with pVAX-hSlo, pSMAA-hSlo or pVAX and the change in gene expression was determined. We also determined whether Vcsa1 expression was changed after pharmacotherapy using tadalafil. Animals treated with vectors expressing hSlo had significantly improved erectile function compared to that in controls, accompanied by changed expression of a subset of genes. Vcsa1 was one of the genes that was most changed in expression (the third of approximately 31,000 with greater than 10-fold up-regulation). Changes in gene expression were different than those observed in corporeal cells transfected in vitro, distinguishing gene expression changes that were a direct effect of hSlo over expression. When tadalafil was administered in retired breeder rats, the Vcsa1 transcript increased 4-fold in corporeal tissue compared to that in untreated controls. Our study identifies a set of genes that are changed in response to improved erectile function, rather than as a direct effect of treatment. We noted Vcsa1 may act as marker of the restoration of erectile function after gene transfer and pharmacotherapy.

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties.

PubMed

Paduano, Francesco; Marrelli, Massimo; Palmieri, Francesca; Tatullo, Marco

2016-10-01

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146 Low and CD146 High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146 Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146 High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146 Low than in CD146 High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.

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.

Grammatical markers switch roles and elicit different electrophysiological responses under shallow and deep semantic requirements.

PubMed

Soshi, Takahiro; Nakajima, Heizo; Hagiwara, Hiroko

2016-10-01

Static knowledge about the grammar of a natural language is represented in the cortico-subcortical system. However, the differences in dynamic verbal processing under different cognitive conditions are unclear. To clarify this, we conducted an electrophysiological experiment involving a semantic priming paradigm in which semantically congruent or incongruent word sequences (prime nouns-target verbs) were randomly presented. We examined the event-related brain potentials that occurred in response to congruent and incongruent target words that were preceded by primes with or without grammatical case markers. The two participant groups performed either the shallow (lexical judgment) or deep (direct semantic judgment) semantic tasks. We hypothesized that, irrespective of the case markers, the congruent targets would reduce centro-posterior N400 activities under the deep semantic condition, which induces selective attention to the semantic relatedness of content words. However, the same congruent targets with correct case markers would reduce lateralized negativity under the shallow semantic condition because grammatical case markers are related to automatic structural integration under semantically unattended conditions. We observed that congruent targets (e.g., 'open') that were preceded by primes with congruent case markers (e.g., 'shutter-object case') reduced lateralized negativity under the shallow semantic condition. In contrast, congruent targets, irrespective of case markers, consistently yielded N400 reductions under the deep semantic condition. To summarize, human neural verbal processing differed in response to the same grammatical markers in the same verbal expressions under semantically attended or unattended conditions.

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.

Characterization and differentiation of human embryonic stem cells.

PubMed

Carpenter, M K; Rosler, E; Rao, M S

2003-01-01

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

The effect of high level natural ionizing radiation on expression of PSA, CA19-9 and CEA tumor markers in blood serum of inhabitants of Ramsar, Iran.

PubMed

Heidari, Mohammad Hassan; Porghasem, Mohsen; Mirzaei, Nazanin; Mohseni, Jafar Hesam; Heidari, Matine; Azargashb, Eznollah; Movafagh, Abolfazl; Heidari, Reihane; Molouki, Aidin; Larijani, Leila

2014-02-01

Since several high level natural radiation areas (HLNRAs) exist on our planet, considerable attention has been drawn to health issues that may develop as the result of visiting or living in such places. City of Ramsar in Iran is an HNLRA, and is a tourist attraction mainly due to its hot spas. However, the growing awareness over its natural radiation sources has prompted widespread scientific investigation at national level. In this study, using an ELISA method, the level of expression of three tumor markers known as carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and carcino antigen 19-9 (CA19-9) in blood serum of 40 local men of Ramsar (subject group) was investigated and compared to 40 men from the city of Noshahr (control group). Noshahr was previously identified as a normal level natural radiation area (NLNRA) that is some 85 km far from Ramsar. According to statistical analysis, there was a significant difference in the levels of PSA and CA19-9 markers between the two groups (p < 0.001) with those of Ramsar being considerably higher. CEA level did not show any difference. Although some of the volunteers tested positive to the markers, they were in good health as confirmed by the physician. Moreover, the high number of positive markers in Noshahr was considerable. Therefore, future study is needed to further validate this result and to determine the level of positivity to tumor markers in both cities. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of fish and krill oil on gene expression in peripheral blood mononuclear cells and circulating markers of inflammation: a randomised controlled trial.

PubMed

Rundblad, Amanda; Holven, Kirsten B; Bruheim, Inge; Myhrstad, Mari C; Ulven, Stine M

2018-01-01

Marine n -3 (omega-3) fatty acids alter gene expression by regulating the activity of transcription factors. Krill oil is a source of marine n -3 fatty acids that has been shown to modulate gene expression in animal studies; however, the effect in humans is not known. Hence, we aimed to compare the effect of intake of krill oil, lean and fatty fish with a similar content of n -3 fatty acids, and high-oleic sunflower oil (HOSO) with added astaxanthin on the expression of genes involved in glucose and lipid metabolism and inflammation in peripheral blood mononuclear cells (PBMC) as well as circulating inflammatory markers. In an 8-week trial, healthy men and women aged 18-70 years with fasting TAG of 1·3-4·0 mmol/l were randomised to receive krill oil capsules ( n 12), HOSO capsules ( n 12) or lean and fatty fish ( n 12). The weekly intakes of marine n -3 fatty acids from the interventions were 4654, 0 and 4103 mg, respectively. The mRNA expression of four genes, PPAR γ coactivator 1A ( PPARGC1A ), steaoryl-CoA desaturase ( SCD ), ATP binding cassette A1 ( ABCA1 ) and cluster of differentiation 40 ( CD40 ), were differently altered by the interventions. Furthermore, within-group analyses revealed that krill oil down-regulated the mRNA expression of thirteen genes, including genes involved in glucose and cholesterol metabolism and β-oxidation. Fish altered the mRNA expression of four genes and HOSO down-regulated sixteen genes, including several inflammation-related genes. There were no differences between the groups in circulating inflammatory markers after the intervention. In conclusion, the intake of krill oil and HOSO with added astaxanthin alter the PBMC mRNA expression of more genes than the intake of fish.

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.

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.

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

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.

Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins.

PubMed

Samalova, Marketa; Fricker, Mark; Moore, Ian

2006-12-01

Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the amino-terminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.

Comparison between retroperitoneal leiomyosarcoma and dedifferentiated liposarcoma.

PubMed

Ishii, Takeaki; Kohashi, Kenichi; Ootsuka, Hiroshi; Iura, Kunio; Maekawa, Akira; Yamada, Yuichi; Bekki, Hirofumi; Yoshimoto, Masato; Yamamoto, Hidetaka; Iwamoto, Yukihide; Oda, Yoshinao

2017-06-01

It is important to distinguish between leiomyosarcoma (LMS) and dedifferentiated liposarcoma (DDLS) in the retroperitoneum. The dedifferentiated component of DDLS shows an LMS-like morphology in some cases; thus, detailed evaluation is necessary to achieve an accurate diagnosis. Immunohistochemically, MDM2 and myogenic markers provide clues for the diagnoses. However, immunoreactivity for MDM2 and myogenic markers has not been well studied in retroperitoneal LMS and DDLS. Here, we compared the clinicopathological data of 20 retroperitoneal tumors initially diagnosed as LMS with that of 36 cases of retroperitoneal DDLS and conducted an immunohistochemical study. Four (20%) of the cases initially diagnosed as LMS were immunoreactive for MDM2. Fifteen cases (41.7%) of DDLS showed positive expression of two or more myogenic markers. The patients with LMS with MDM2 overexpression were older than the patients with LMS without MDM2 overexpression (P=0.0328). LMS with MDM2 overexpression showed a worse prognosis than DDLS (P=0.0408). No significant difference in prognosis was found between LMS without MDM2 overexpression and DDLS with myogenic differentiation. In conclusion, we recommend that systemic MDM2 expression analysis be performed in cases of retroperitoneal sarcoma. Overdependence on the expression of myogenic markers could lead to misdiagnosis in distinguishing LMS from DDLS. Copyright © 2017 Elsevier GmbH. All rights reserved.

Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells.

PubMed

Rivera, Francisco J; Sierralta, Walter D; Minguell, Jose J; Aigner, Ludwig

2006-10-02

Bone marrow-derived mesenchymal stem cells (MSCs) are not restricted in their differentiation fate to cells of the mesenchymal lineage. They acquire a neural phenotype in vitro and in vivo after transplantation in the central nervous system. Here we investigated whether soluble factors derived from different brain regions are sufficient to induce a neuronal phenotype in MSCs. We incubated bone marrow-derived MSCs in conditioned medium (CM) derived from adult hippocampus (HCM), cortex (CoCM) or cerebellum (CeCM) and analyzed the cellular morphology and the expression of neuronal and glial markers. In contrast to muscle derived conditioned medium, which served as control, conditioned medium derived from the different brain regions induced a neuronal morphology and the expression of the neuronal markers GAP-43 and neurofilaments in MSCs. Hippocampus derived conditioned medium had the strongest activity. It was independent of NGF or BDNF; and it was restricted to the neuronal differentiation fate, since no induction of the astroglial marker GFAP was observed. The work indicates that soluble factors present in the brain are sufficient to induce a neuronal phenotype in MSCs.

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.

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis

PubMed Central

1985-01-01

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full- length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain- specific expression of V kappa Ser-associated phenotypic markers are discussed. PMID:3926938

Expression of neuronal antigens and related ventral and dorsal proteins in the normal spinal cord and a surgically induced open neural tube defect of the spine in chick embryos: an immunohistochemical study.

PubMed

Lee, Do-Hun; Phi, Ji Hoon; Chung, You-Nam; Lee, Yun-Jin; Kim, Seung-Ki; Cho, Byung-Kyu; Kim, Dong Won; Park, Moon-Sik; Wang, Kyu-Chang

2010-05-01

The aims of this study were to elucidate the processes of neuronal differentiation and ventrodorsal patterning in the spinal cord of the chick embryo from embryonic day (E) 3 to E17 and to study the effect of a prenatal spinal open neural tube defect (ONTD) on these processes. Expression patterns of neuronal antigens (neuronal nuclear antigen, neurofilament-associated protein (NAP), and synaptophysin) and related ventral markers [sonic hedgehog, paired box gene (PAX)6, and islet-1], and dorsal markers (bone morphogenetic protein, Notch homolog 1, and PAX7) were investigated in the normal spinal cord and in a surgically induced spinal ONTD in chick embryos. Four normal and ONTD chick embryos were used for each antigen group. There were no differences in the expression of neuronal and ventrodorsal markers between the control and ONTD groups. NAP and synaptophysin were useful for identifying dorsal structures in the distorted anatomy of the ONTD chicks.

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.

Expression of Kv3.1b potassium channel is widespread in macaque motor cortex pyramidal cells: A histological comparison between rat and macaque.

PubMed

Soares, David; Goldrick, Isabelle; Lemon, Roger N; Kraskov, Alexander; Greensmith, Linda; Kalmar, Bernadett

2017-06-15

There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration "thin" spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons. © 2017 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Normal and PPP-affected palmoplantar sweat gland express neuroendocrine markers chromogranins and synaptophysin differently.

PubMed

Hagforsen, Eva; Michaëlsson, Gerd; Stridsberg, Mats

2010-11-01

Earlier findings indicate the acrosyringium as the target for the inflammation in the chronic and intensely inflammatory skin disease palmoplantar pustulosis (PPP). The sweat gland apparatus seems to be an immune-competent structure that probably contributes to the defence of the skin. Furthermore, the sweat gland and duct may be a hitherto unrecognized neuroendocrine organ because it expresses cholineacetyl-transferase and acetylcholinesterase, nicotinic receptors, beta-adrenergic and angiotensin receptors. The aim of this study was to obtain further information about neuroendocrine properties of the sweat gland apparatus by examining the expression of common neuroendocrine markers synaptophysin and chromogranins A and B in healthy palmar skin and in PPP skin. Synaptophysin and chromogranins were expressed in the sweat glands and ducts with some variation in the pattern and intensity of the expression. In PPP skin the expression differed, being higher and lower, depending on the part of the sweat duct. Chromogranins were further expressed in the epidermis, endothelium and inflammatory cells, but its intensity was weaker in epidermis than in the sweat gland apparatus. In most cases, chromogranins in epidermis in involved PPP were weakly expressed compared to healthy controls. The presence of synaptophysin and chromogranins in palmoplantar skin may have marked neuroendocrine effects, and the palmoplantar skin is likely to have important neuroimmuno-endocrine properties. Moreover, the altered chromogranin expression in PPP skin might influence both the neuroendocrine and neuroimmunologic properties of palmoplantar skin in these patients. These results indicate important neuroendocrine properties of the palmoplantar skin.

Bone sialoprotein is predictive of bone metastases in resectable non-small-cell lung cancer: a retrospective case-control study.

PubMed

Papotti, Mauro; Kalebic, Thea; Volante, Marco; Chiusa, Luigi; Bacillo, Elisa; Cappia, Susanna; Lausi, Paolo; Novello, Silvia; Borasio, Piero; Scagliotti, Giorgio V

2006-10-20

Bone metastases (BM) in non-small-cell lung cancer (NSCLC) may be detected at diagnosis or during the course of the disease, and are associated with a worse prognosis. Currently, there are no predictive or diagnostic markers to identify high-risk patients for metastatic bone dissemination. Thirty patients with resected NSCLC who subsequently developed BM were matched for clinicopathologic parameters to 30 control patients with resected NSCLC without any metastases and 26 patients with resected NSCLC and non-BM lesions. Primary tumors were investigated by immunohistochemistry for 10 markers involved in bone resorption or development of metastases. Differences among groups were estimated by chi2 test, whereas the prognostic impact of clinicopathologic parameters and marker expression was evaluated by univariate (Wilcoxon and Mantel-Cox tests) and multivariate (Cox proportional hazards regression model) analyses. The presence of bone sialoprotein (BSP) was strongly associated with bone dissemination (P < .001) and, independently, with worse outcome (P = .02, Mantel-Cox test), as defined by overall survival. To evaluate BSP protein expression in nonselected NSCLC, a series of 120 consecutive resected lung carcinomas was added to the study, and BSP prevalence reached 40%. No other markers showed a statistically significant difference among the three groups or demonstrated a prognostic impact, in terms of both overall survival and time interval to metastases. BSP protein expression in the primary resected NSCLC is strongly associated with BM progression and could be useful in identifying high-risk patients who could benefit from novel modalities of surveillance and preventive treatment.

Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

PubMed Central

Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

2012-01-01

Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

Potential use of TIA-1, MFF, microRNA-200a-3p, and microRNA-27 as a novel marker for hepatocellular carcinoma.

PubMed

Tak, Hyosun; Kang, Hoin; Ji, Eunbyul; Hong, Youlim; Kim, Wook; Lee, Eun Kyung

2018-03-18

Precise and early diagnosis is critical to improve the survival rate of hepatocellular carcinoma (HCC) patients. Although several genetic and protein markers have been developed and are currently used for diagnosis, prognosis, risk stratification, and therapeutic monitoring, application of these markers still needs to be improved for better specificity and efficacy. In this study, we investigated the relative expression of mitochondrial dynamics-regulating factors including T-cell intercellular antigen protein-1 (TIA-1), mitochondrial fission factor (MFF), microRNA (miR)-200a-3p, and miR-27a/b in the liver tissues from HCC patients. The expressions of TIA-1 and MFF were augmented in the cancerous liver tissues compared to the corresponding non-tumor tissues at mRNA and protein level, while the levels of miR-200a-3p and miR-27a/b were relatively lower in the cancerous liver tissues. In addition, high levels of TIA-1 and MFF mRNA were related to the poor survival rate of HCC patients. Our results indicated that the expressions of TIA-1, MFF, miR-200a-3p, and miR-27a/b in the cancerous liver tissues differed to these in non-cancerous tissues of HCC patients, demonstrating that these gene expressions could be potential markers for the diagnosis and prognosis of HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

PubMed

Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

2012-06-01

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

Immunohistochemistry predictive markers for primary colorectal cancer tumors: where are we and where are we going?

PubMed

Bărbălan, Alexandru; Nicolaescu, Andrei Cristian; Măgăran, Antoanela Valentina; Mercuţ, Răzvan; Bălăşoiu, Maria; Băncescu, Gabriela; Şerbănescu, Mircea Sebastian; Lazăr, Octavian Fulger; Săftoiu, Adrian

2018-01-01

The aim of our study is to highlight and organize the recently published immunohistochemistry (IHC) predictive biomarkers of primary colorectal cancers (CRCs) that could lead to practical implementation. We reviewed articles that examined CRC samples with significant statistic correlation between the IHC marker expression and disease progression over time, relationships with the available clinical features and those who detect the prognosis of drug effects. Our analysis showed that nine markers could correlate with medical treatment response of CRCs in different stages. When using better overall survival (OS) and better disease-free survival (DFS) as a grouping factor, there were 14 markers that could be used in assessing CRC prognosis. By using poor prognostic for the OS and the DFS as a grouping factor, we found 43 markers. Subgroup analysis was also performed based on the 32 markers recently confirmed to predict metastasis evolution or the recurrence risks. Venous invasion could be predictable for tumors, statistically significant metastasis susceptibility was observed for markers and also the capacity to evaluate recurrence. CRCs integrate a variety of localizations and there are proofs that distinguish the sites of tumors. The studies reporting data specifically for rectal cancer separating it from colon cancer contained seven IHC markers. In order to be able to implement a predictive biomarker in clinical practice, it must comply with certain criteria as clinical value and analytical proof. Unique biological signature of CRC can be distinguished by identifying biomarkers expression. Several markers have shown potential, but the majority still need to render clinical utility.

Isolated tumor endothelial cells maintain specific character during long-term culture

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

Matsuda, Kohei; Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586; Oral and Maxillofacial Surgery, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, wemore » have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.« less

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes

PubMed Central

Chittiprol, Seetharamaiah; Chen, Phylip; Petrovic-Djergovic, Danica; Eichler, Tad

2011-01-01

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR >> KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines. PMID:21632959

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes.

PubMed

Chittiprol, Seetharamaiah; Chen, Phylip; Petrovic-Djergovic, Danica; Eichler, Tad; Ransom, Richard F

2011-09-01

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR > KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.

Expression of proliferation marker Ki67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas.

PubMed

Protzel, C; Knoedel, J; Zimmermann, U; Woenckhaus, C; Poetsch, M; Giebel, J

2007-11-01

Clinical outcome of penile squamous cell carcinoma (PSCC) largely depends on the presence of lymph node metastasis. In search of a valuable marker predicting the risk for metastasis, the expression of Ki67 was investigated immunohistochemically in primary tumors and compared to presence of inguinal lymph node metastasis. As human papilloma virus (HPV) is thought to affect Ki67 expression, we evaluated whether occurrence of HPV DNA correlates to Ki67 score or metastatic potential. Samples originated from patients subjected to resection of invasive SCC of penis. Immunohistochemistry was done on paraffin-embedded sections using a monoclonal antibody against Ki67. After DNA isolation from paraffin embedded tissue the presence of HPV 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Statistical analysis was done using two tail unpaired t test and Chi-square test. Four of 28 patients showed a weak Ki67 expression, without displaying lymph node metastasis. Among 17 patients showing an intermediate Ki67 index, eight exhibited metastases while in all seven patients with a strong expression of Ki67 lymph node metastases were found. The median Ki67 expression in metastastic lesions was significantly different (50.3%) from tumors without lymph node metastasis (31.8%) (p=0.024). Furthermore, a correlation between presence of HPV DNA and strong Ki67 expression was determined (p=0.009). Since our study demonstrated a strong Ki67 labeling index significantly associated to positive lymph nodes, we suggest Ki67 expression as a prognostic marker for lymph node metastasis in penile squamous carcinoma.

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.

[The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells].

PubMed

Lyu, Jianmei; He, Yanjin; Xie, Lianfeng; Liu, Xun; Zhu, Limin

2015-10-01

To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties. Experimental study. Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed. Cells were divided into two groups, the LACC-CSC experimental group and the LACC control group. The flow cytometry instrument was used to detect the expression of classical stem cell markers CD133 and ABCG2. Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells. The tumorigenic force in vitro xenotransplantation were applied. LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days. Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67 ± 6.86)%, significantly different to LACC with (0.46 ± 0.48)%, (t = 8.867, P < 0.05). Similarly, the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99 ± 4.54)%, significantly different to LACC with (6.75 ± 1.34)%, (t = -9.932, P < 0.05). In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60 ± 8.79)/HP contrary to LACC with average (10.20 ± 2.77)/HP after 24 hours, showing statistically significance (t = 5.433, P < 0.05) between the two groups. After training for 48 hours, the difference between two groups was still obvious (t = 5.779, P < 0.05) with LACC-CSC average (62.60 ± 4.83)/HP to LACC (44.00 ± 5.34)/HP. When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34. Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate. LACC-CSC can be obtained through serum-free culture method. LACC-CSC grew suspensively and expressed classical stem cell markers. LACC-CSC were identified as cancer stem cells with stronger migration and invasion. LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.

PCL-PDMS-PCL copolymer-based microspheres mediate cardiovascular differentiation from embryonic stem cells

NASA Astrophysics Data System (ADS)

Song, Liqing

Poly-epsilon-caprolactone (PCL) based copolymers have received much attention as drug or growth factor delivery carriers and tissue engineering scaffolds due to their biocompatibility, biodegradability, and tunable biophysical properties. Copolymers of PCL and polydimethylsiloxane (PDMS) also have shape memory behaviors and can be made into thermoresponsive shape memory polymers for various biomedical applications such as smart sutures and vascular stents. However, the influence of biophysical properties of PCL-PDMS-PCL copolymers on stem cell lineage commitment is not well understood. In this study, PDMS was used as soft segments of varying length to tailor the biophysical properties of PCL-based co-polymers. While low elastic modulus (<10 kPa) of the tri-block copolymer PCL-PDMS-PCL affected cardiovascular differentiation of embryonic stem cells, the range of 60-100 MPa PCL-PDMS-PCL showed little influence on the differentiation. Then different size (30-140 mum) of microspheres were fabricated from PCL-PDMS-PCL copolymers and incorporated within embryoid bodies (EBs). Mesoderm differentiation was induced using bone morphogenetic protein (BMP)-4 for cardiovascular differentiation. Differential expressions of mesoderm progenitor marker KDR and vascular markers CD31 and VE-cadherin were observed for the cells differentiated from EBs incorporated with microspheres of different size, while little difference was observed for cardiac marker alpha-actinin expression. Small size of microspheres (30 mum) resulted in higher expression of KDR while medium size of microspheres (94 mum) resulted in higher CD31 and VE-cadherin expression. This study indicated that the biophysical properties of PCL-based copolymers impacted stem cell lineage commitment, which should be considered for drug delivery and tissue engineering applications.

Diagnostic markers for germ cell neoplasms: from placental-like alkaline phosphatase to micro-RNAs.

PubMed

Rajpert-De Meyts, Ewa; Nielsen, John E; Skakkebaek, Niels E; Almstrup, Kristian

2015-01-01

This concise review summarises tissue and serum markers useful for differential diagnosis of germ cell tumours (GCT), with focus on the most common testicular GCT (TGCT). GCT are characterised by phenotypic heterogeneity due to largely retained embryonic pluripotency and aberrant somatic differentiation. TGCT that occur in young men are divided into two main types, seminoma and nonseminoma, both derived from a pre-invasive germ cell neoplasia in situ (GCNIS), which originates from transformed foetal gonocytes. In severely dysgenetic gonads, a GCNIS-resembling lesion is called gonadoblastoma. GCT occur rarely in young children (infantile GCT) in whom the pathogenesis is different (no GCNIS/gonadoblastoma stage) but the histopathological features are similar to the adult GCT. The rare spermatocytic tumour of older men is derived from post-pubertal spermatogonia that clonally expand due to gain-of function mutations in survival-promoting genes (e.g. FGFR3, HRAS), thus this tumour has a different expression profile than GCNIS-derived TGCT. Clinically most informative immunohistochemical markers for GCT, except teratoma, are genes expressed in primordial germ cells/gonocytes and embryonic pluripotency-related factors, such as placental-like alkaline phosphatase (PLAP), OCT4 (POU5F1), NANOG, AP-2γ (TFAP2C) and LIN28, which are not expressed in normal adult germ cells. Some of these markers can also be used for immunocytochemistry to detect GCNIS or incipient tumours in semen samples. Gene expression in GCT is regulated in part by DNA and histone modifications, and the epigenetic profile of these tumours is characterised by genome-wide demethylation, except nonseminomas. In addition, a recently discovered mechanism of post-genomic gene expression regulation involves small non-coding RNAs, predominantly micro-RNA (miR). Testicular GCT display micro-RNA profiles similar to embryonic stem cells. Targeted miRNA-based blood tests for miR-371-3 and miR-367 clusters are currently under development and hold a great promise for the future. In some patients miR-based tests may be even more sensitive than the classical serum tumour markers, β -chorio-gonadotrophin (β-hCG), α-fetoprotein (AFP) and lactate dehydrogenase (LDH), which are currently used in the clinic. In summary, research advances have provided clinicians with a panel of molecular markers, which allow specific diagnosis of various subtypes of GCT and are very useful for early detection at the precursor stage and for monitoring of patients during the follow-up.

Molecular profiling identifies prognostic markers of stage IA lung adenocarcinoma.

PubMed

Zhang, Jie; Shao, Jinchen; Zhu, Lei; Zhao, Ruiying; Xing, Jie; Wang, Jun; Guo, Xiaohui; Tu, Shichun; Han, Baohui; Yu, Keke

2017-09-26

We previously showed that different pathologic subtypes were associated with different prognostic values in patients with stage IA lung adenocarcinoma (AC). We hypothesize that differential gene expression profiles of different subtypes may be valuable factors for prognosis in stage IA lung adenocarcinoma. We performed microarray gene expression profiling on tumor tissues micro-dissected from patients with acinar and solid predominant subtypes of stage IA lung adenocarcinoma. These patients had undergone a lobectomy and mediastinal lymph node dissection at the Shanghai Chest Hospital, Shanghai, China in 2012. No patient had preoperative treatment. We performed the Gene Set Enrichment Analysis (GSEA) analysis to look for gene expression signatures associated with tumor subtypes. The histologic subtypes of all patients were classified according to the 2015 WHO lung Adenocarcinoma classification. We found that patients with the solid predominant subtype are enriched for genes involved in RNA polymerase activity as well as inactivation of the p53 pathway. Further, we identified a list of genes that may serve as prognostic markers for stage IA lung adenocarcinoma. Validation in the TCGA database shows that these genes are correlated with survival, suggesting that they are novel prognostic factors for stage IA lung adenocarcinoma. In conclusion, we have uncovered novel prognostic factors for stage IA lung adenocarcinoma using gene expression profiling in combination with histopathology subtyping.

The alteration of mRNA expression of SOD and GPX genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles.

PubMed

Alharby, Hesham F; Metwali, Ehab M R; Fuller, Michael P; Aldhebiani, Amal Y

2016-11-01

Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L -1 ) and ZnO-NPs (0, 15 and 30 mg L -1 ). Treatments with NaCl at both 3 and 6 g L -1 suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS-PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts

PubMed Central

Shirasawa, Kenta; Hand, Melanie L.; Henderson, Steven T.; Okada, Takashi; Johnson, Susan D.; Taylor, Jennifer M.; Spriggs, Andrew; Siddons, Hayley; Hirakawa, Hideki; Isobe, Sachiko; Tabata, Satoshi; Koltunow, Anna M. G.

2015-01-01

Background and Aims Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. Methods RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. Key Results A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. Conclusions A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations. PMID:25538115

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

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.

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.

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

Prognostic value of proliferating cell nuclear antigen in parotid gland cancer.

PubMed

Stenner, Markus; Demgensky, Ariane; Molls, Christoph; Hardt, Aline; Luers, Jan C; Grosheva, Maria; Huebbers, Christian U; Klussmann, Jens P

2012-04-01

Although cell proliferation is related to tumour aggressiveness and prognosis, there are few studies describing the expression of proliferative markers in salivary gland cancer. Our aim was to assess the long-term prognostic value of the proliferating cell nuclear antigen (PCNA) in a large group of histologically different salivary gland cancers. We analysed the expression of PCNA in 159 patients with parotid gland cancer by means of immunohistochemistry. The mean follow-up time was 56.6 months. A high expression of PCNA showed a significant correlation to the patients' pathological lymph node stage (p = 0.004). A high PCNA expression significantly indicated a poor 5-year disease-free (p = 0.046) and overall survival rate (p = 0.018). The PCNA expression was the only prognostic factor for a worse 5-year disease-free and overall survival in acinic cell carcinomas (p = 0.004, p = 0.022). The correlation between PCNA expression and survival probabilities of salivary gland cancer might make proliferation markers helpful tools in patient follow-up, prognosis and targeted therapy in salivary gland cancer in future.

Neighbor of Punc E 11: expression pattern of the new hepatic stem/progenitor cell marker during murine liver development.

PubMed

Schievenbusch, Stephanie; Sauer, Elisabeth; Curth, Harald-Morten; Schulte, Sigrid; Demir, Münevver; Toex, Ulrich; Goeser, Tobias; Nierhoff, Dirk

2012-09-20

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

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.

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.

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

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Finniss, Susan; Bögler, Oliver; Duchrow, Michael

2004-04-15

The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions. Copyright 2004 Wiley-Liss, Inc.

Deracemization of bilirubin as the marker of the chirality of micellar aggregates.

PubMed

Sorrenti, Alessandro; Altieri, Barbara; Ceccacci, Francesca; Di Profio, Pietro; Germani, Raimondo; Giansanti, Luisa; Savelli, Gianfranco; Mancini, Giovanna

2012-01-01

The deracemization of bilirubin in micellar aggregates of structurally correlated chiral surfactants was studied by circular dichroism experiments and exploited as the marker of the expression of chirality of the aggregates. The obtained results suggest that the hydrophobic interactions control the transfer of chirality from the monomers to the aggregates, and that different regions of the same aggregate might feature opposite enantiorecognition capabilities. Copyright © 2011 Wiley-Liss, Inc.

Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits.

PubMed

Zhang, Yanghai; Cui, Yang; Zhang, Xuelian; Wang, Yimin; Gao, Jiayang; Yu, Ting; Lv, Xiaoyan; Pan, Chuanying

2018-05-31

Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P < 0.01); among different types of testicular cells, the StAR mRNA expression level was significantly higher in LCs than others (P < 0.05). Furthermore, three splice variants, StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P < 0.01), suggesting StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P < 0.05), and we speculated that the allele "D" of StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum.

PubMed

Ren, Jiaqiang; Ward, Dawn; Chen, Steven; Tran, Katherine; Jin, Ping; Sabatino, Marianna; Robey, Pamela G; Stroncek, David F

2018-03-14

Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

Gene Expression (mRNA) Markers for Differentiating between Malignant and Benign Follicular Thyroid Tumours

PubMed Central

Wojtas, Bartosz; Pfeifer, Aleksandra; Oczko-Wojciechowska, Malgorzata; Krajewska, Jolanta; Czarniecka, Agnieszka; Kukulska, Aleksandra; Eszlinger, Markus; Musholt, Thomas; Stokowy, Tomasz; Swierniak, Michal; Stobiecka, Ewa; Chmielik, Ewa; Rusinek, Dagmara; Tyszkiewicz, Tomasz; Halczok, Monika; Hauptmann, Steffen; Lange, Dariusz; Jarzab, Michal; Paschke, Ralf; Jarzab, Barbara

2017-01-01

Distinguishing between follicular thyroid cancer (FTC) and follicular thyroid adenoma (FTA) constitutes a long-standing diagnostic problem resulting in equivocal histopathological diagnoses. There is therefore a need for additional molecular markers. To identify molecular differences between FTC and FTA, we analyzed the gene expression microarray data of 52 follicular neoplasms. We also performed a meta-analysis involving 14 studies employing high throughput methods (365 follicular neoplasms analyzed). Based on these two analyses, we selected 18 genes differentially expressed between FTA and FTC. We validated them by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent set of 71 follicular neoplasms from formaldehyde-fixed paraffin embedded (FFPE) tissue material. We confirmed differential expression for 7 genes (CPQ, PLVAP, TFF3, ACVRL1, ZFYVE21, FAM189A2, and CLEC3B). Finally, we created a classifier that distinguished between FTC and FTA with an accuracy of 78%, sensitivity of 76%, and specificity of 80%, based on the expression of 4 genes (CPQ, PLVAP, TFF3, ACVRL1). In our study, we have demonstrated that meta-analysis is a valuable method for selecting possible molecular markers. Based on our results, we conclude that there might exist a plausible limit of gene classifier accuracy of approximately 80%, when follicular tumors are discriminated based on formalin-fixed postoperative material. PMID:28574441

Gene Expression (mRNA) Markers for Differentiating between Malignant and Benign Follicular Thyroid Tumours.

PubMed

Wojtas, Bartosz; Pfeifer, Aleksandra; Oczko-Wojciechowska, Malgorzata; Krajewska, Jolanta; Czarniecka, Agnieszka; Kukulska, Aleksandra; Eszlinger, Markus; Musholt, Thomas; Stokowy, Tomasz; Swierniak, Michal; Stobiecka, Ewa; Chmielik, Ewa; Rusinek, Dagmara; Tyszkiewicz, Tomasz; Halczok, Monika; Hauptmann, Steffen; Lange, Dariusz; Jarzab, Michal; Paschke, Ralf; Jarzab, Barbara

2017-06-02

Distinguishing between follicular thyroid cancer (FTC) and follicular thyroid adenoma (FTA) constitutes a long-standing diagnostic problem resulting in equivocal histopathological diagnoses. There is therefore a need for additional molecular markers. To identify molecular differences between FTC and FTA, we analyzed the gene expression microarray data of 52 follicular neoplasms. We also performed a meta-analysis involving 14 studies employing high throughput methods (365 follicular neoplasms analyzed). Based on these two analyses, we selected 18 genes differentially expressed between FTA and FTC. We validated them by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent set of 71 follicular neoplasms from formaldehyde-fixed paraffin embedded (FFPE) tissue material. We confirmed differential expression for 7 genes ( CPQ , PLVAP , TFF3 , ACVRL1 , ZFYVE21 , FAM189A2 , and CLEC3B ). Finally, we created a classifier that distinguished between FTC and FTA with an accuracy of 78%, sensitivity of 76%, and specificity of 80%, based on the expression of 4 genes ( CPQ , PLVAP , TFF3 , ACVRL1 ). In our study, we have demonstrated that meta-analysis is a valuable method for selecting possible molecular markers. Based on our results, we conclude that there might exist a plausible limit of gene classifier accuracy of approximately 80%, when follicular tumors are discriminated based on formalin-fixed postoperative material.

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.

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

PubMed

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A

2014-02-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

PubMed Central

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

2014-01-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation

PubMed Central

De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

2013-01-01

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology ‘reverse engineering’ approaches. We ‘reverse engineered’ an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression (‘hubs’). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central ‘hub’ of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation. PMID:23180766

Neurochemical differences between target-specific populations of rat dorsal raphe projection neurons.

PubMed

Prouty, Eric W; Chandler, Daniel J; Waterhouse, Barry D

2017-11-15

Serotonin (5-HT)-containing neurons in the dorsal raphe (DR) nucleus project throughout the forebrain and are implicated in many physiological processes and neuropsychiatric disorders. Diversity among these neurons has been characterized in terms of their neurochemistry and anatomical organization, but a clear sense of whether these attributes align with specific brain functions or terminal fields is lacking. DR 5-HT neurons can co-express additional neuroactive substances, increasing the potential for individualized regulation of target circuits. The goal of this study was to link DR neurons to a specific functional role by characterizing cells according to both their neurotransmitter expression and efferent connectivity; specifically, cells projecting to the medial prefrontal cortex (mPFC), a region implicated in cognition, emotion, and responses to stress. Following retrograde tracer injection, brainstem sections from Sprague-Dawley rats were immunohistochemically stained for markers of serotonin, glutamate, GABA, and nitric oxide (NO). 98% of the mPFC-projecting serotonergic neurons co-expressed the marker for glutamate, while the markers for NO and GABA were observed in 60% and less than 1% of those neurons, respectively. To identify potential target-specific differences in co-transmitter expression, we also characterized DR neurons projecting to a visual sensory structure, the lateral geniculate nucleus (LGN). The proportion of serotonergic neurons co-expressing NO was greater amongst cells targeting the mPFC vs LGN (60% vs 22%). The established role of 5-HT in affective disorders and the emerging role of NO in stress signaling suggest that the impact of 5-HT/NO co-localization in DR neurons that regulate mPFC circuit function may be clinically relevant. Copyright © 2017 Elsevier B.V. All rights reserved.

Lamin A/C might be involved in the EMT signalling pathway.

PubMed

Zuo, Lingkun; Zhao, Huanying; Yang, Ronghui; Wang, Liyong; Ma, Hui; Xu, Xiaoxue; Zhou, Ping; Kong, Lu

2018-07-15

We have previously reported a heterogeneous expression pattern of the nuclear membrane protein lamin A/C in low- and high-Gleason score (GS) prostate cancer (PC) tissues, and we have now found that this change is not associated with LMNA mutations. This expression pattern appears to be similar to the process of epithelial to mesenchymal transition (EMT) or to that of mesenchymal to epithelial transition (MET). The role of lamin A/C in EMT or MET in PC remains unclear. Therefore, we first investigated the expression levels of and the associations between lamin A/C and several common EMT markers, such as E-cadherin, N-cadherin, β-catenin, snail, slug and vimentin in PC tissues with different GS values and in different cell lines with varying invasion abilities. Our results suggest that lamin A/C might constitute a type of epithelial marker that better signifies EMT and MET in PC tissue, since a decrease in lamin A/C expression in GS 4 + 5 cases is likely associated with the EMT process, while the re-expression of lamin A/C in GS 5 + 4 cases is likely linked with MET. The detailed GS better exhibited the changes in lamin A/C and the EMT markers examined. Lamin A/C overexpression or knockdown had an impact on EMT biomarkers in a cell model by direct regulation of β-catenin. Hence, we suggest that lamin A/C might serve as a reliable epithelial biomarker for the distinction of PC cell differentiation and might also be a fundamental factor in the occurrence of EMT or MET in PC. Copyright © 2018. Published by Elsevier B.V.

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.

CD44 expression in curettage and postoperative specimens of endometrial cancer.

PubMed

Wojciechowski, Michał; Krawczyk, Tomasz; Śmigielski, Janusz; Malinowski, Andrzej

2015-02-01

Adhesive molecules like CD44 are well defined key players in the metastatic cascade in many cancers, including endometrial cancer. They could play a role of markers of invasion, metastasis and prognostic factors. The aim of the study is to assess a possible role of the CD44 as a marker of invasion in endometrial cancer, both at the moment of preoperative workup and final staging. Available for analysis were archival specimens of 51 patients who had underwent curettage and surgery between 2002 and 2007. An immunohistochemical study for CD44 expression was performed in curettage and postoperative specimens. Normal endometrium of 20 randomly chosen patients was used as a control group. In endometrial cancer the expression of CD44 was significantly more intensive than in normal endometrium. In postoperative specimens, the CD44 expression was weaker in serous than in endometrioid cancer. There was no significant correlation between the adhesion molecule expression and clinicopathological features: grade,depth of invasion, cervical involvement, serosal and adnexal involvement, lymph-vascular space involvement, lymph node and distant metastases nor FIGO stage. An increased expression of CD44 in endometrial cancer suggests its possible role in pathogenesis of this disease, however, it doesn't seem to be crucial. Different expression of the CD44 in endometrioid and papillary-serous type may reflect different pathogenesis of these types of cancer. No statistically proved relation between the investigated molecule expression and clinicopathological parameters suggests scepticism about its use in diagnostic process of endometrial cancer.

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.

FAP-1 and NF-κB expressions in oral squamous cell carcinoma as potential markers for chemo-radio sensitivity and prognosis.

PubMed

Nariai, Y; Mishima, K; Yoshimura, Y; Sekine, J

2011-04-01

This study was designed to investigate the feasibility of using Fas-associated phosphatase-1 (FAP-1), nuclear factor kappa B (NF-κB) and p53 as markers for chemo-radio sensitivity in oral squamous cell carcinoma (OSCC). FAP-1 plays a role as an anti-apoptotic factor through Fas-dependent apoptosis after chemo-radiotherapy. NF-κB and p53 might be involved in modulation of FAP-1 expression. FAP-1, NF-κB and p53 expression were immunohistochemically examined using biopsy specimens in 50 OSCC patients treated with chemotherapy and/or radiotherapy. FAP-1 was expressed in 52%, NF-κB in 52% and p53 in 46% of patients. There was no significant difference in FAP-1, p53 or NF-κB expression according to the clinicopathological features. No correlation was found among FAP-1, p53 or NF-κB expression. FAP-1-positive cases showed a poorer survival rate than FAP-1-negative cases (P = 0.0409) and NF-κB-positive cases showed a poorer survival rate than NF-κB-negative cases (P = 0.0018). Multivariate analysis showed that FAP-1 expression, NF-κB expression, clinical stage and age were significant independent variables for survival (clinical stage: P = 0.0016; age: P = 0.0016; NF-κB: P = 0.0314; FAP-1: P = 0.0366). These results suggest that FAP-1 and NF-κB might play a role as chemo-radioresistant factor during chemo-radiotherapy, and FAP-1 and NF-κB expression in OSCC would be feasible markers for chemo-radio sensitivity and prognosis. Copyright © 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

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.

CD56 Expression in Odontogenic Cysts and Tumors.

PubMed

Jaafari-Ashkavandi, Zohreh; Dehghani-Nazhvani, Ali; Razmjouyi, Faranak

2014-01-01

Background and aims. Odontogenic cysts and tumors have a wide spectrum of clinical characteristics that lead to the different management strategies. Since definite diagnosis is difficult in some cases, it has been suggested that CD56 may be a candidate marker for definitive diagnosis of some odontogenic tumors. The present study was designed to examine CD56 expression in lesions with histopathological similarities. Materials and methods. In this cross-sectional, analytical study the subjects were 22 ameloblastomas, 13 dentigerous cysts, 10 keratocystic odontogenic tumors (KCOT), 4 adenomatoid odontogenic tumors (AOT), 3 orthokeratinized odonto-genic cysts, 3 calcifying odontogenic cysts (COC) and one glandular odontogenic cyst (GOC). All the samples were examined for CD56 immunoreactivity. Data were analyzed using chi-square test. Results. Twenty cases (91%) of ameloblastomas, 3 (75%) AOT, 4 (40%) KCOT and one case of GOC were positive for CD56. None of the dentigerous cysts, COC and orthokeratinized odontogenic cysts was CD56-positive. There was a significant difference in the CD56 expression between ameloblastoma and dentigerous cyst, as well as COC. Also, KCOT showed significantly higher expression than orthokeratinized odontogenic cyst. Conclusion. In this study CD56 expression was limited to the odontogenic tumors and more aggressive cystic lesions. This marker can be a useful aid for distinguishing cysts and tumors from similar lesions.

Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1.

PubMed

Darwish, Noureldien H E; Sudha, Thangirala; Godugu, Kavitha; Elbaz, Osama; Abdelghaffar, Hasan A; Hassan, Emad E A; Mousa, Shaker A

2016-09-06

Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.

A 3-Protein Expression Signature of Neuroblastoma for Outcome Prediction.

PubMed

Xie, Yi; Xu, Hua; Fang, Fang; Li, Zhiheng; Zhou, Huiting; Pan, Jian; Guo, Wanliang; Zhu, Xueming; Wang, Jian; Wu, Yi

2018-05-22

Neuroblastoma (NB) is the most common extracranial solid tumor in children with contrasting outcomes. Precise risk assessment contributes to prognosis prediction, which is critical for treatment strategy decisions. In this study, we developed a 3-protein predictor model, including the neural stem cell marker Msi1, neural differentiation marker ID1, and proliferation marker proliferating cell nuclear antigen (PCNA), to improve clinical risk assessment of patients with NB. Kaplan-Meier analysis in the microarray data (GSE16476) revealed that low expression of ID1 and high expression of Msi1 and PCNA were associated with poor prognosis in NB patients. Combined application of these 3 markers to constitute a signature further stratified NB patients into different risk subgroups can help obtain more accurate prediction performance. Survival prognostic power of age and Msi1_ID1_PCNA signature by receiver operating characteristics analysis showed that this signature predicted more effectively and sensitively compared with classic risk stratification system, compensating for the deficiency of the prediction function of the age. Furthermore, we validated the expressions of these 3 proteins in neuroblastic tumor spectrum tissues by immunohistochemistry revealed that Msi1 and PCNA exhibited increased expression in NB compared with intermedial ganglioneuroblastoma and benign ganglioneuroma, whereas ID1 levels were reduced in NB. In conclusion, we established a robust risk assessment predictor model based on simple immunohistochemistry for therapeutic decisions of NB patients.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

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

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays

PubMed Central

Chan, Robin F.; Lewellyn, Lara; DeLoyht, Jacqueline M.; Sennett, Kristyn; Coffman, Scarlett; Hewitt, Matthew; Bettinger, Jill C.; Warrick, John M.; Grotewiel, Mike

2014-01-01

Background The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white, a derivative of the endogenous gene white. Whether the mini-white transgenic marker or the endogenous white gene influence behavioral responses to acute ethanol exposure in flies has not been systematically investigated. Methods We manipulated mini-white and white expression via (i) transposons marked with mini-white, (ii) RNAi against mini-white and white and (iii) a null allele of white. We assessed ethanol sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of ethanol) and an assay based on ethanol-induced sedation. Results In eRING assays, ethanol-induced impairment of climbing correlated inversely with expression of the mini-white marker from a series of transposon insertions. Additionally, flies harboring a null allele of white or flies with RNAi-mediated knockdown of mini-white were significantly more sensitive to ethanol in eRING assays than controls expressing endogenous white or the mini-white marker. In contrast, ethanol sensitivity and rapid tolerance measured in the ethanol sedation assay were not affected by decreased expression of mini-white or endogenous white in flies. Conclusions Ethanol sensitivity measured in the eRING assay is noticeably influenced by white and mini-white, making eRING problematic for studies on ethanol-related behavior in Drosophila using transgenes marked with mini-white. In contrast, the ethanol sedation assay described here is a suitable behavioral paradigm for studies on ethanol sedation and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-white. PMID:24890118

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

Stem-Like Cell Characteristics from Breast Milk of Mothers with Preterm Infants as Compared to Mothers with Term Infants.

PubMed

Briere, Carrie-Ellen; Jensen, Todd; McGrath, Jacqueline M; Young, Erin E; Finck, Christine

2017-04-01

Breast milk stem cells are hypothesized to be involved in infant health and development. Our research team is the first known team to enroll mothers of hospitalized preterm infants during the first few weeks of lactation and compare stem cell phenotypes and gene expression to mothers of healthy full-term infants. Participants were recruited from a Level IV Neonatal Intensive Care Unit (preterm dyads) and the community (full-term dyads) in the northeastern United States. Mothers of hospitalized preterm infants (<37 weeks gestational age at birth) and mothers of healthy full-term infants (>39 weeks gestational age at birth). Breast milk stem-like cell populations were identified in both preterm and full-term breast milk samples. The data suggest variability in the proportion of stem cell phenotypes present, as well as statistically significant differential expression (both over- and underexpression) of stem cell-specific genetic markers when comparing mothers' milk for preterm and full-term births. Our findings indicate that (1) stem cells are present in preterm breast milk; (2) differential expression of stem cell-specific markers can be detected in preterm and full-term breast milk samples; and (3) the percentage of cells expressing the various stem cell-specific markers differs when preterm and full-term breast milk samples are compared.

The effects of tumor location on diagnostic criteria for canine malignant peripheral nerve sheath tumors (MPNSTs) and the markers for distinction between canine MPNSTs and canine perivascular wall tumors.

PubMed

Suzuki, S; Uchida, K; Nakayama, H

2014-07-01

Canine malignant peripheral nerve sheath tumors (MPNSTs) occur not only in the peripheral nervous system (PNS) but also in soft tissue and various organs (non-PNS). The most important diagnostic criterion is proof of peripheral nerve sheath origin. This is difficult in non-PNS MPNSTs, and its differential diagnosis is challenging. Canine perivascular wall tumors (PWTs) also commonly arise in soft tissue. Their histopathological features are quite similar to those of canine MPNSTs, making their differential diagnosis challenging. To elucidate whether the morphological features are applicable to diagnose non-PNS MPNSTs and to demonstrate useful markers for distinction between canine MPNSTs and PWTs, the authors examined 30 canine MPNSTs and 31 PWTs immunohistochemically for S100, nestin, NGFR, Olig2, claudin-1, CD57, PRX, α-SMA, desmin, and calponin. Among canine MPNSTs, the PNS tumors displayed significantly higher S100 and Olig2 expression than the non-PNS tumors. The expression levels of the other markers did not differ significantly, suggesting that the same morphological diagnostic criteria are applicable regardless of their location. The PWT cells displayed significantly weaker immunoreactivity than MPNSTs to markers used except α-SMA and desmin. Cluster analysis sorted most canine MPNSTs and PWTs into 2 distinctly different clusters, whereas 3 MPNSTs and 6 PWTs were assigned to the opposing cluster. These 3 MPNSTs were negative for almost all markers, while these 6 PWTs were positive for only neuronal markers. In particular, NGFR and Olig2 were almost negative in the rest of PWT cases. These findings suggest that NGFR and Olig2 are useful to distinguish these 2 tumors. © The Author(s) 2013.

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.

Distribution of glutamatergic, GABAergic, and glycinergic neurons in the auditory pathways of macaque monkeys.

PubMed

Ito, T; Inoue, K; Takada, M

2015-12-03

Macaque monkeys use complex communication calls and are regarded as a model for studying the coding and decoding of complex sound in the auditory system. However, little is known about the distribution of excitatory and inhibitory neurons in the auditory system of macaque monkeys. In this study, we examined the overall distribution of cell bodies that expressed mRNAs for VGLUT1, and VGLUT2 (markers for glutamatergic neurons), GAD67 (a marker for GABAergic neurons), and GLYT2 (a marker for glycinergic neurons) in the auditory system of the Japanese macaque. In addition, we performed immunohistochemistry for VGLUT1, VGLUT2, and GAD67 in order to compare the distribution of proteins and mRNAs. We found that most of the excitatory neurons in the auditory brainstem expressed VGLUT2. In contrast, the expression of VGLUT1 mRNA was restricted to the auditory cortex (AC), periolivary nuclei, and cochlear nuclei (CN). The co-expression of GAD67 and GLYT2 mRNAs was common in the ventral nucleus of the lateral lemniscus (VNLL), CN, and superior olivary complex except for the medial nucleus of the trapezoid body, which expressed GLYT2 alone. In contrast, the dorsal nucleus of the lateral lemniscus, inferior colliculus, thalamus, and AC expressed GAD67 alone. The absence of co-expression of VGLUT1 and VGLUT2 in the medial geniculate, medial superior olive, and VNLL suggests that synaptic responses in the target neurons of these nuclei may be different between rodents and macaque monkeys. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

DOEpatents

Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

2007-02-27

The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

Immunohistochemical Expression Of Ezrin In Oral Potentially Malignant Disorders-A Descriptive Study.

PubMed

Mohanraj, Raghini; Ramani, Pratibha; Premkumar, Priya; Natesan, Anuja; Sherlin, Herald J; Sukumaran, Gheena

2017-11-01

Ezrin, also known as cytovillin, is a member of the ERM family of protein. Ezrin cross-links actin filament with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility, and membrane trafficking. Recent analysis reveals their involvement in signaling pathways. Ezrin is highly expressed in several types of human cancers, and correlation between its immunoreactivity and histopathological data as well as the patient outcome has previously been studied. The objective of the study was to analyze the immunohistochemical expression pattern of ezrin in oral potentially malignant disorders (OPMDs), namely, oral submucous fibrosis (OSMF) with different grades and clinically leucoplakia (hyperkeratosis with various degree of dysplasia) and its use as a predictive marker for malignant transformation. Sample size n = 43, histopathologically confirmed cases of OPMDs (13 cases of OSMF with different grades and 30 cases of clinically leukoplakia) were retrieved from the Department of Oral and Maxillofacial Pathology. Immunohistochemistry was done using anti-ezrin antibody, and the expression was graded in terms of proportion and intensity. There was a significant expression of ezrin in OPMDs, and its cytoplasmic shift can be used as a predictive marker for malignant transformation. The findings of the current study revealed that the expression of ezrin in OPMDs may be related to the progression of the disease.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

Molecular classification and molecular forecasting of breast cancer: ready for clinical application?

PubMed

Brenton, James D; Carey, Lisa A; Ahmed, Ahmed Ashour; Caldas, Carlos

2005-10-10

Profiling breast cancer with expression arrays has become common, and it has been suggested that the results from early studies will lead to understanding of the molecular differences between clinical cases and allow individualization of care. We critically review two main applications of expression profiling; studies unraveling novel breast cancer classifications and those that aim to identify novel markers for prediction of clinical outcome. Breast cancer may now be subclassified into luminal, basal, and HER2 subtypes with distinct differences in prognosis and response to therapy. However, profiling studies to identify predictive markers have suffered from methodologic problems that prevent general application of their results. Future work will need to reanalyze existing microarray data sets to identify more representative sets of candidate genes for use as prognostic signatures and will need to take into account the new knowledge of molecular subtypes of breast cancer when assessing predictive effects.

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.

Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof.

PubMed

Linardi, Renata L; Megee, Susan O; Mainardi, Sarah R; Senoo, Makoto; Galantino-Homer, Hannah L

2015-08-01

The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. To the best of the author's knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses. © 2015 ESVD and ACVD.

Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

PubMed

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

2013-08-01

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

Lymphangiogenesis assessed using three methods is related to tumour grade, breast cancer subtype and expression of basal marker.

PubMed

Niemiec, Joanna; Adamczyk, Agnieszka; Ambicka, Aleksandra; Mucha-Małecka, Anna; Wysocki, Wojciech; Mituś, Jerzy; Ryś, Janusz

2012-11-01

Lymphangiogenesis is a potential indicator of cancer patients' survival. However, there is no standardisation of methodologies applied to the assessment of lymphatic vessel density. In 156 invasive ductal breast cancers (T  1/N+/M0), lymphatic and blood vessels were visualised using podoplanin and CD34, respectively. Based on these markers expression, four parameters were assessed: (i) distribution of podoplanin-stained vessels (DPV) - the percentage of fields with at least one lymphatic vessel (a simple method proposed by us), (ii) lymphatic vessel density (LVD), (iii) LVD to microvessel density ratio (LVD/MVD) and (iv) the expression of podoplanin in cancer-associated fibroblasts. Next, we estimated relations between the above-mentioned parameters and: (i) breast cancer subtype, (ii) tumour grade, and (iii) basal markers expression. We found that intensive lymphangiogenesis, assessed using all studied methods, is positively related to high tumour grade, triple negative or HER2 subtype and expression of basal markers. Whereas, the absence of podoplanin expression in fibroblasts of cancer stroma is related to luminal A subtype, low tumour grade or lack of basal markers expression. Distribution of podoplanin-stained vessels, assessed by a simple method proposed by us (indicating the percentage of fields with at least one lymphatic vessel), might be used instead of the "hot-spot" method.

Transcriptional mechanisms of resistance to anti-PD-1 therapy

PubMed Central

Ascierto, Maria L.; Makohon-Moore, Alvin; Lipson, Evan J.; Taube, Janis M.; McMiller, Tracee L.; Berger, Alan E.; Fan, Jinshui; Kaunitz, Genevieve J.; Cottrell, Tricia R.; Kohutek, Zachary A.; Favorov, Alexander; Makarov, Vladimir; Riaz, Nadeem; Chan, Timothy A.; Cope, Leslie; Hruban, Ralph H.; Pardoll, Drew M.; Taylor, Barry S.; Solit, David B.; Iacobuzio-Donahue, Christine A; Topalian, Suzanne L.

2017-01-01

Purpose To explore factors associated with response and resistance to anti-PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Materials and Methods Twenty-six melanoma specimens (four pre-mortem, 22 post-mortem) were subjected to whole-exome sequencing. Candidate immunologic markers and gene expression were assessed in ten cutaneous metastases showing response or progression during therapy. Results The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with over-expression of genes associated with extracellular matrix and neutrophil function. Conclusions Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti-PD-1 outcomes. PMID:28193624

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

PubMed

Peltzer, J; Colman, L; Cebrian, J; Musa, H; Peckham, M; Keller, A

2008-05-01

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Clear cell and endometrioid carcinomas: are their differences attributable to distinct cells of origin?

PubMed

Cochrane, Dawn R; Tessier-Cloutier, Basile; Lawrence, Katherine M; Nazeran, Tayyebeh; Karnezis, Anthony N; Salamanca, Clara; Cheng, Angela S; McAlpine, Jessica N; Hoang, Lien N; Gilks, C Blake; Huntsman, David G

2017-09-01

Endometrial epithelium is the presumed tissue of origin for both eutopic and endometriosis-derived clear cell and endometrioid carcinomas. We had previously hypothesized that the morphological, biological and clinical differences between these carcinomas are due to histotype-specific mutations. Although some mutations and genomic landscape features are more likely to be found in one of these histotypes, we were not able to identify a single class of mutations that was exclusively present in one histotype and not the other. This lack of genomic differences led us to an alternative hypothesis that these cancers could arise from distinct cells of origin within endometrial tissue, and that it is the cellular context that accounts for their differences. In a proteomic screen, we identified cystathionine γ-lyase (CTH) as a marker for clear cell carcinoma, as it is expressed at high levels in clear cell carcinomas of the ovary and endometrium. In the current study, we analysed normal Müllerian tissues, and found that CTH is expressed in ciliated cells of endometrium (both eutopic endometrium and endometriosis) and fallopian tubes. We then demonstrated that other ciliated cell markers are expressed in clear cell carcinomas, whereas endometrial secretory cell markers are expressed in endometrioid carcinomas. The same differential staining of secretory and ciliated cells was demonstrable in a three-dimensional organoid culture system, in which stem cells were stimulated to differentiate into an admixture of secretory and ciliated cells. These data suggest that endometrioid carcinomas are derived from cells of the secretory cell lineage, whereas clear cell carcinomas are derived from, or have similarities to, cells of the ciliated cell lineage. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oxytocin Modulates Expression of Neuron and Glial Markers in the Rat Hippocampus.

PubMed

Havránek, T; Lešťanová, Z; Mravec, B; Štrbák, V; Bakoš, J; Bačová, Z

2017-01-01

Neuropeptides including oxytocin belong to the group of factors that may play a role in the control of neuronal cell survival, proliferation and differentiation. The aim of the present study was to investigate potential contribution of oxytocin to neuronal differentiation by measuring gene and protein expression of specific neuron and glial markers in the brain. Neonatal and adult oxytocin administration was used to reveal developmental and/or acute effects of oxytocin in Wistar rats. Gene and protein expression of neuron-specific enolase (NSE) in the hippocampus was increased in 21-day and 2-month old rats in response to neonatal oxytocin administration. Neonatal oxytocin treatment induced a significant increase of gene and protein expression of the marker of astrocytes - glial fibrillary acid protein (GFAP). Oxytocin treatment resulted in a decrease of oligodendrocyte marker mRNA - 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) - in 21-day and 2-month old rats, while no change of CD68 mRNA, marker of microglia, was observed. Central oxytocin administration in adult rats induced a significant increase of gene expression of NSE and CNPase. The present study provides the first data revealing the effect of oxytocin on the expression of neuron and glial markers in the brain. It may be suggested that the oxytocin system is involved in the regulation of development of neuronal precursor cells in the brain.

Serglycin as a potential biomarker for glioma: association of serglycin expression, extent of mast cell recruitment and glioblastoma progression

PubMed Central

Roy, Ananya; Attarha, Sanaz; Weishaupt, Holger; Edqvist, Per-Henrik; Swartling, Fredrik J.; Bergqvist, Michael; Siebzehnrubl, Florian A.; Smits, Anja; Pontén, Fredrik; Tchougounova, Elena

2017-01-01

Serglycin is an intracellular proteoglycan with a unique ability to adopt highly divergent structures by glycosylation with variable types of glycosaminoglycans (GAGs) when expressed by different cell types. Serglycin is overexpressed in aggressive cancers suggesting its protumorigenic role. In this study, we explored the expression of serglycin in human glioma and its correlation with survival and immune cell infiltration. We demonstrate that serglycin is expressed in glioma and that increased expression predicts poor survival of patients. Analysis of serglycin expression in a large cohort of low- and high-grade human glioma samples reveals that its expression is grade dependent and is positively correlated with mast cell (MC) infiltration. Moreover, serglycin expression in patient-derived glioma cells is significantly increased upon MC co-culture. This is also accompanied by increased expression of CXCL12, CXCL10, as well as markers of cancer progression, including CD44, ZEB1 and vimentin. In conclusion, these findings indicate the importance of infiltrating MCs in glioma by modulating signaling cascades involving serglycin, CD44 and ZEB1. The present investigation reveals serglycin as a potential prognostic marker for glioma and demonstrates an association with the extent of MC recruitment and glioma progression, uncovering potential future therapeutic opportunities for patients. PMID:28445977

Indian hedgehog contributes to human cartilage endplate degeneration.

PubMed

Wang, Shaowei; Yang, Kun; Chen, Shuai; Wang, Jiying; Du, Guoqing; Fan, Shunwu; Wei, Lei

2015-08-01

To determine the role of Indian hedgehog (Ihh) signaling in human cartilage endplate (CEP) degeneration. CEP-degenerated tissues from patients with Modic I or II changes (n = 9 and 45, respectively) and normal tissues from vertebral burst fracture patients (n = 17) were collected. Specimens were either cut into slices for organ culture ex vivo or digested to isolate chondrocytes for cell culture in vitro. Ihh expression and the effect of Ihh on cartilage degeneration were determined by investigating degeneration markers in this study. Ihh expression and cartilage degeneration markers significantly increased in the Modic I and II groups. The expression of cartilage degeneration markers was positively correlated with degeneration severity. Gain-of-function for Ihh promoted expression of cartilage degeneration markers in vitro, while loss-of-function for Ihh inhibited their expression both in vitro and ex vivo. These findings demonstrated that Ihh promotes CEP degeneration. Blocking Ihh pathway has potential clinical usage for attenuating CEP degeneration.

Puromycin and Methotrexate Resistance Cassettes and Optimized cre-recombinase Expression Plasmids for use in Yeast

PubMed Central

MacDonald, Chris; Piper, Robert C.

2015-01-01

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy to use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR) that confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites allowing for their excision from the genome following expression of cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers. PMID:25688547

Modulatory efficacy of green tea polyphenols on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis-A therapeutic approach.

PubMed

Srinivasan, Periasamy; Sabitha, Kuruvimalai Ekambaram; Shyamaladevi, Chennam Srinivasulu

2006-08-25

Green tea polyphenols (GTP) has been used as a chemopreventive agent world wide against chemically induced cancer. The present study is aimed to understand the therapeutic action of GTP on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide (4-NQO)-induced oral cancer over a period of 30 days at 200mg/kg, p.o., Oral cancer was induced by painting 4-NQO for 8 weeks followed by administration of GTP after 22 weeks, for 30 days. Glycoconjugates such as hexose, hexosamine, sialicacid, fucose and mucoprotein were analysed. Expression of glycoconjugates was examined through histology and SDS-PAGE. Immunological markers such as circulating immune complex and mast cell density were studied. Oral cancer-induced animals showed a significant increase in levels of glycoconjugates and its expression, similar to that observed for immunological markers. Treatment with GTP altered the expression of glycoconjugates as well as immunological markers. The results suggest that GTP modulates both the expression of glycoconjugates and immunological markers resulting in regression of oral cancer.

Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

PubMed

Garnache-Ottou, Francine; Chaperot, Laurence; Biichle, Sabeha; Ferrand, Christophe; Remy-Martin, Jean-Paul; Deconinck, Eric; de Tailly, Patrick Darodes; Bulabois, Bénédicte; Poulet, Jacqueline; Kuhlein, Emilienne; Jacob, Marie-Christine; Salaun, Véronique; Arock, Michel; Drenou, Bernard; Schillinger, Françoise; Seilles, Estelle; Tiberghien, Pierre; Bensa, Jean-Claude; Plumas, Joel; Saas, Philippe

2005-02-01

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

Investigation of apoptosis-related gene expression levels in preimplantation biopsies as predictors of delayed kidney graft function.

PubMed

Goncalves-Primo, Amador; Mourão, Tuíla B; Andrade-Oliveira, Vinícius; Campos, Erika F; Medina-Pestana, José O; Tedesco-Silva, Hélio; Gerbase-DeLima, Maria

2014-06-27

The purpose of this study was to investigate the expression of the gene coding for the antiapoptotic molecule Bcl-2, the proapoptotic molecule Bax, and the apoptosis executor enzyme caspase-3 in preimplantation renal biopsies (PIB) as markers for delayed graft function. In this prospective single-center study, gene expression levels were evaluated using real-time TaqMan polymerase chain reaction in PIB of kidneys from 72 deceased donors (DDs) and 18 living donors (LDs). CASP3 and BAX expression levels were higher, whereas those of BCL2 were lower, in DD than in LD PIB. In biopsies from DD, BCL2 levels were lower in cases with DGF, whereas no differences were observed concerning CASP3 and BAX. The BAX/BCL2 gene expression ratio greater than 2.29 associated with DGF with an odds ratio of 2.00. A multiple regression analysis including data of TLR4 expression in the first day posttransplant PB from a previous study of our group conducted in the same patients revealed a very strong association of the combination of BAX/BCL2 greater than 2.3 in PIB and TLR4 of 0.95 uRE or lesser in PB with the occurrence of DGF, with OR of 120 and positive and negative predictive values of 91% and 92%, respectively. The power to predict DGF of the combination of high BAX/BCL2 expression in PIB and low TLR4 expression in the first day posttransplant peripheral blood observed in the present study is extremely high, in comparison to any other marker or combinations of markers so far published in the literature.

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

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.

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.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers.

PubMed

Robinson, Philip C; Lau, Eugene; Keith, Patricia; Lau, Max C; Thomas, Gethin P; Bradbury, Linda A; Brown, Matthew A; Kenna, Tony J

2015-11-01

Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. There was no significant difference in HLA-class I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Large differences were not seen in HLA-B27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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.

Optimal tumor sampling for immunostaining of biomarkers in breast carcinoma

PubMed Central

2011-01-01

Introduction Biomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidence-based standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau. Methods Two collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set. Results The optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement. Conclusions The optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis. PMID:21592345

Small RNA-based prediction of hybrid performance in maize.

PubMed

Seifert, Felix; Thiemann, Alexander; Schrag, Tobias A; Rybka, Dominika; Melchinger, Albrecht E; Frisch, Matthias; Scholten, Stefan

2018-05-21

Small RNA (sRNA) sequences are known to have a broad impact on gene regulation by various mechanisms. Their performance for the prediction of hybrid traits has not yet been analyzed. Our objective was to analyze the relation of parental sRNA expression with the performance of their hybrids, to develop a sRNA-based prediction approach, and to compare it to more common SNP and mRNA transcript based predictions using a factorial mating scheme of a maize hybrid breeding program. Correlation of genomic differences and messenger RNA (mRNA) or sRNA expression differences between parental lines with hybrid performance of their hybrids revealed that sRNAs showed an inverse relationship in contrast to the other two data types. We associated differences for SNPs, mRNA and sRNA expression between parental inbred lines with the performance of their hybrid combinations and developed two prediction approaches using distance measures based on associated markers. Cross-validations revealed parental differences in sRNA expression to be strong predictors for hybrid performance for grain yield in maize, comparable to genomic and mRNA data. The integration of both positively and negatively associated markers in the prediction approaches enhanced the prediction accurary. The associated sRNAs belong predominantly to the canonical size classes of 22- and 24-nt that show specific genomic mapping characteristics. Expression profiles of sRNA are a promising alternative to SNPs or mRNA expression profiles for hybrid prediction, especially for plant species without reference genome or transcriptome information. The characteristics of the sRNAs we identified suggest that association studies based on breeding populations facilitate the identification of sRNAs involved in hybrid performance.

ASTROCYTE PATHOLOGY IN MAJOR DEPRESSIVE DISORDER: INSIGHTS FROM HUMAN POSTMORTEM BRAIN TISSUE

PubMed Central

Rajkowska, Grazyna; Stockmeier, Craig A.

2013-01-01

The present paper reviews astrocyte pathology in major depressive disorder (MDD) and proposes that reductions in astrocytes and related markers are key features in the pathology of MDD. Astrocytes are the most numerous and versatile of all types of glial cells. They are crucial to the neuronal microenvironment by regulating glucose metabolism, neurotransmitter uptake (particularly for glutamate), synaptic development and maturation and the blood brain barrier. Pathology of astrocytes has been consistently noted in MDD as well as in rodent models of depressive-like behavior. This review summarizes evidence from human postmortem tissue showing alterations in the expression of protein and mRNA for astrocyte markers such as glial fibrillary acidic protein (GFAP), gap junction proteins (connexin 40 and 43), the water channel aquaporin-4 (AQP4), a calcium-binding protein S100B and glutamatergic markers including the excitatory amino acid transporters 1 and 2 (EAAT1, EAAT2) and glutamine synthetase. Moreover, preclinical studies are presented that demonstrate the involvement of GFAP and astrocytes in animal models of stress and depressive-like behavior and the influence of different classes of antidepressant medications on astrocytes. In light of the various astrocyte deficits noted in MDD, astrocytes may be novel targets for the action of antidepressant medications. Possible functional consequences of altered expression of astrocytic markers in MDD are also discussed. Finally, the unique pattern of cell pathology in MDD, characterized by prominent reductions in the density of astrocytes and in the expression of their markers without obvious neuronal loss, is contrasted with that found in other neuropsychiatric and neurodegenerative disorders. PMID:23469922

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.

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.

Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis.

PubMed

Dharmaraj, N; Chapela, P J; Morgado, M; Hawkins, S M; Lessey, B A; Young, S L; Carson, D D

2014-08-01

Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or β-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to β-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare. © The Author 2014. 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.

Different Levels of Expression of the Clock Protein PER and the Glial Marker REPO in Ensheathing and Astrocyte-Like Glia of the Distal Medulla of Drosophila Optic Lobe

PubMed Central

Krzeptowski, Wojciech; Walkowicz, Lucyna; Płonczyńska, Alicja; Górska-Andrzejak, Jolanta

2018-01-01

Circadian plasticity of the visual system of Drosophila melanogaster depends on functioning of both the neuronal and glial oscillators. The clock function of the former is already quite well-recognized. The latter, however, is much less known and documented. In this study we focus on the glial oscillators that reside in the distal part of the second visual neuropil, medulla (dMnGl), in vicinity of the PIGMENT-DISPERSING FACTOR (PDF) releasing terminals of the circadian clock ventral Lateral Neurons (LNvs). We reveal the heterogeneity of the dMnGl, which express the clock protein PERIOD (PER) and the pan-glial marker REVERSED POLARITY (REPO) at higher (P1) or lower (P2) levels. We show that the cells with stronger expression of PER display also stronger expression of REPO, and that the number of REPO-P1 cells is bigger during the day than during the night. Using a combination of genetic markers and immunofluorescent labeling with anti PER and REPO Abs, we have established that the P1 and P2 cells can be associated with two different types of the dMnGl, the ensheathing (EnGl), and the astrocyte-like glia (ALGl). Surprisingly, the EnGl belong to the P1 cells, whereas the ALGl, previously reported to play the main role in the circadian rhythms, display the characteristics of the P2 cells (express very low level of PER and low level of REPO). Next to the EnGl and ALGl we have also observed another type of cells in the distal medulla that express PER and REPO, although at very low levels. Based on their morphology we have identified them as the T1 interneurons. Our study reveals the complexity of the distal medulla circadian network, which appears to consist of different types of glial and neuronal peripheral clocks, displaying molecular oscillations of higher (EnGl) and lower (ALGl and T1) amplitudes. PMID:29695973

Different Levels of Expression of the Clock Protein PER and the Glial Marker REPO in Ensheathing and Astrocyte-Like Glia of the Distal Medulla of Drosophila Optic Lobe.

PubMed

Krzeptowski, Wojciech; Walkowicz, Lucyna; Płonczyńska, Alicja; Górska-Andrzejak, Jolanta

2018-01-01

Circadian plasticity of the visual system of Drosophila melanogaster depends on functioning of both the neuronal and glial oscillators. The clock function of the former is already quite well-recognized. The latter, however, is much less known and documented. In this study we focus on the glial oscillators that reside in the distal part of the second visual neuropil, medulla (dMnGl), in vicinity of the PIGMENT-DISPERSING FACTOR (PDF) releasing terminals of the circadian clock ventral Lateral Neurons (LNvs). We reveal the heterogeneity of the dMnGl, which express the clock protein PERIOD (PER) and the pan-glial marker REVERSED POLARITY (REPO) at higher (P1) or lower (P2) levels. We show that the cells with stronger expression of PER display also stronger expression of REPO, and that the number of REPO-P1 cells is bigger during the day than during the night. Using a combination of genetic markers and immunofluorescent labeling with anti PER and REPO Abs, we have established that the P1 and P2 cells can be associated with two different types of the dMnGl, the ensheathing (EnGl), and the astrocyte-like glia (ALGl). Surprisingly, the EnGl belong to the P1 cells, whereas the ALGl, previously reported to play the main role in the circadian rhythms, display the characteristics of the P2 cells (express very low level of PER and low level of REPO). Next to the EnGl and ALGl we have also observed another type of cells in the distal medulla that express PER and REPO, although at very low levels. Based on their morphology we have identified them as the T1 interneurons. Our study reveals the complexity of the distal medulla circadian network, which appears to consist of different types of glial and neuronal peripheral clocks, displaying molecular oscillations of higher (EnGl) and lower (ALGl and T1) amplitudes.

4PBA strongly attenuates endoplasmic reticulum stress, fibrosis, and mitochondrial apoptosis markers in cyclosporine treated human gingival fibroblasts.

PubMed

Ranga Rao, Suresh; Subbarayan, Rajasekaran; Ajitkumar, Supraja; Murugan Girija, Dinesh

2018-01-01

Cyclosporine induces overgrowth of human gingiva. Previously we have shown (i) cyclosporine-inducing ER stress in human gingival fibroblasts (HGF), (ii) increased matrix protein expression, and (iii) interference with mitochondrial pro- and anti-apoptotic factors. This study was undertaken to assess the effects of melatonin (an antioxidant), 4PBA (an ER stress inhibitor), and simvastatin on the expression of ER Stress markers as well as on matrix and mitochondrial markers. HGF incubated with cyclosporine, or without melatonin/4PBA/statin. After 24 hr of incubation, mRNA expression of ER stress markers (GRP78, CHOP, XBP1, and XBPs) and matrix protein markers (like α-SMA, VEGF, TGF-β, CTGF), and mitochondrial apoptosis markers estimated and compared with housekeeping gene GAPDH. Compared to the control cyclosporine significantly augmented ER Stress and matrix proteins, which decreased significantly with the use of melatonin, 4PBA, and simvastatin. The mitochondrial proapoptotic molecule cyclophilin D, as well as Bcl2 expression also decreased after PBA treatment, paralleling an increase in cytochrome c expression. The effect of 4PBA was much more pronounced than the influence of other two. In conclusion, 4PBA could be a viable therapeutic option for drug-induced gingival overgrowth. © 2017 Wiley Periodicals, Inc.

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

Two EST-derived marker systems for cultivar identification in tree peony.

PubMed

Zhang, J J; Shu, Q Y; Liu, Z A; Ren, H X; Wang, L S; De Keyser, E

2012-02-01

Tree peony (Paeonia suffruticosa Andrews), a woody deciduous shrub, belongs to the section Moutan DC. in the genus of Paeonia of the Paeoniaceae family. To increase the efficiency of breeding, two EST-derived marker systems were developed based on a tree peony expressed sequence tag (EST) database. Using target region amplification polymorphism (TRAP), 19 of 39 primer pairs showed good amplification for 56 accessions with amplicons ranging from 120 to 3,000 bp long, among which 99.3% were polymorphic. In contrast, 7 of 21 primer pairs demonstrated adequate amplification with clear bands for simple sequence repeats (SSRs) developed from ESTs, and a total of 33 alleles were found in 56 accessions. The similarity matrices generated by TRAP and EST-SSR markers were compared, and the Mantel test (r = 0.57778, P = 0.0020) showed a moderate correlation between the two types of molecular markers. TRAP markers were suitable for DNA fingerprinting and EST-SSR markers were more appropriate for discriminating synonyms (the same cultivars with different names due to limited information exchanged among different geographic areas). The two sets of EST-derived markers will be used further for genetic linkage map construction and quantitative trait locus detection in tree peony.

High density DNA microarrays: algorithms and biomedical applications.

PubMed

Liu, Wei-Min

2004-08-01

DNA microarrays are devices capable of detecting the identity and abundance of numerous DNA or RNA segments in samples. They are used for analyzing gene expressions, identifying genetic markers and detecting mutations on a genomic scale. The fundamental chemical mechanism of DNA microarrays is the hybridization between probes and targets due to the hydrogen bonds of nucleotide base pairing. Since the cross hybridization is inevitable, and probes or targets may form undesirable secondary or tertiary structures, the microarray data contain noise and depend on experimental conditions. It is crucial to apply proper statistical algorithms to obtain useful signals from noisy data. After we obtained the signals of a large amount of probes, we need to derive the biomedical information such as the existence of a transcript in a cell, the difference of expression levels of a gene in multiple samples, and the type of a genetic marker. Furthermore, after the expression levels of thousands of genes or the genotypes of thousands of single nucleotide polymorphisms are determined, it is usually important to find a small number of genes or markers that are related to a disease, individual reactions to drugs, or other phenotypes. All these applications need careful data analyses and reliable algorithms.

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

DTIC Science & Technology

2007-12-01

CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with... surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the DCs...cell surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the

Differential expression of miRNAs in the seminal plasma and serum of testicular cancer patients.

PubMed

Pelloni, Marianna; Coltrinari, Giulia; Paoli, Donatella; Pallotti, Francesco; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

2017-09-01

Various microRNAs from the miR-371-3 and miR-302a-d clusters have recently been proposed as markers for testicular germ cell tumours. Upregulation of these miRNAs has been found in both the tissue and serum of testicular cancer patients, but they have never been studied in human seminal plasma. The aim of this study was, therefore, to assess the differences in the expression of miR-371-3 and miR-302a-d between the seminal plasma and serum of testicular cancer patients, and to identify new potential testicular cancer markers in seminal plasma. We investigated the serum and seminal plasma of 28 pre-orchiectomy patients subsequently diagnosed with testicular cancer, the seminal plasma of another 20 patients 30 days post-orchiectomy and a control group consisting of 28 cancer-free subjects attending our centre for an andrological check-up. Serum microRNA expression was analysed using RT-qPCR. TaqMan Array Card 3.0 platform was used for microRNA profiling in the seminal plasma of cancer patients. Results for both miR-371-3 and the miR-302 cluster in the serum of testicular cancer patients were in line with literature reports, while miR-371and miR-372 expression in seminal plasma showed the opposite trend to serum. On array analysis, 37 miRNAs were differentially expressed in the seminal plasma of cancer patients, and the upregulated miR-142 and the downregulated miR-34b were validated using RT-qPCR. Our study investigated the expression of miRNAs in the seminal plasma of patients with testicular cancer for the first time. Unlike in serum, miR-371-3 cannot be considered as markers in seminal plasma, whereas miR-142 levels in seminal plasma may be a potential marker for testicular cancer.

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

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

Mantovani, Cristina; Department of Integrative Medical Biology and Surgical and Perioperative Science, Umea University, Umea; Department of Surgical and Perioperative Science, Umea University, Umea

2012-10-01

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 atmore » similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker{sup Registered-Sign} staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration. -- Highlights: Black-Right-Pointing-Pointer Aged MSC and ASC differentiated into Schwann-like cells support axon regeneration. Black-Right-Pointing-Pointer p53 expression does not appreciably influence the biology of Schwann or stem cells. Black-Right-Pointing-Pointer Notch 2 expression was similar in cells derived from animals of different ages. Black-Right-Pointing-Pointer Proliferation rates of dMSC varied little over time or with animal age.« less

Six stroma-based RNA markers diagnostic for prostate cancer in European-Americans validated at the RNA and protein levels in patients in China

PubMed Central

Zhu, Jianguo; Pan, Cong; Jiang, Jun; Deng, Mingsen; Gao, Hengjun; Men, Bozhao; McClelland, Michael; Mercola, Dan; Zhong, Wei-De; Jia, Zhenyu

2015-01-01

We previously analyzed human prostate tissue containing stroma near to tumor and from cancer-negative tissues of volunteers. Over 100 candidate gene expression differences were identified and used to develop a classifier that could detect nearby tumor with an accuracy of 97% (sensitivity = 98% and specificity = 88%) based on 364 independent test cases from primarily European American cases. These stroma-based gene signatures have the potential to identify cancer patients among those with negative biopsies. In this study, we used prostate tissues from Chinese cases to validate six of these markers (CAV1, COL4A2, HSPB1, ITGB3, MAP1A and MCAM). In validation by real-time PCR, four genes (COL4A2, HSPB1, ITGB3, and MAP1A) demonstrated significantly lower expression in tumor-adjacent stroma compared to normal stroma (p value ≤ 0.05). Next, we tested whether these expression differences could be extended to the protein level. In IHC assays, all six selected proteins showed lower expression in tumor-adjacent stroma compared to the normal stroma, of which COL4A2, HSPB1 and ITGB3 showed significant differences (p value ≤ 0.05). These results suggest that biomarkers for diagnosing prostate cancer based on tumor microenvironment may be applicable across multiple racial groups. PMID:26158290

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

No harmful effect of different Coca-cola beverages after 6 months of intake on rat testes.

PubMed

Tóthová, Lubomíra; Hodosy, Július; Mettenburg, Kathryn; Fábryová, Helena; Wagnerová, Alexandra; Bábíčková, Janka; Okuliarová, Monika; Zeman, Michal; Celec, Peter

2013-12-01

Our laboratory recently reported that a 3-month exposure of rats to cola-like beverages induced sex hormone changes. The aim of the study was to investigate the effects of various types of Coca-cola intake with different composition for 6 months on oxidative status in testes and testosterone in adult male rats. Fifty adult male Wistar rats were divided into control group drinking water, and groups drinking different Coca-cola beverages (regular Coca-cola, Coca-cola caffeine-free, Coca-cola Light and Coca-cola Zero). Oxidative and carbonyl stress markers were measured in the testicular tissue to assess oxidative status together with testicular and plasma testosterone. StAR expression in testes as a marker of steroidogenesis was quantified. No significant differences were found between the groups in any of the measured parameters. In conclusion, oxidative and carbonyl stress in testicular tissue were not influenced by drinking any type of Coca-cola. Additionally, testosterone in testes and in plasma, as well as testicular StAR expression were comparable among the groups. Copyright © 2013 Elsevier 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

Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

PubMed Central

Muthamilarasan, Mehanathan; Venkata Suresh, B.; Pandey, Garima; Kumari, Kajal; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei's average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species. PMID:24086082

Survivin expression in head and neck squamous cell carcinomas is frequent and correlates with clinical parameters and treatment outcomes.

PubMed

Münscher, Adrian; Prochnow, Sebastian; Gulati, Amit; Sauter, Guido; Lörincz, Balazs; Blessmann, Marco; Hanken, Henning; Böttcher, Arne; Clauditz, Till Sebastian

2018-04-18

Strong expression of survivin is associated with worse survival in many different tumours, and in cell culture, a correlation between radiation resistance and survivin expression can be seen. The potential of survivin expression as a prognostic/predictive marker or therapeutic target has not been examined in head and neck squamous cell carcinomas (HNSCC) yet. Retrospective study of 452 tissue samples and clinical data from patients with squamous cell carcinomas of the larynx/hypopharynx (LSCC), oral cavity (OSCC) and oropharynx (OPSCC) treated in the University Medical Centre Hamburg-Eppendorf between 2002 and 2006. The expression patterns were detected by tissue microarray technique and correlated with clinical parameters (sex, age, tumour location, TNM 7th edition, grading, recurrence-free and overall survival). 222 OSCC, 126 OPSCC and 105 LSCC tumours of 118 females and 335 males with a mean follow-up of 41.3 months were examined. Survivin expression correlates with pN, cM, pT and overall survival. The potential of survivin as a prognostic/predictive marker is very high. The findings have to be confirmed in a larger cohort of HNSCC esp. in those tumours treated primarily with radio/radiochemotherapy.

African Americans with pancreatic ductal adenocarcinoma exhibit gender differences in Kaiso expression

PubMed Central

Mukherjee, Angana; Jones, Jacqueline; Karanam, Balasubramanyam; Davis, Melissa; Jaynes, Jesse; Reams, R. Renee; Dean-Colomb, Windy; Yates, Clayton

2016-01-01

Kaiso, a bi-modal transcription factor, regulates gene expression, and is elevated in breast, prostate, and colon cancers. Depletion of Kaiso in other cancer types leads to a reduction in markers for the epithelial–mesenchymal transition (EMT) (Jones et al., 2014), however its clinical implications in pancreatic ductal adenocarcinoma (PDCA) have not been widely explored. PDCA is rarely detected at an early stage but is characterized by rapid progression and invasiveness. We now report the significance of the subcellular localization of Kaiso in PDCAs from African Americans. Kaiso expression is higher in the cytoplasm of invasive and metastatic pancreatic cancers. In males, cytoplasmic expression of Kaiso correlates with cancer grade and lymph node positivity. In male and female patients, cytoplasmic Kaiso expression correlates with invasiveness. Also, nuclear expression of Kaiso increases with increased invasiveness and lymph node positivity. Further, analysis of the largest PDCA dataset available on ONCOMINE shows that as Kaiso increases, there is an overall increase in Zeb1, which is the inverse for E-cadherin. Hence, these findings suggest a role for Kaiso in the progression of PDCAs, involving the EMT markers, E-cadherin and Zeb1. PMID:27424525

Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

PubMed

Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

2014-12-01

Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

PubMed Central

2013-01-01

Background Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. Results In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. Conclusion The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression. PMID:23768159

Stage-specific effects of FGF2 on the differentiation of dental pulp cells

PubMed Central

Sagomonyants, Karen; Mina, Mina

2015-01-01

Dentinogenesis is a complex and multistep process, which is regulated by various growth factors, including members of the Fibroblast Growth Factor (FGF) family. Both positive and negative effects of FGFs on dentinogenesis have been reported but the underlying mechanisms of these conflicting results are still unclear. To gain better insight into the role of FGF2 in dentinogenesis, we used dental pulp cells from various transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast differentiation. Our results showed that continuous exposure of pulp cells to FGF2 inhibited mineralization and revealed both stimulatory and inhibitory effects of FGF2 on expression of markers of dentinogenesis and various transgenes. During the proliferation phase of in vitro growth FGF2 increased expression of markers of dentinogenesis and the percentages of DMP1-GFP+ functional odontoblasts and DSPP-Cerulean+ odontoblasts. Additional exposure to FGF2 during the differentiation/mineralization phase of in vitro growth decreased the extent of mineralization, expression of markers of dentinogenesis, and expression of DMP1-GFP and DSPP-Cerulean transgenes. Recovery experiments showed that the inhibitory effects of FGF2 on dentinogenesis were related to the blocking of differentiation of cells into mature odontoblasts. These observations together showed stage-specific effects of FGF2 on dentinogenesis by dental pulp cells and provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration. PMID:25823776

Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

2009-04-01

Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

PubMed

Sternberg, Hal; Jiang, Jianjie; Sim, Pamela; Kidd, Jennifer; Janus, Jeffrey; Rinon, Ariel; Edgar, Ron; Shitrit, Alina; Larocca, David; Chapman, Karen B; Binette, Francois; West, Michael D

2014-01-01

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Micropost microenvironments for studying luminal-basal lineage commitment of breast cancer cells

NASA Astrophysics Data System (ADS)

Kesavaraju, Anand; Qing, Bo; Jabart, Eric; Labarge, Mark; Sohn, Lydia

2013-03-01

MCF-7 breast cancer cells were plated onto polydimethylsiloxane (PDMS) microposts in order to examine the effects of the microenvironment on cell lineage. Different stiffnesses and sizes of the microposts are postulated to impact cell surface marker expression levels. We will provide preliminary results analyzing CD271 and focal adhesion markers such as vinculin. 3D shear flow will also be applied to the microposts to study how external mechanical stimuli affect cancer cells within their microenvironment.

Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts.

PubMed

Baquero, Maria T; Lostritto, Karen; Gustavson, Mark D; Bassi, Kimberly A; Appia, Franck; Camp, Robert L; Molinaro, Annette M; Harris, Lyndsay N; Rimm, David L

2011-11-02

Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy.

Granulysin-Expressing CD4+ T Cells as Candidate Immune Marker for Tuberculosis during Childhood and Adolescence

PubMed Central

Mueller, Henrik; Faé, Kellen C.; Magdorf, Klaus; Ganoza, Christian A.; Wahn, Ulrich; Guhlich, Ute; Feiterna-Sperling, Cornelia; Kaufmann, Stefan H. E.

2011-01-01

Background Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB. Methods Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4+ CD45RO+ memory T cells. Results CD4+ CD45RO+ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSElowCD4+CD45RO+) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4+ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells. Conclusions Our data suggest granulysin expression by CD4+ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence. PMID:22216262

Liver damage, proliferation, and progenitor cell markers in experimental necrotizing enterocolitis.

PubMed

Miyake, Hiromu; Li, Bo; Lee, Carol; Koike, Yuhki; Chen, Yong; Seo, Shogo; Pierro, Agostino

2018-05-01

Necrotizing enterocolitis (NEC) is a disease known to cause injury to multiple organs including the liver. Liver regeneration is essential for the recovery after NEC-induced liver injury. Our aim was to investigate hepatic proliferation and progenitor cell marker expression in experimental NEC. Following ethical approval (#32238), NEC was induced in mice by hypoxia, gavage feeding of hyperosmolar formula, and lipopolysaccharide. Breastfed pups were used as control. We analyzed serum ALT level, liver inflammatory cytokines, liver proliferation markers, and progenitor cell marker expression. Comparison was made between NEC and controls. Serum ALT level was higher in NEC (p<0.05). The mRNA expression of inflammatory cytokines in the liver was also higher in NEC (IL6: p<0.05, TNF-α: p<0.01). Conversely, mRNA expression of proliferation markers in the liver was lower in NEC (Ki67; p<0.01, PCNA: p<0.01). LGR5 expression was also significantly decreased in NEC as demonstrated by mRNA (p<0.05) and protein (p<0.01) levels. Inflammatory injury was present in the liver during experimental NEC. Proliferation and LGR5 expression were impaired in the NEC liver. Modulation of progenitor cell expressing LGR5 may result in stimulation of liver regeneration in NEC-induced liver injury and improved clinical outcome. Level IV. Copyright © 2018. Published by Elsevier Inc.

p16 expression in follicular dendritic cell sarcoma: a potential mimicker of human papillomavirus-related oropharyngeal squamous cell carcinoma.

PubMed

Zhang, Lingxin; Yang, Chen; Lewis, James S; El-Mofty, Samir K; Chernock, Rebecca D

2017-08-01

Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm that most commonly occurs in cervical lymph nodes. It has histologic and clinical overlap with the much more common p16-positive human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx, which characteristically has nonkeratinizing morphology and often presents as an isolated neck mass. Not surprisingly, follicular dendritic cell sarcomas are commonly misdiagnosed as squamous cell carcinoma. Immunohistochemistry is helpful in separating the 2 entities. Follicular dendritic cell sarcoma expresses dendritic markers such as CD21 and CD23 and is almost always cytokeratin negative. However, in many cases of HPV-related oropharyngeal carcinoma, only p16 immunohistochemistry as a prognostic and surrogate marker for HPV is performed. p16 expression in follicular dendritic cell sarcoma has not been characterized. Here, we investigate the expression of p16 in follicular dendritic cell sarcoma and correlate it with retinoblastoma protein expression. A pilot study of dendritic marker expression in HPV-related oropharyngeal squamous cell carcinoma was also performed. We found that 4 of 8 sarcomas expressed p16 with strong and diffuse staining in 2 cases. In 2 of the 4 cases, p16 expression corresponded to loss of retinoblastoma protein expression. Dendritic marker expression (CD21 and CD23) was not found in HPV-related oropharyngeal squamous cell carcinomas. As such, positive p16 immunohistochemistry cannot be used as supportive evidence for the diagnosis of squamous cell carcinoma as strong and diffuse p16 expression may also occur in follicular dendritic cell sarcoma. Cytokeratins and dendritic markers are critical in separating the two tumor types. Copyright © 2017 Elsevier Inc. All rights reserved.

[Pathological and immunohistochemical analysis of giant cells of pancreas].

PubMed

Miyake, T; Suda, K; Yamamura, A; Tada, Y

1997-10-01

Multinucleated giant cells in the pancreas (five giant cell carcinomas, a mucinous cystadenocarcinoma attended with many osteoclast-like giant cells, 42 invasive ductal carcinomas and 29 chronic pancreatitises) were examined. Three types of multinucleated giant cell were identified: epithelial type, coexpressive type, mesenchymal type. Epithelial type expressed epithelial markers, such as keratin and EMA in 23 ductal carcinomas. Coexpressive type expressed both epithelial markers and mesenchymal marker vimentin was in four ductal carcinomas. Mesenchymal type expressed mesenchymal markers, vimentin and CD68 in four osteoclastoid type giant cell carcinomas, the mucinous cystadenocarcinoma, six ductal carcinomas and ten chronic pancreatitises. Epithelial and coexpressive type were considered to be epithelial neoplastic origin, those had bizarre appearance and transitional area from definite adenocarcinoma area. Vimentin expression is associated with sarcomatous proliferation. Mesenchymal type was considered to be nonneoplastic and a certain type of macrophage polykaryons.

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

Desmocollin 2 is a new immunohistochemical marker indicative of squamous differentiation in urothelial carcinoma.

PubMed

Hayashi, Tetsutaro; Sentani, Kazuhiro; Oue, Naohide; Anami, Katsuhiro; Sakamoto, Naoya; Ohara, Shinya; Teishima, Jun; Noguchi, Tsuyoshi; Nakayama, Hirofumi; Taniyama, Kiyomi; Matsubara, Akio; Yasui, Wataru

2011-10-01

Urothelial carcinoma (UC) with squamous differentiation tends to present at higher stages than pure UC. To distinguish UC with squamous differentiation from pure UC, a sensitive and specific marker is needed. Desmocollin 2 (DSC2) is a protein localized in desmosomal junctions of stratified epithelium, but little is known about its biological significance in bladder cancer. We examined the utility of DSC2 as a diagnostic marker. We analysed the immunohistochemical characteristics of DSC2, and studied the relationship of DSC2 expression with the expression of the known markers uroplakin III (UPIII), cytokeratin (CK)7, CK20, epidermal growth factor receptor (EGFR), and p53. DSC2 staining was detected in 24 of 25 (96%) cases of UC with squamous differentiation, but in none of 85 (0%) cases of pure UC. DSC2 staining was detected only in areas of squamous differentiation. DSC2 expression was mutually exclusive of UPIII expression, and was correlated with EGFR expression. Furthermore, DSC2 expression was correlated with higher stage (P = 0.0314) and poor prognosis (P = 0.0477). DSC2 staining offers high sensitivity (96%) and high specificity (100%) for the detection of squamous differentiation in UC. DSC2 is a useful immunohistochemical marker for separation of UC with squamous differentiation from pure UC. 2011 Blackwell Publishing Limited.

Gene Expression Profiling Reveals Novel Candidate Markers of Ovarian Carcinoma Intraperitoneal Metastasis.

PubMed

Elsnerova, Katerina; Bartakova, Alena; Tihlarik, Josef; Bouda, Jiri; Rob, Lukas; Skapa, Petr; Hruda, Martin; Gut, Ivan; Mohelnikova-Duchonova, Beatrice; Soucek, Pavel; Vaclavikova, Radka

2017-01-01

Epithelial ovarian cancer (EOC) has the highest mortality among gynecological carcinomas. The lack of specific markers for prognostic determination of EOC progression hinders the search for novel effective therapies. The aim of the present study was (i) to explore differences in expressions of ATP-binding cassette (ABC) and solute carrier (SLC) transporter genes, genes associated with drug metabolism and cell cycle regulation between control ovarian tissues (n = 14), primary EOCs (n = 44) and intraperitoneal metastases (n = 29); (ii) to investigate associations of gene expression levels with prognosis of patients with intraperitoneal metastases. In all tissue samples, transcript levels of the above target genes were assessed using quantitative real-time PCR. Gene expression levels were compared between particular tissue types and evaluated with regard to progression-free survival (PFS) and drug-resistance status of patients with metastases. Gene expression of ABCA7 significantly increased and that of ESR2 decreased in the order control ovarian tissues - primary EOCs - metastases. High expressions of ABCA2 / 8 / 9 / 10 , ABCB1 , ABCC9 , ABCG2 , ATP7A , SLC16A14 , and SOD3 genes were significantly associated with longer progression-free survival of patients. In intraperitoneal metastases, expression of all of these genes highly correlated and indicated prognostic profile. Transporters from the ABCA family, ABCG2, and ESR2 are involved mainly in lipid metabolism, membrane transport, and cell proliferation. These processes are thus probably the most important for EOC progression. Based on these results, we have proposed novel markers of ovarian carcinoma progression and metastatic spread which might be potentially useful as therapeutic targets. Their significance should be further explored on a larger independent set of patients.

AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum

PubMed Central

Cost, Hoa N.; Noratel, Elizabeth F.; Blumberg, Daphne D.

2013-01-01

The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell–cell and cell–substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell–cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level. PMID:23911723

New markers of pancreatic cancer identified through differential gene expression analyses: claudin 18 and annexin A8.

PubMed

Karanjawala, Zarir E; Illei, Peter B; Ashfaq, Raheela; Infante, Jeffrey R; Murphy, Kathleen; Pandey, Akhilesh; Schulick, Richard; Winter, Jordan; Sharma, Rajni; Maitra, Anirban; Goggins, Michael; Hruban, Ralph H

2008-02-01

New markers to distinguish benign reactive glands from infiltrating ductal adenocarcinoma of the pancreas are needed. The gene expression patterns of 24 surgically resected primary infiltrating ductal adenocarcinomas of the pancreas were compared with 18 non-neoplastic samples using the Affymetrix U133 Plus 2.0 Arrays and the Gene Logic GeneExpress Software System. Gene fragments from 4 genes (annexin A8, claudin 18, CXCL5, and S100 A2) were selected from the fragments found to be highly expressed in infiltrating adenocarcinomas when compared with normal tissues. The protein expression of these genes was examined using immunohistochemical labeling of tissue microarrays. Claudin 18 labeled infiltrating carcinomas in a membranous pattern. When compared with normal and reactive ducts, claudin 18 was overexpressed, at least focally, in 159 of 166 evaluable carcinomas (96%). Strong and diffuse claudin 18 overexpression was most often seen in well-differentiated carcinomas (P=0.02). Claudin 18 was overexpressed in 51 of 52 cases (98%) of pancreatic intraepithelial neoplasia. Annexin A8 was at least focally overexpressed in 149 of 154 evaluable infiltrating carcinomas (97%). S100 A2 was at least focally overexpressed in 118 of 154 evaluable infiltrating carcinomas (77%). Non-neoplastic glands also frequently expressed S100 A2 diminishing its potential diagnostic utility. Immunolabeling with antibodies directed against CXCL5 did not reveal any significant differences in protein expression between infiltrating adenocarcinomas and normal pancreatic ducts. Claudin 18 and annexin A8 are frequently highly overexpressed in infiltrating ductal adenocarcinomas when compared with normal reactive ducts, suggesting a role for these molecules in pancreatic ductal adenocarcinomas. Furthermore, these may serve as diagnostic markers, as screening tests and as therapeutic targets.

ESR1, ERBB2, and Ki67 mRNA expression predicts stage and grade of non-muscle-invasive bladder carcinoma (NMIBC).

PubMed

Breyer, Johannes; Wirtz, Ralph M; Laible, Mark; Schlombs, Kornelia; Erben, Philipp; Kriegmair, Maximilian Christian; Stoehr, Robert; Eidt, Sebastian; Denzinger, Stefan; Burger, Maximilian; Hartmann, Arndt; Otto, Wolfgang

2016-11-01

Pathological staging and grading are crucial for risk assessment in non-muscle-invasive bladder cancer (NMIBC). Molecular grading might support pathological evaluation and minimize interobserver variability. In this study, the well-established breast cancer markers ESR1, PGR, ERBB2, and MKI67 were evaluated as potential molecular markers to support grading and staging in NMIBC. We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with NMIBC. Messenger RNA (mRNA) expression of the aforementioned markers was measured by single-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using RNA-specific TaqMan assays. Relative gene expression was determined by normalization to two reference genes (CALM2 and B2M) using the 40 -ΔΔCT method and correlated to histopathological stage and grade. Pathological assessment was performed by an experienced uropathologist. Statistical analysis was performed using the SAS software JMP 9.0.0 version and GraphPad Prism 5.04. Of 381 cases of NMIBC, samples of 100 pTa and 255 pT1 cases were included in the final study. Spearman rank correlation revealed significant correlations between grade and expression of MKI67 (r = 0.52, p < 0.0001), ESR1 (r = 0.25, p < 0.0001), and ERBB2 (r = 0.18, p = 0.0008). In Mann-Whitney tests, MKI67 was significantly different between all grades (p < 0.0001), while ESR1 (p = 0.0006) and ERBB2 (p = 0.027) were significantly different between G2 and G3. Higher expression of MKI67 (r = 0.49; p < 0.0001), ERBB2 (r = 0.22; p < 0.0001), and ESR1 (r = 0.18; p = 0.0009) mRNA was positively correlated with higher stage. MKI67 (p < 0.0001), ERBB2 (p = 0.0058), and PGR (p = 0.0007) were significantly different between pTa and pT1. In NMIBC expression of ESR1, ERBB2 and MKI67 are significantly different between stage and grade. This potentially provides objective parameters for pathological evaluation.

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Left Ventricular Hypertrophy: An allometric comparative analysis of different ECG markers

NASA Astrophysics Data System (ADS)

Bonomini, M. P.; Ingallina, F.; Barone, V.; Valentinuzzi, M. E.; Arini, P. D.

2011-12-01

Allometry, in general biology, measures the relative growth of a part in relation to the whole living organism. Left ventricular hypertrophy (LVH) is the heart adaptation to excessive load (systolic or diastolic). The increase in left ventricular mass leads to an increase in the electrocardiographic voltages. Based on clinical data, we compared the allometric behavior of three different ECG markers of LVH. To do this, the allometric fit AECG = δ + β (VM) relating left ventricular mass (estimated from ecocardiographic data) and ECG amplitudes (expressed as the Cornell-Voltage, Sokolow and the ECG overall voltage indexes) were compared. Besides, sensitivity and specifity for each index were analyzed. The more sensitive the ECG criteria, the better the allometric fit. In conclusion: The allometric paradigm should be regarded as the way to design new and more sensitive ECG-based LVH markers.

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

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

PubMed

Hsieh, Chin-Hsuan; Hsiung, Shih-Chieh; Yeh, Chi-Tai; Yen, Chih-Feng; Chou, Yah-Huei Wu; Lei, Wei-Yi; Pang, See-Tong; Chuang, Cheng-Keng; Liao, Shuen-Kuei

2017-02-28

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson’s Disease

PubMed Central

Viereckel, Thomas; Dumas, Sylvie; Smith-Anttila, Casey J. A.; Vlcek, Bianca; Bimpisidis, Zisis; Lagerström, Malin C.; Konradsson-Geuken, Åsa; Wallén-Mackenzie, Åsa

2016-01-01

The ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain are associated with Parkinson’s disease (PD), schizophrenia, mood disorders and addiction. Based on the recently unraveled heterogeneity within the VTA and SNc, where glutamate, GABA and co-releasing neurons have been found to co-exist with the classical dopamine neurons, there is a compelling need for identification of gene expression patterns that represent this heterogeneity and that are of value for development of human therapies. Here, several unique gene expression patterns were identified in the mouse midbrain of which NeuroD6 and Grp were expressed within different dopaminergic subpopulations of the VTA, and TrpV1 within a small heterogeneous population. Optogenetics-coupled in vivo amperometry revealed a previously unknown glutamatergic mesoaccumbal pathway characterized by TrpV1-Cre-expression. Human GRP was strongly detected in non-melanized dopaminergic neurons within the SNc of both control and PD brains, suggesting GRP as a marker for neuroprotected neurons in PD. This study thus unravels markers for distinct subpopulations of neurons within the mouse and human midbrain, defines unique anatomical subregions within the VTA and exposes an entirely new glutamatergic pathway. Finally, both TRPV1 and GRP are implied in midbrain physiology of importance to neurological and neuropsychiatric disorders. PMID:27762319

Basal cell carcinoma: CD56 and cytokeratin 5/6 staining patterns in the differential diagnosis with Merkel cell carcinoma.

PubMed

Panse, Gauri; McNiff, Jennifer M; Ko, Christine J

2017-06-01

Basal cell carcinoma (BCC) can resemble Merkel cell carcinoma (MCC) on histopathological examination and while CK20 is a useful marker in this differential, it is occasionally negative in MCC. CD56, a sensitive marker of neuroendocrine differentiation, is sometimes used to identify MCC, but has been reportedly variably positive in BCC as well. In contrast, CK5/6 consistently labels BCC but is not expressed in neuroendocrine tumors. We evaluated 20 cases of BCC for the pattern of CD56 and cytokeratin 5/6 (CK5/6) staining, hypothesizing that these 2 stains could differentiate BCC from MCC in difficult cases. Seventeen cases of MCC previously stained with CD56 were also examined. All BCCs showed patchy expression of CD56 except for 2 cases, which showed staining of greater than 70% of tumor. CK5/6 was diffusely positive in all cases of BCC. Fifteen of 17 MCCs were diffusely positive for CD56. The difference in the pattern of CD56 expression between MCC and BCC (diffuse vs patchy, respectively) was statistically significant (P < .05). BCC typically shows patchy CD56 expression and diffuse CK5/6 positivity. These 2 markers can be used to distinguish between BCC and MCC in challenging cases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

PubMed

Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

2010-06-01

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emergedmore » cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.« less

Bladder Cancer-associated Protein, a Potential Prognostic Biomarker in Human Bladder Cancer*

PubMed Central

Moreira, José M. A.; Ohlsson, Gita; Gromov, Pavel; Simon, Ronald; Sauter, Guido; Celis, Julio E.; Gromova, Irina

2010-01-01

It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Interpatient and intratumor heterogeneity provides overwhelming odds against the existence of such an ideal marker. With this in mind, our laboratory has been applying a long term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network, we have taken some of the most promising candidate proteins into postdiscovery validation studies, and here we report on the characterization of one such biomarker, the bladder cancer-associated protein (BLCAP), formerly termed Bc10. To characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and urothelial carcinomas (UCs), we used two independent sets of samples from different patient cohorts: a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies) and a validation set consisting of 2,108 retrospectively collected UCs with long term clinical follow-up. We could categorize the UCs examined into four groups based on levels of expression and subcellular localization of BLCAP protein and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and adipocyte-type fatty acid-binding protein, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed. PMID:19783793

Molecular mapping and marker development for the Triticum dicoccoides-derived stripe rust resistance gene YrSM139-1B in bread wheat cv. Shaanmai 139.

PubMed

Zhang, Hong; Zhang, Lu; Wang, Changyou; Wang, Yajuan; Zhou, Xinli; Lv, Shikai; Liu, Xinlun; Kang, Zhensheng; Ji, Wanquan

2016-02-01

YrSM139-1B maybe a new gene for effective resistance to stripe rust and useful flanking markers for marker-assisted selection were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. Two dominant stripe rust resistant genes YrSM139-1B and YrSM139-2D were pyramided in bread wheat cultivar Shaanmai 139; one from wild emmer and the other from Thinopyrum intermedium. Three near-isogenic F7:8 line pairs (contrasting RILs), N122-1013R/S, N122-185R/S, and N122-1812R/S, independently derived from different F2 plants and differing at the YrSM139-1B locus were generated from the cross Shaanmai 139 × Hu 901-19 through marker-assisted selection. A large F2:3 population from cross N122-1013R × N122-1013S tested for stripe rust response and subjected to analysis with markers in the 1BS10-0.5 bin region using SSR expressed sequence tags (EST) and site-specific sequence markers developed from the 90 K Illumina iSelect SNP array. Five EST-STS markers and four allele-specific PCR markers were mapped to the YrSM139-1B region. The 30.5 cM genetic map for YrSM139-1B consisted of nine markers, two of which were closer to YrSM139-1B than Xgwm273, which was used in producing the contrasting RIL pairs. Race response data and allelism tests showed that YrSM139-1B is different from Yr10, Yr15, and Yr24/26/CH42.

Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects.

PubMed

Neubauer, Elisa; Wirtz, Ralph M; Kaemmerer, Daniel; Athelogou, Maria; Schmidt, Lydia; Sänger, Jörg; Lupp, Amelie

2016-07-05

The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In this regard, we compared the expression of three proliferation markers, Ki-67, Topoisomerase II alpha (TOP2A), and RacGAP1, in a series of tumor samples from 104 BP-NEN patients (24 TC, 21 AC, 52 SCLC, 7 LCNEC) using different evaluation methods (immunohistochemistry (IHC): Average evaluation, Hotspot evaluation, digital image analysis; RT-qPCR).The results indicated that all three markers had increased protein and mRNA expression with poorer differentiation and correlated well with each other, as well as with grading, staging, and poor survival. Compared with Ki-67 and TOP2A, RacGAP1 allowed for a clearer prognostic statement. The cut-off limits obtained for Ki-67-Average (IHC) were TC-AC 1.5, AC-SCLC 19, and AC-LCNEC 23.5. The Hotspot evaluation generated equal to higher, the digital image analysis generally lower between-entity cut-off limits.All three markers enabled a clear-cut differentiation between the BP-NEN entities, and all methods evaluated were suitable for marker assessment. However, to define optimal cut-off limits, the Ki-67 evaluation methods should be standardized. RacGAP1 appeared to be a new marker with great potential.

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts.

PubMed

Shirasawa, Kenta; Hand, Melanie L; Henderson, Steven T; Okada, Takashi; Johnson, Susan D; Taylor, Jennifer M; Spriggs, Andrew; Siddons, Hayley; Hirakawa, Hideki; Isobe, Sachiko; Tabata, Satoshi; Koltunow, Anna M G

2015-03-01

Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Skeletal Site-Specific Role of Connective Tissue Growth Factor in Prenatal Osteogenesis

PubMed Central

Lambi, Alex G.; Pankratz, Talia L.; Mundy, Christina; Gannon, Maureen; Barbe, Mary F.; Richtsmeier, Joan T.; Popoff, Steven N.

2013-01-01

Background Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted. Results We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype. Conclusions We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation. PMID:23073844

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.

GTA: a game theoretic approach to identifying cancer subnetwork markers.

PubMed

Farahmand, S; Goliaei, S; Ansari-Pour, N; Razaghi-Moghadam, Z

2016-03-01

The identification of genetic markers (e.g. genes, pathways and subnetworks) for cancer has been one of the most challenging research areas in recent years. A subset of these studies attempt to analyze genome-wide expression profiles to identify markers with high reliability and reusability across independent whole-transcriptome microarray datasets. Therefore, the functional relationships of genes are integrated with their expression data. However, for a more accurate representation of the functional relationships among genes, utilization of the protein-protein interaction network (PPIN) seems to be necessary. Herein, a novel game theoretic approach (GTA) is proposed for the identification of cancer subnetwork markers by integrating genome-wide expression profiles and PPIN. The GTA method was applied to three distinct whole-transcriptome breast cancer datasets to identify the subnetwork markers associated with metastasis. To evaluate the performance of our approach, the identified subnetwork markers were compared with gene-based, pathway-based and network-based markers. We show that GTA is not only capable of identifying robust metastatic markers, it also provides a higher classification performance. In addition, based on these GTA-based subnetworks, we identified a new bonafide candidate gene for breast cancer susceptibility.

The effect of different collagen modifications for titanium and titanium nitrite surfaces on functions of gingival fibroblasts.

PubMed

Ritz, U; Nusselt, T; Sewing, A; Ziebart, T; Kaufmann, K; Baranowski, A; Rommens, P M; Hofmann, Alexander

2017-01-01

Targeted modifications of the bulk implant surfaces using bioactive agents provide a promising tool for improvement of the long-term bony and soft tissue integration of dental implants. In this study, we assessed the cellular responses of primary human gingival fibroblasts (HGF) to different surface modifications of titanium (Ti) and titanium nitride (TiN) alloys with type I collagen or cyclic-RGDfK-peptide in order to define a modification improving long-term implants in dental medicine. Employing Ti and TiN implants, we compared the performance of simple dip coating and anodic immobilization of type I collagen that provided collagen layers of two different thicknesses. HGF were seeded on the different coated implants, and adhesion, proliferation, and gene expression were analyzed. Although there were no strong differences in initial cell adhesion between the groups at 2 and 4 hours, we found that all surface modifications induced higher proliferation rates as compared to the unmodified controls. Consistently, gene expression levels of cell adhesion markers (focal adhesion kinase (FAK), integrin beta1, and vinculin), cell differentiation markers (FGFR1, TGFb-R1), extracellular protein markers (type I collagen, vimentin), and cytoskeletal protein marker aktinin-1 were consistently higher in all surface modification groups at two different time points of investigation as compared to the unmodified controls. Our results indicate that simple dip coating of Ti and TiN with collagen is sufficient to induce in vitro cellular responses that are comparable to those of more reliable coating methods like anodic adsorption, chemical cross-linking, or RGD coating. TiN alloys do not possess any positive or adverse effects on HGF. Our results demonstrate a simple, yet effective, method for collagen coating on titanium implants to improve the long term integration and stability of dental implants.

Fascin and EMMPRIN expression in primary mucinous tumors of ovary: a tissue microarray study.

PubMed

Alici, Omer; Kefeli, Mehmet; Yildiz, Levent; Baris, Sancar; Karagoz, Filiz; Kandemir, Bedri

2014-12-01

The aim of this study was to compare the expressions of fascin and EMMPRIN in primary malignant, borderline and benign mucinous ovarian tumors, and to investigate the relationship of these markers with tumor progression and their applicability to differential diagnosis. An immunohistochemical study was performed for fascin and EMMPRIN using the tissue microarray technique. Eighty-one cases were included in the study; there were 37 benign, 25 borderline and 19 malignant primary mucinous ovarian tumors. For each case, a total staining score was determined, consisting of scores for extent of staining and intensity of staining. The cases were allocated to negative, weakly positive and strongly positive staining categories, according to the total staining score. Both of the markers were significantly negative in benign tumors as compared with borderline and malignant tumors. There was no significant difference between borderline and malignant groups for both markers. Sixty-eight percent of malignant tumors were stained positive by fascin, while this rate was 40% for borderline mucinous tumors. All malignant tumors were strongly stained positive for EMMPRIN, while this rate was 92% for borderline mucinous tumors. The rest of the cases stained weakly positive. No significant difference in staining score was found between fascin and EMMPRIN expression. In ovarian primary mucinous tumors, fascin and EMMPRIN may play an important role in tumor progression from benign tumor to carcinoma. In that context, EMMPRIN and fascin expression may have potential application in the differential diagnosis of some diagnostically problematic mucinous ovarian tumors. However, the differential diagnostic applicability of EMMPRIN appears to be more limited than that of fascin due to its wide spectrum of staining in mucinous ovarian tumors. Copyright © 2014 Elsevier GmbH. All rights reserved.

Aberrant expression of epithelial leucine-rich repeat containing G protein-coupled receptor 5-positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis.

PubMed

Vallvé-Juanico, Júlia; Suárez-Salvador, Elena; Castellví, Josep; Ballesteros, Agustín; Taylor, Hugh S; Gil-Moreno, Antonio; Santamaria, Xavier

2017-11-01

To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5 + ) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5 + cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5 + cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5 + cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5 + versus LGR5 - cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5 + cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5 + cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes. Copyright © 2017. Published by Elsevier Inc.

A single measurement of biochemical markers of bone turnover has limited utility in the individual person.

PubMed

Beck-Jensen, J E; Kollerup, G; Sørensen, H A; Pors Nielsen, S; Sørensen, O H

1997-07-01

Biochemical markers of bone turnover are used to estimate the rate of bone loss in the individual osteoporotic patient. During recent years it has become increasingly clear that the biological variability of biochemical bone markers has to be taken into consideration in the evaluation of their usefulness in the clinical setting. Eleven premenopausal, 8 perimenopausal and 11 postmenopausal healthy women were included. We assessed the analytical and the biological components of variation for a number of resorptive and formative bone markers: u-hydroxyproline, u-pyridinoline, and u-deoxypyridinoline together with u-calcium and u-creatinine, s-total alkaline phosphatases and s-osteocalcin. Blood and urine samples were collected five times with 7-day intervals. Urinary parameters were expressed as outputs and corrected for creatinine in fasting night urines and second void fasting morning urines. The absolute values differed with a tendency towards increasing values in the postmenopausal women, but the biological variations in relation to menopausal status were not different. The biological variability was much higher for the urinary resorptive markers than for the formative markers in the blood. The critical difference expressing the difference needed between two serial results from the same person to be significant at a 5% level was 15% for s-alkaline phosphatases, 18% for s-osteocalcin, and lowest in the second void fasting morning urines with values of 28% and 34% for u-pyridinoline/creatinine and u-deoxypyridinoline/creatinine, and 50% and 112% for u-hydroxyproline/creatinine and u-calcium/creatinine, respectively. The index of individuality, denoting the individual variation divided by the variation between subjects, was in the range from 0.19 for s-alkaline phosphatases to 1.23 for u-hydroxyproline/minute in second void fasting morning urine making the use of conventional reference intervals difficult. Low indices, however, indicate high test performance and offer the possibility of stratification of persons within a range. The number of samples required to determine the true individual mean value +/- 5% for the single person, ranged from 5 for s-total alkaline phosphatases, 6 for s-osteocalcin, 23 for u-deoxypyridinoline/creatinine in the fasting morning urine to over two hundred for u-calcium analytes. It is concluded that, due to high biological variation, a single measurement of biochemical markers of bone turnover is of limited utility in the individual person. We recommend that routine clinical use of biochemical markers should be restricted until further evidence justifies it.

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces.

PubMed

Sevillano, Laura; Díaz, Margarita; Santamaría, Ramón I

2017-09-26

The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes.

PubMed

Isoler-Alcaraz, J; Fernández-Pérez, D; Larriba, E; Del Mazo, J

2017-10-18

Recently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development. Testicular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches. The method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations. In vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed that as long as cultures progress, the proportion of microRNAs declined respect to piwi-interactingRNAs levels that increased, showing a similar propensity than which happens in in vivo spermatogenesis. Our study allows to improve and potentially to control the ex vivo spermatogenesis development, opening new perspectives in the reproductive biology fields including male fertility.

The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment.

PubMed

Segev, Hana; Fishman, Betina; Schulman, Rita; Itskovitz-Eldor, Joseph

2012-07-01

Even before the first appearance of the developing pancreas, glucose is the major substrate in the growing embryo. The transport of glucose across cell membranes is facilitated by a family of membranal glucose transporters (GLUT). We analyzed changes in expression of class 1 glucose transporters (GLUT1-4) during human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiation, from undifferentiated cells to 28-day-old embryoid bodies (EBs). We also examined the potential use of GLUT2 as a marker for differentiating pancreatic progenitor cells. Using quantitative real time polymerase chain reaction (qPCR), western blot, and immunofluorescence, we observed enhanced expression of GLUT1 and GLUT2 during differentiation, but only minor change in GLUT3 expression. GLUT4 expression was found to be very low both at the RNA and in the protein levels. Expression of the early pancreatic transcription factor, pancreatic duodenal homeobox gene 1 (PDX1), correlated with GLUT2 expression, suggesting the potential use of GLUT2 as a surface marker for tracking pancreatic precursor cells. After sorting EBs according to their membranal GLUT2 expression, GLUT2 and PDX1 expression were found elevated, as was expression of other endodermal markers such as PAX4, NGN3, CXCR4, and SOX17. This simple method may be used to differentiate embryonic stem cells and to isolate from them, using GLUT2 as a surface marker, an enriched pancreatic progenitor cell population in order to achieve insulin-producing cells. The sorted GLUT2 cells may potentially be used in the future as insulin-producing cells for beta cell therapies.

Tissue distribution of mesenchymal stem cell marker Stro-1.

PubMed

Lin, Guiting; Liu, Gang; Banie, Lia; Wang, Guifang; Ning, Hongxiu; Lue, Tom F; Lin, Ching-Shwun

2011-10-01

Stro-1 is the best-known mesenchymal stem cell marker. However, despite its bone marrow origin, its localization in bone marrow has never been demonstrated. By immunofluorescence staining, we show here that ∼ 0.74% of nucleated bone marrow cells expressed Stro-1. We also found that ∼ 8.7% of CD34-expressing cells expressed Stro-1, and more than 20% of Stro-1-expressing cells did not express CD34. In adipose tissue Stro-1 expression was identified in the endothelium of arterioles and capillaries. Stro-1 was also localized in the endothelium of some but not all adipose tissue veins. Endothelial expression of Stro-1 was also identified in blood vessels in penis and in leg muscles, but not in other tested tissues. In these other tissues, Stro-1 was scantly expressed near but not in blood vessels. These variable and endothelial expression patterns of Stro-1 point to a need to re-examine published data that relied on Stro-1 as a mesenchymal stem cell marker.

Prostate cancer antigen 3 gene expression in peripheral blood and urine sediments from prostate cancer and benign prostatic hyperplasia patients versus healthy individuals.

PubMed

Moradi Sardareh, Hemen; Goodarzi, Mohammad Taghi; Yadegar-Azari, Reza; Poorolajal, Jalal; Mousavi-Bahar, Seyed Habibollah; Saidijam, Massoud

2014-11-30

To determine the expression of prostate cancer antigen 3 (PCA3) gene in peripheral blood and urine sediments from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) and normal subjects. A total number of 48 patients [24 with biopsy proven prostate cancer (PCa) and 24 with benign prostate hyperplasia (BPH)] were studied. Twenty-four healthy individuals were also recruited as control group. After blood and urine sampling, total RNA was extracted and cDNA was synthesized. Expression of PCA3 gene was assessed by quantitative reverse transcription polymerase chain reaction. Comparison of PCA3 gene expression between control and BPH groups indicated no statistically significant differences in both urine and blood samples. Patients with PCa demonstrated an increased PCA3 gene expression rate compared to control and BPH groups (10.64 and 7.17 folds, respectively). The rate of fold increased PCA3 gene expression in urine was 20.90, 20.90, and 20.35 in patients with PCa, BPH and normal subjects, respectively. Evaluation of PCA3 gene expression can be considered as a reliable marker for detection of PCa. Increased level of this marker in urine sediments is more sensitive than blood for distinguishing between cancerous and non-cancerous groups. 

High-frequency stimulation of the subthalamic nucleus modifies the expression of vesicular glutamate transporters in basal ganglia in a rat model of Parkinson's disease.

PubMed

Favier, Mathieu; Carcenac, Carole; Drui, Guillaume; Boulet, Sabrina; El Mestikawy, Salah; Savasta, Marc

2013-12-05

It has been suggested that glutamatergic system hyperactivity may be related to the pathogenesis of Parkinson's disease (PD). Vesicular glutamate transporters (VGLUT1-3) import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission. Both VGLUT1 and VGLUT2 have been identified as definitive markers of glutamatergic neurons, but VGLUT 3 is also expressed by non glutamatergic neurons. VGLUT1 and VGLUT2 are thought to be expressed in a complementary manner in the cortex and the thalamus (VL/VM), in glutamatergic neurons involved in different physiological functions. Chronic high-frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced PD. STN-HFS is highly effective, but its mechanisms of action remain unclear. This study examines the effect of STN-HFS on VGLUT1-3 expression in different brain nuclei involved in motor circuits, namely the basal ganglia (BG) network, in normal and 6-hydroxydopamine (6-OHDA) lesioned rats. Here we report that: 1) Dopamine(DA)-depletion did not affect VGLUT1 and VGLUT3 expression but significantly decreased that of VGLUT2 in almost all BG structures studied; 2) STN-HFS did not change VGLUT1-3 expression in the different brain areas of normal rats while, on the contrary, it systematically induced a significant increase of their expression in DA-depleted rats and 3) STN-HFS reversed the decrease in VGLUT2 expression induced by the DA-depletion. These results show for the first time a comparative analysis of changes of expression for the three VGLUTs induced by STN-HFS in the BG network of normal and hemiparkinsonian rats. They provide evidence for the involvement of VGLUT2 in the modulation of BG cicuits and in particular that of thalamostriatal and thalamocortical pathways suggesting their key role in its therapeutic effects for alleviating PD motor symptoms.

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.

De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers.

PubMed

Xanthopoulou, Aliki; Ganopoulos, Ioannis; Psomopoulos, Fotis; Manioudaki, Maria; Moysiadis, Theodoros; Kapazoglou, Aliki; Osathanunkul, Maslin; Michailidou, Sofia; Kalivas, Apostolos; Tsaftaris, Athanasios; Nianiou-Obeidat, Irini; Madesis, Panagiotis

2017-07-30

The genetic basis of fruit size and shape was investigated for the first time in Cucurbita species and genetic loci associated with fruit morphology have been identified. Although extensive genomic resources are available at present for tomato (Solanum lycopersicum), cucumber (Cucumis sativus), melon (Cucumis melo) and watermelon (Citrullus lanatus), genomic databases for Cucurbita species are limited. Recently, our group reported the generation of pumpkin (Cucurbita pepo) transcriptome databases from two contrasting cultivars with extreme fruit sizes. In the current study we used these databases to perform comparative transcriptome analysis in order to identify genes with potential roles in fruit morphology and fruit size. Differential Gene Expression (DGE) analysis between cv. 'Munchkin' (small-fruit) and cv. 'Big Moose' (large-fruit) revealed a variety of candidate genes associated with fruit morphology with significant differences in gene expression between the two cultivars. In addition, we have set the framework for generating EST-SSR markers, which discriminate different C. pepo cultivars and show transferability to related Cucurbitaceae species. The results of the present study will contribute to both further understanding the molecular mechanisms regulating fruit morphology and furthermore identifying the factors that determine fruit size. Moreover, they may lead to the development of molecular marker tools for selecting genotypes with desired morphological traits. Copyright © 2017. Published by Elsevier B.V.

Cells involved in extracellular matrix remodeling after acute myocardial infarction

PubMed Central

Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

2015-01-01

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

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

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

PubMed

Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

2011-12-01

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

miR15a and miR16 in Chilean type 1 diabetes patients: possible association with apoptosis, inflammatory, or autoimmunity markers.

PubMed

Garcia-Diaz, D F; Camacho-Guillén, P; Codner, E; Pérez-Bravo, F

2018-01-31

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the progressive destruction of β cells, mediated by the interaction between T cells and several cytokines. The pathogenesis of T1D has established its possible relationship with miRNAs. In this study, we analyze the expression profile of miR-15a and miR-16 in peripheral blood mononuclear cells (PBMCs) and their possible association with apoptosis, inflammation, or autoimmunity markers. 38 T1D patients and 41 control subjects were recruited. mRNAs were analyzed by means of qPCR and TaqMan probes. PBMCs were treated with different concentrations of glucose (baseline, 11 and 25 mM) with or without an inflammatory stimulus as TNF-α (10 ng/ml). A decrease in the levels of the miR-15a expression in basal conditions is observed in T1D patients compared to healthy control subjects (relative units 0.5 vs. 1.8, p < 0.05). This change in miR-15a and miR-16 is not affected by the addition of TNF-α. No association is observed with inflammatory markers (IL-6, TNF-α, vCAM) or apoptosis (bcl2 expression). The relationship with immunological markers shows an interaction effect between miR16 and IA-2 (p < 0.03). TNF-α does not affect the expression profile of miR-15a and miR16 in PBMCs. A weak correlation is observed between miR-16 and with the autoimmunity profile (IA-2 autoantibody).

miR15a and miR16 in Chilean type 1 diabetes patients: possible association with apoptosis, inflammatory, or autoimmunity markers.

PubMed

Garcia-Diaz, D F; Camacho-Guillén, P; Codner, E; Pérez-Bravo, F

2018-01-30

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the progressive destruction of β cells, mediated by the interaction between T cells and several cytokines. The pathogenesis of T1D has established its possible relationship with miRNAs. In this study, we analyze the expression profile of miR-15a and miR-16 in peripheral blood mononuclear cells (PBMCs) and their possible association with apoptosis, inflammation, or autoimmunity markers. 38 T1D patients and 41 control subjects were recruited. mRNAs were analyzed by means of qPCR and TaqMan probes. PBMCs were treated with different concentrations of glucose (baseline, 11 and 25 mM) with or without an inflammatory stimulus as TNF-α (10 ng/ml). A decrease in the levels of the miR-15a expression in basal conditions is observed in T1D patients compared to healthy control subjects (relative units 0.5 vs. 1.8, p < 0.05). This change in miR-15a and miR-16 is not affected by the addition of TNF-α. No association is observed with inflammatory markers (IL-6, TNF-α, vCAM) or apoptosis (bcl2 expression). The relationship with immunological markers shows an interaction effect between miR16 and IA-2 (p < 0.03). TNF-α does not affect the expression profile of miR-15a and miR16 in PBMCs. A weak correlation is observed between miR-16 and with the autoimmunity profile (IA-2 autoantibody).

The role of epithelial-mesenchymal transition in squamous cell carcinoma of the oral cavity.

PubMed

Zidar, Nina; Boštjančič, Emanuela; Malgaj, Marija; Gale, Nina; Dovšak, Tadej; Didanovič, Vojko

2018-02-01

Epithelial-mesenchymal transition (EMT) has emerged as a possible mechanism of cancer metastasizing, but strong evidence for EMT involvement in human cancer is lacking. Our aim was to compare oral spindle cell carcinoma (SpCC) as an example of EMT with oral conventional squamous cell carcinoma (SCC) with and without nodal metastases to test the hypothesis that EMT contributes to metastasizing in oral SCC. Thirty cases of oral SCC with and without nodal metastasis and 15 cases of SpCC were included. Epithelial (cytokeratin, E-cadherin), mesenchymal (vimentin, N-cadherin), and stem cell markers (ALDH-1, CD44, Nanog, Sox-2) and transcription repressors (Snail, Slug, Twist) were analyzed immunohistochemically. We also analyzed the expression of microRNAs miR-141, miR-200 family, miR-205, and miR-429. SpCC exhibited loss of epithelial markers and expression of mesenchymal markers or coexpression of both up-regulation of transcription repressors and down-regulation of the investigated microRNAs. SCC showed only occasional focal expression of mesenchymal markers at the invasive front. No other differences were observed between SCC with and without nodal metastases except for a higher expression of ALDH-1 in SCC with metastases. Our results suggest that SpCC is an example of true EMT but do not support the hypothesis that EMT is involved in metastasizing of conventional SCC. Regarding oral SCC progression and metastasizing, we have been facing a shift from the initial enthusiasm for the EMT concept towards a more critical approach with "EMT-like" and "partial EMT" concepts. The real question, though, is, is there no EMT at all?

Isolation, Characterization, and Differentiation of Dental Pulp Stem Cells in Ferrets.

PubMed

Homayounfar, Negar; Verma, Prashant; Nosrat, Ali; El Ayachi, Ikbale; Yu, Zongdong; Romberg, Elaine; Huang, George T-J; Fouad, Ashraf F

2016-03-01

The ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential. Dental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance. fDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005). fDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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.

Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications

PubMed Central

Lange, Sara E.S.; Zheleznyak, Alex; Studer, Matthew; O'Shannessy, Daniel J.; Lapi, Suzanne E.; Van Tine, Brian A.

2016-01-01

Purpose The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab. Results Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs. Experimental Design Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed. Conclusion 89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy. PMID:26909615

Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth.

PubMed

Carter, Yvette M; Kunnimalaiyaan, Selvi; Chen, Herbert; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2014-05-01

Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma. NGP and SH-5Y-SY cells were treated with 0-20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot. Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P<0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P<0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression. Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.

Interleukin-6 controls uterine Th9 cells and CD8(+) T regulatory cells to accelerate parturition in mice.

PubMed

Gomez-Lopez, Nardhy; Olson, David M; Robertson, Sarah A

2016-01-01

Interleukin-6 (IL6) is a determinant of the timing of parturition and birth in mice. We previously demonstrated that genetic IL6 deficiency delays parturition by ~24 h, and this is restored by administration of exogenous IL6. In this study, we have investigated whether IL6 influences the number or phenotypes of T cells or other leukocytes in uterine decidual tissue at the maternal-fetal interface. In late gestation, decidual leukocytes in Il6 null mutant (Il6(-/-)) mice exhibit an altered profile, characterized by reduced numbers of cells expressing the monocyte/macrophage marker F4/80 or the T-cell marker CD4, increased cells expressing the natural killer (NK) cell marker CD49b or the dendritic cell marker CD11c, but no change in cells expressing the neutrophil marker Ly6G. These changes are specific to late pregnancy, as similar differences in decidual leukocytes were not evident in mid-gestation Il6(-/-) mice. The IL6-regulated changes in decidual NK and dendritic cells appear secondary to local recruitment, as no comparable changes occurred in peripheral blood of Il6(-/-) mice. When exogenous IL6 was administered to restore normal timing of parturition, a partial reversal of the altered leukocyte profile was observed, with a 10% increase in the proportion of decidual CD4(+) T cells, a notable 60% increase in CD8(+) T cells including CD8(+)CD25(+)Foxp3(+) regulatory T cells and a 60% reduction in CD4(+)IL9(+) Th9 cells. Together these findings suggest that IL6-controlled accumulation of decidual CD4(+) T cells and CD8(+) regulatory T cells, with an associated decline in decidual Th9 cells, is instrumental for progressing parturition in mice.

The autophagy-related marker LC3 can predict prognosis in human hepatocellular carcinoma.

PubMed

Lee, Yoo Jin; Hah, Yu Jin; Ha, Yu Jin; Kang, Yu Na; Kang, Koo Jeong; Hwang, Jae Seok; Chung, Woo Jin; Cho, Kwang Bum; Park, Kyung Sik; Kim, Eun Soo; Seo, Hye-Young; Kim, Mi-Kyung; Park, Keun-Gyu; Jang, Byoung Kuk

2013-01-01

Defects of autophagy and endoplasmic reticulum (ER) stress are related to many diseases and tumors. However, only a few studies have examined hepatocellular carcinoma (HCC) as related to these processes. Therefore, in this study, we investigated the expression and extent of autophagy and ER stress-related markers in HCC and their influence on clinical characteristics and prognosis for each protein. The expression of autophagy-related markers (LC3 and Beclin-1) and ER stress-related markers (GRP78 and CHOP) was analyzed by immunohistochemistry on tissues from completely resected specimens of 190 HCC patients. Their influence on clinicopathologic features and prognosis were evaluated using the chi-square test and Kaplan-Meier analysis. Correlations of each protein were determined by Spearman's correlation analysis. LC3 expression was not correlated with TNM, BCLC stage, or Edmonson-Steiner grading, whereas it was correlated with longer overall survival (OS) (p = 0.039) and tended to be related with longer time to recurrence (TTR) (p=0.068) although it did not show statistical significance. Multivariate analysis indicated that LC3 expression was a significantly independent prognostic factor of OS (HR, 0.42; 95% CI, 0.22-0.80; p-value=0.009) and TTR (HR, 0.54; 95% CI, 0.33-0.90; p=0.017). Expression of LC3 in advanced stages of TNM (III) (p=0.045) and Edmonson-Steiner Grades (III and IV) (p=0.043) was correlated with longer survival, but not in the early stages. A positive correlation was not observed between the expression of autophagy-related markers and ER stress-related markers. Our results suggest that the expression and extent of LC3 might be a strong prognostic factor of HCC, especially in patients with surgical resection.

Consumption of protein-enriched milk has minor effects on inflammation in older adults-A 12-week double-blind randomized controlled trial.

PubMed

Gjevestad, Gyrd O; Ottestad, Inger; Biong, Anne Sofie; Iversen, Per Ole; Retterstøl, Kjetil; Raastad, Truls; Skålhegg, Bjørn S; Ulven, Stine M; Holven, Kirsten B

2017-03-01

Aging is associated with increased levels of circulating inflammatory markers and reduced muscle mass and strength. We investigated whether intake of protein-enriched milk for 12 weeks would influence markers of inflammation among adults ≥70years of age with reduced physical strength. In a double-blind randomized controlled intervention study, subjects were randomly allocated into two groups, receiving a protein-enriched milk (2×20g protein/d, n=14, mean (±SD) age 76.9±4.9 yrs) or an isocaloric carbohydrate drink (n=17, age 77.7±4.8 yrs) for 12 weeks. We measured serum and mRNA expression levels of inflammatory markers in PBMCs. Significant differences in the mRNA expression of nuclear receptor subfamily, group H, member 3 (NR1H3, encoding the LXRα transcription factor) and interferon gamma (INFG) were observed between groups. The mRNA level of TNFRSF1A was significantly reduced, while the mRNA level of dipeptidyl-peptidase 4 (DPP4) was significantly increased, in the control group. The serum level of TNFα increased significantly in the control group, while sTNFRSF1A increased significantly in both groups, but with no significant differences between groups. Consumption of a low-fat, protein-enriched milk for 12 weeks had minor effects on inflammatory related markers in older adults compared to an isocaloric carbohydrate drink. Copyright © 2017 Elsevier B.V. All rights reserved.

High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

PubMed Central

Fabian, Gabriella; Farago, Nora; Feher, Liliana Z.; Nagy, Lajos I.; Kulin, Sandor; Kitajka, Klara; Bito, Tamas; Tubak, Vilmos; Katona, Robert L.; Tiszlavicz, Laszlo; Puskas, Laszlo G.

2011-01-01

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment. PMID:22016648

Graft Growth and Podocyte Dedifferentiation in Donor-Recipient Size Mismatch Kidney Transplants.

PubMed

Müller-Deile, Janina; Bräsen, Jan Hinrich; Pollheimer, Marion; Ratschek, Manfred; Haller, Hermann; Pape, Lars; Schiffer, Mario

2017-10-01

Kidney transplantation is the treatment choice for patients with end-stage renal diseases. Because of good long-term outcome, pediatric kidney grafts are also accepted for transplantation in adult recipients despite a significant mismatch in body size and age between donor and recipient. These grafts show a remarkable ability of adaptation to the recipient body and increase in size in a very short period, presumably as an adaptation to hyperfiltration. We investigated renal graft growth as well as glomerular proliferation and differentiation markers Kiel-67, paired box gene 2 and Wilms tumor protein (WT1) expression in control biopsies from different transplant constellations: infant donor for infant recipient, infant donor for child recipient, infant donor for adult recipient, child donor for child recipient, child donor for adult recipient, and adult donor for an adult recipient. We detected a significant increase in kidney graft size after transplantation in all conditions with a body size mismatch, which was most prominent when an infant donated for a child. Podocyte WT1 expression was comparable in different transplant conditions, whereas a significant increase in WT1 expression could be detected in parietal epithelial cells, when a kidney graft from a child was transplanted into an adult. In kidney grafts that were relatively small for the recipients, we could detect reexpression of podocyte paired box gene 2. Moreover, the proliferation marker Kiel-67 was expressed in glomerular cells in grafts that increased in size after transplantation. Kidney grafts rapidly adapt to the recipient size after transplantation if they are transplanted in a body size mismatch constellation. The increase in transplant size is accompanied by an upregulation of proliferation and dedifferentiation markers in podocytes. The different examined conditions exclude hormonal factors as the key trigger for this growth so that most likely hyperfiltration is the key trigger inducing the rapid growth response.

Graft Growth and Podocyte Dedifferentiation in Donor-Recipient Size Mismatch Kidney Transplants

PubMed Central

Müller-Deile, Janina; Bräsen, Jan Hinrich; Pollheimer, Marion; Ratschek, Manfred; Haller, Hermann; Pape, Lars; Schiffer, Mario

2017-01-01

Background Kidney transplantation is the treatment choice for patients with end-stage renal diseases. Because of good long-term outcome, pediatric kidney grafts are also accepted for transplantation in adult recipients despite a significant mismatch in body size and age between donor and recipient. These grafts show a remarkable ability of adaptation to the recipient body and increase in size in a very short period, presumably as an adaptation to hyperfiltration. Methods We investigated renal graft growth as well as glomerular proliferation and differentiation markers Kiel-67, paired box gene 2 and Wilms tumor protein (WT1) expression in control biopsies from different transplant constellations: infant donor for infant recipient, infant donor for child recipient, infant donor for adult recipient, child donor for child recipient, child donor for adult recipient, and adult donor for an adult recipient. Results We detected a significant increase in kidney graft size after transplantation in all conditions with a body size mismatch, which was most prominent when an infant donated for a child. Podocyte WT1 expression was comparable in different transplant conditions, whereas a significant increase in WT1 expression could be detected in parietal epithelial cells, when a kidney graft from a child was transplanted into an adult. In kidney grafts that were relatively small for the recipients, we could detect reexpression of podocyte paired box gene 2. Moreover, the proliferation marker Kiel-67 was expressed in glomerular cells in grafts that increased in size after transplantation. Conclusions Kidney grafts rapidly adapt to the recipient size after transplantation if they are transplanted in a body size mismatch constellation. The increase in transplant size is accompanied by an upregulation of proliferation and dedifferentiation markers in podocytes. The different examined conditions exclude hormonal factors as the key trigger for this growth so that most likely hyperfiltration is the key trigger inducing the rapid growth response. PMID:29026873

SMAD4 is a potential prognostic marker in human breast carcinomas

PubMed Central

Liu, Nan-nan; Xi, Yue; Callaghan, Michael U.; Fribley, Andrew; Moore-Smith, Lakisha; Zimmerman, Jacquelyn W.; Pasche, Boris

2014-01-01

SMAD4 is a downstream mediator of transforming growth factor beta. While its tumor suppressor function has been investigated as a prognostic biomarker in several human malignancies, its role as a prognostic marker in breast carcinoma is still undefined. We investigated SMAD4 expression in breast carcinoma samples of different histologic grades to evaluate the association between SMAD4 and outcome in breast cancer. We also investigated the role of SMAD4 expression status in MDA-MB-468 breast cancer cells in responding to TGF-β stimulation. SMAD4 expression was assessed in 53 breast ductal carcinoma samples and in the surrounding normal tissue from 50 of the samples using immunohistochemistry, Western blot, and real-time PCR. TGF-β-SMAD and non-SMAD signaling was assessed by Western blot in MDA-MB-468 cells with and without SMAD4 restoration. SMAD4 expression was reduced in ductal breast carcinoma as compared to surrounding uninvolved ductal breast epithelia (p <0.05). SMAD4 expression levels decreased from Grade 1 to Grade 3 ductal breast carcinoma as assessed by immunohistochemistry (p <0.05). Results were recapitulated by tissue array. In addition, immunohistochemistry results were further confirmed at the protein and mRNA level. We then found that non-SMAD MEK/MAPK signaling was significantly different between SMAD4 expressing MDA-MB-468 cells and SMAD4-null MDA-MB-468 cells. This is the first study indicating that SMAD4 plays a key role in shifting MAPK signaling. Further, we have demonstrated that SMAD4 has a potential role in the development of breast carcinoma and SMAD4 was a potential prognostic marker of breast carcinoma. Our findings further support the role of SMAD4 in breast carcinoma development. In addition, we observed an inverse relationship between SMAD4 levels and breast carcinoma histological grade. Our finding indicated that SMAD4 expression level in breast cancer cells played a role in responding non-SMAD signaling but not the canonic SMAD signaling. Further mechanistic studies are necessary to establish the role of SMAD4 in breast carcinoma prognosis and potential specific targeting. PMID:23975369

Sex differences in the effects of adolescent stress on adult brain inflammatory markers in rats

PubMed Central

Pyter, Leah M.; Kelly, Sean D.; Harrell, Constance S.; Neigh, Gretchen N.

2013-01-01

Both basic and clinical research indicates that females are more susceptible to stress-related affective disorders than males. One of the mechanisms by which stress induces depression is via inflammatory signaling in the brain. Stress during adolescence, in particular, can also disrupt the activation and continued development of both the hypothalamic–pituitary–adrenal (HPA) and –gonadal (HPG) axes, both of which modulate inflammatory pathways and brain regions involved in affective behavior. Therefore, we tested the hypothesis that adolescent stress differentially alters brain inflammatory mechanisms associated with affective-like behavior into adulthood based on sex. Male and female Wistar rats underwent mixed-modality stress during adolescence (PND 37–48) and were challenged with lipopolysaccharide (LPS; 250 μg/kg, i.p.) or saline 4.5 weeks later (in adulthood). Hippocampal inflammatory marker gene expression and circulating HPA and HPG axes hormone concentrations were then determined. Despite previous studies indicating that adolescent stress induces affective-like behaviors in female rats only, this study demonstrated that adolescent stress increased hippocampal inflammatory responses to LPS in males only, suggesting that differences in neuroinflammatory signaling do not drive the divergent affective-like behaviors. The sex differences in inflammatory markers were not associated with differences in corticosterone. In females that experienced adolescent stress, LPS increased circulating estradiol. Estradiol positively correlated with hippocampal microglial gene expression in control female rats, whereas adolescent stress negated this relationship. Thus, estradiol in females may potentially protect against stress-induced increases in neuroinflammation. PMID:23348027

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.

The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes.

PubMed

Orozco, Carlos A; Acevedo, Andrés; Cortina, Lazaro; Cuellar, Gina E; Duarte, Mónica; Martín, Liliana; Mesa, Néstor M; Muñoz, Javier; Portilla, Carlos A; Quijano, Sandra M; Quintero, Guillermo; Rodriguez, Miriam; Saavedra, Carlos E; Groot, Helena; Torres, María M; López-Segura, Valeriano

2013-01-01

A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

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.

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

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

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE PAGES

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna; ...

2018-05-25

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

PubMed Central

El-Sayed, Karim M Fawzy; Paris, Sebastian; Graetz, Christian; Kassem, Neemat; Mekhemar, Mohamed; Ungefroren, Hendrick; Fändrich, Fred; Dörfer, Christof

2015-01-01

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells. PMID:25257881

MUC4 immunohistochemistry is useful in distinguishing epithelioid mesothelioma from adenocarcinoma and squamous cell carcinoma of the lung.

PubMed

Mawas, Amany Sayed; Amatya, Vishwa Jeet; Kushitani, Kei; Kai, Yuichiro; Miyata, Yoshihiro; Okada, Morihito; Takeshima, Yukio

2018-01-09

The differential diagnosis of epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma requires the positive and negative immunohistochemical markers of mesothelioma. The IMIG guideline has suggested the use of Calretinin, D2-40, WT1, and CK5/6 as mesothelial markers, TTF-1, Napsin-A, Claudin 4, CEA as lung adenocarcinoma markers p40, p63, CK5/6, MOC-31 as squamous cell markers. However, use of other immunohistochemical markers is still necessary. We evaluated 65 epithelioid mesotheliomas, 60 adenocarcinomas, and 57 squamous cell carcinomas of the lung for MUC4 expression by immunohistochemistry and compared with the previously known immunohistochemical markers. MUC4 expression was not found in any of 65 cases of epithelioid mesothelioma. In contrast, MUC4 expression was observed in 50/60(83.3%) cases of lung adenocarcinoma and 50/56(89.3%) cases of lung squamous cell carcinoma. The negative MUC4 expression showed 100% sensitivity, 86.2% specificity and accuracy rate of 91.2% to differentiate epithelioid mesothelioma from lung carcinoma. The sensitivity, specificity, and accuracy of MUC4 are comparable to that of previously known markers of lung adenocarcinoma and squamous cell carcinoma, namely CEA, Claudin 4 and better than that of MOC-31. In conclusion, MUC4 immunohistochemistry is useful for differentiation of epithelioid mesothelioma from lung carcinoma, either adenocarcinoma or squamous cell carcinoma.

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.

The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells.

PubMed

Jeffery, N; Richardson, S; Beall, C; Harries, L W

2017-12-15

Interaction between islet cell subtypes and the extracellular matrix influences beta-cell function in mammals. The tissue architecture of rodent islets is very different to that of human islets; cell-to-cell communication and interaction with the extracellular matrix may vary between species. In this work, we have compared the responses of the human EndoC-βH1 cell line to non-human and human-derived growth matrices in terms of growth morphology, gene expression and glucose-stimulated insulin secretion (GSIS). EndoC-βH1 cells demonstrated a greater tendency to form cell clusters when cultured in a human microenvironment and exhibited reduced alpha cell markers at the mRNA level; mean expression difference - 0.23 and - 0.51; p = 0.009 and 0.002 for the Aristaless-related homeobox (ARX) and Glucagon (GCG) genes respectively. No differences were noted in the protein expression of mature beta cell markers such as Pdx1 and NeuroD1 were noted in EndoC-βH1 cells grown in a human microenvironment but cells were however more sensitive to glucose (4.3-fold increase in insulin secretion following glucose challenge compared with a 1.9-fold increase in cells grown in a non-human microenvironment; p = 0.0003). Our data suggests that the tissue origin of the cellular microenvironment has effects on the function of EndoC-βH1 cells in vitro, and the use of a more human-like culture microenvironment may bring benefits in terms of increased physiological relevance. Copyright © 2017 Elsevier Inc. All rights reserved.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.

PubMed

Shirasawa, Madoka; Fujiwara, Naoyuki; Hirabayashi, Susumu; Ohno, Hideki; Iida, Junko; Makita, Koshi; Hata, Yutaka

2004-02-01

Lung alveolar epithelial cells are comprised of type I (ATI) and type II (ATII) cells. ATI cells are polarized, although they have very flat morphology. The identification of marker proteins for apical and basolateral membranes of ATI cells is important to investigate into the differentiation of ATI cells. In this paper, we characterized receptor for advanced glycation end-products (RAGE) as a marker for ATI cells. RAGE was localized on basolateral membranes of ATI cells in the immunoelectron microscopy and its expression was enhanced in a parallel manner to the differentiation of ATI cells in vivo and in primary cultures of ATII cells. RAGE and T1 alpha, a well-known ATI marker protein, were targeted to basolateral and apical membranes, respectively, when expressed in polarized Madine Darby canine kidney cells. Moreover, RAGE was expressed in ATI cells after T1 alpha in vivo and in ex in vivo organ cultures. In conclusion, RAGE is a marker for basolateral membranes of well-differentiated ATI cells. ATI cells require some signal provided by the in vivo environment to express RAGE.

Plasmacytoid dendritic cell leukaemia/lymphoma: towards a well defined entity?

PubMed

Garnache-Ottou, Francine; Feuillard, Jean; Saas, Philippe

2007-02-01

CD4(+)/CD56(+) haematodermic neoplasm or 'early' plasmacytoid dendritic cell leukaemia/lymphoma (pDCL) was described as a disease entity in the last World Health Organisation/European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas. These leukaemia/lymphomas co-express CD4 and CD56 without any other lineage-specific markers and have been identified as arising from plasmacytoid dendritic cells. Despite a fairly homogeneous pattern of markers expressed by most pDCL, numerous distinctive features (e.g. cytological aspects and aberrant marker expression) have been reported. This may be related to the 'lineage-independent developmental' programme of dendritic cells, which may be able to develop from either immature or already committed haematopoietic progenitors. This highlights the need for specific validated markers to diagnose such aggressive leukaemia. Here, we propose--among others (e.g. T-cell leukaemia 1)--blood dendritic cell antigen-2 and high levels of CD123 expression as potential markers. In addition, we propose a multidisciplinary approach including several fields of haematology to improve pDCL diagnosis.

ESTs and EST-linked polymorphisms for genetic mapping and phylogenetic reconstruction in the guppy, Poecilia reticulata

PubMed Central

Dreyer, Christine; Hoffmann, Margarete; Lanz, Christa; Willing, Eva-Maria; Riester, Markus; Warthmann, Norman; Sprecher, Andrea; Tripathi, Namita; Henz, Stefan R; Weigel, Detlef

2007-01-01

Background The guppy, Poecilia reticulata, is a well-known model organism for studying inheritance and variation of male ornamental traits as well as adaptation to different river habitats. However, genomic resources for studying this important model were not previously widely available. Results With the aim of generating molecular markers for genetic mapping of the guppy, cDNA libraries were constructed from embryos and different adult organs to generate expressed sequence tags (ESTs). About 18,000 ESTs were annotated according to BLASTN and BLASTX results and the sequence information from the 3' UTRs was exploited to generate PCR primers for re-sequencing of genomic DNA from different wild type strains. By comparison of EST-linked genomic sequences from at least four different ecotypes, about 1,700 polymorphisms were identified, representing about 400 distinct genes. Two interconnected MySQL databases were built to organize the ESTs and markers, respectively. A robust phylogeny of the guppy was reconstructed, based on 10 different nuclear genes. Conclusion Our EST and marker databases provide useful tools for genetic mapping and phylogenetic studies of the guppy. PMID:17686157

Identification of markers for quiescent pancreatic stellate cells in the normal human pancreas.

PubMed

Nielsen, Michael Friberg Bruun; Mortensen, Michael Bau; Detlefsen, Sönke

2017-10-01

Pancreatic stellate cells (PSCs) play a central role as source of fibrogenic cells in pancreatic cancer and chronic pancreatitis. In contrast to quiescent hepatic stellate cells (qHSCs), a specific marker for quiescent PSCs (qPSCs) that can be used in formalin-fixed and paraffin embedded (FFPE) normal human pancreatic tissue has not been identified. The aim of this study was to identify a marker enabling the identification of qPSCs in normal human FFPE pancreatic tissue. Immunohistochemical (IHC), double-IHC, immunofluorescence (IF) and double-IF analyses were carried out using a tissue microarray consisting of cores with normal human pancreatic tissue. Cores with normal human liver served as control. Antibodies directed against adipophilin, α-SMA, CD146, CRBP-1, cytoglobin, desmin, GFAP, nestin, S100A4 and vinculin were examined, with special emphasis on their expression in periacinar cells in the normal human pancreas and perisinusoidal cells in the normal human liver. The immunolabelling capacity was evaluated according to a semiquantitative scoring system. Double-IF of the markers of interest together with markers for other periacinar cells was performed. Moreover, the utility of histochemical stains for the identification of human qPSCs was examined, and their ultrastructure was revisited by electron microscopy. Adipophilin, CRBP-1, cytoglobin and vinculin were expressed in qHSCs in the liver, whereas cytoglobin and adipophilin were expressed in qPSCs in the pancreas. Adipophilin immunohistochemistry was highly dependent on the preanalytical time interval (PATI) from removal of the tissue to formalin fixation. Cytoglobin, S100A4 and vinculin were expressed in periacinar fibroblasts (FBs). The other examined markers were negative in human qPSCs. Our data indicate that cytoglobin and adipophilin are markers of qPSCs in the normal human pancreas. However, the use of adipophilin as a qPSC marker may be limited due to its high dependence on optimal PATI. Cytoglobin, on the other hand, is a sensitive marker for qPSCs but is expressed in FBs as well.

Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.

PubMed

Petersen, B E; Goff, J P; Greenberger, J S; Michalopoulos, G K

1998-02-01

Hepatic oval cells (HOC) are a small subpopulation of cells found in the liver when hepatocyte proliferation is inhibited and followed by some type of hepatic injury. HOC can be induced to proliferate using a 2-acetylaminofluorene (2-AAF)/hepatic injury (i.e., CCl4, partial hepatectomy [PHx]) protocol. These cells are believed to be bipotential, i.e., able to differentiate into hepatocytes or bile ductular cells. In the past, isolation of highly enriched populations of these cells has been difficult. Thy-1 is a cell surface marker used in conjunction with CD34 and lineage-specific markers to identify hematopoietic stem cells. Thy-1 antigen is not normally expressed in adult liver, but is expressed in fetal liver, presumably on the hematopoietic cells. We report herein that HOC express high levels of Thy-1. Immunohistochemistry revealed that the cells expressing Thy-1 were indeed oval cells, because they also expressed alpha-fetoprotein (AFP), gamma-glutamyl transpeptidase (GGT), cytokeratin 19 (CK-19), OC.2, and OV-6, all known markers for oval cell identification. In addition, the Thy-1+ cells were negative for desmin, a marker specific for Ito cells. Using Thy-1 antibody as a new marker for the identification of oval cells, a highly enriched population was obtained. Using flow cytometric methods, we isolated a 95% to 97% pure Thy-1+ oval cell population. Our results indicate that cell sorting using Thy-1 could be an attractive tool for future studies, which would facilitate both in vivo and in vitro studies of HOC.

ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

PubMed Central

Valabrega, G; Fagioli, F; Corso, S; Madon, E; Brach del Prever, A; Biasin, E; Linari, A; Aglietta, M; Giordano, S

2003-01-01

Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker. PMID:12569382

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

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

PubMed

Guzmán-Uribe, Daniela; Alvarado-Estrada, Keila Neri; Pierdant-Pérez, Mauricio; Torres-Álvarez, Bertha; Sánchez-Aguilar, Jesus Martin; Rosales-Ibáñez, Raúl

2017-01-01

The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis

PubMed Central

Dharmaraj, N.; Chapela, P.J.; Morgado, M.; Hawkins, S.M.; Lessey, B.A.; Young, S.L.; Carson, D.D.

2014-01-01

STUDY QUESTION Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? SUMMARY ANSWER This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). WHAT IS KNOWN ALREADY Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. STUDY DESIGN, SIZE, DURATION This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT–PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or β-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. MAIN RESULTS AND THE ROLE OF CHANCE qRT–PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. LIMITATIONS, REASONS FOR CAUTION qRT–PCR analysis was the main method used for mucin detection. Additional studies with stage III–IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to β-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. STUDY FUNDING This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare. PMID:24939955

Squamous Cell Carcinoma Antigen-encoding Genes SERPINB3/B4 as Potentially Useful Markers for the Stratification of HNSCC Tumours.

PubMed

Saidak, Zuzana; Morisse, Mony Chenda; Chatelain, Denis; Sauzay, Chloé; Houessinon, Aline; Guilain, Nelly; Soyez, Marion; Chauffert, Bruno; Dakpé, Stéphanie; Galmiche, Antoine

2018-03-01

The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context. We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA). HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status. Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A study on OPG/RANK/RANKL axis in osteoporotic bile duct-ligated rats and the involvement of nitrergic and opioidergic systems.

PubMed

Doustimotlagh, Amir Hossein; Dehpour, Ahmad Reza; Etemad-Moghadam, Shahroo; Alaeddini, Mojgan; Ostadhadi, Sattar; Golestani, Abolfazl

2018-06-01

Chronic liver disease (CLD) affects millions of people and its impact on bone loss has become a subject of interest. Nitric oxide and endogenous opioids are suggested to increase during cholestasis/cirrhosis and may impact bone resorption by different mechanisms. The receptor activator of nuclear factor-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) signaling pathway regulates bone resorption, but its role in metabolic bone disease subsequent to CLD is unknown. We aimed to investigate the involvement of nitrergic and opioidergic systems in bone loss relative to the RANK/RANKL/OPG pathway, in bile duct-ligated (BDL) rats. Eighty BDL/sham-operated (SO) rats received injections of 3 mg/kg/day Nω-Nitro-L-arginine methyl ester ± naltrexone (10 mg/kg/day) or saline for 28 days. Plasma bone turnover markers, OPG, RANK, and RANKL along with mRNA expression levels of the latter three were assessed. Plasma bone turnover markers and OPG level increased, but RANKL decreased in the BDL group compared with their SO controls (both: P ≤ 0.05). Administration of naltrexone reduced bone turnover markers and OPG level while increased RANKL content in comparison to BDL rats ( P ≤ 0.05). As compared to untreated BDL rats, nitric oxide inhibition showed no effect on bone turnover marker i.e. OPG, RANK, and RANKL levels. BDL significantly increased RANK mRNA, but had no significant effect on RANKL and OPG mRNA expression. The lack of association between plasma levels and quantitative gene expression of RANKL and OPG suggests an indirect function of these markers in BDL rats. Considering that opioid receptor blockage by naltrexone in BDL animals caused a significant decrease in OPG and an increase in RANKL plasma contents, it could be postulated that the opioidergic system may have a regulatory effect on these bone markers.

Keratins 17 and 19 expression as prognostic markers in oral squamous cell carcinoma.

PubMed

Coelho, B A; Peterle, G T; Santos, M; Agostini, L P; Maia, L L; Stur, E; Silva, C V M; Mendes, S O; Almança, C C J; Freitas, F V; Borçoi, A R; Archanjo, A B; Mercante, A M C; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

2015-11-25

Five-year survival rates for oral squamous cell carcinoma (OSCC) are 30% and the mortality rate is 50%. Immunohistochemistry panels are used to evaluate proliferation, vascularization, apoptosis, HPV infection, and keratin expression, which are important markers of malignant progression. Keratins are a family of intermediate filaments predominantly expressed in epithelial cells and have an essential role in mechanical support and cytoskeleton formation, which is essential for the structural integrity and stability of the cell. In this study, we analyzed the expressions of keratins 17 and 19 (K17 and K19) by immunohistochemistry in tumoral and non-tumoral tissues from patients with OSCC. The results show that expression of these keratins is higher in tumor tissues compared to non-tumor tissues. Positive K17 expression correlates with lymph node metastasis and multivariate analysis confirmed this relationship, revealing a 6-fold increase in lymph node metastasis when K17 is expressed. We observed a correlation between K17 expression with disease-free survival and disease-specific death in patients who received surgery and radiotherapy. Multivariate analysis revealed that low expression of K17 was an independent marker for early disease relapse and disease-specific death in patients treated with surgery and radiotherapy, with an approximately 4-fold increased risk when compared to high K17 expression. Our results suggest a potential role for K17 and K19 expression profiles as tumor prognostic markers in OSCC patients.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

An essential role for LPA signalling in telencephalon development.

PubMed

Geach, Timothy J; Faas, Laura; Devader, Christelle; Gonzalez-Cordero, Anai; Tabler, Jacqueline M; Brunsdon, Hannah; Isaacs, Harry V; Dale, Leslie

2014-02-01

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Characterization of an acute molecular marker of nongenotoxic rodent hepatocarcinogenesis by gene expression profiling in a long term clofibric acid study.

PubMed

Michel, Cécile; Roberts, Ruth A; Desdouets, Chantal; Isaacs, Kevin R; Boitier, Eric

2005-04-01

Evaluation of the nongenotoxic potential early during the development of a drug presents a major challenge. Recently, two genes were identified as potential molecular markers of rodent hepatic carcinogenesis: transforming growth factor-beta stimulated clone 22 (TSC-22) and NAD(P)H cytochrome P450 oxidoreductase (CYP-R) (1). They were identified after comparing the gene expression profiles obtained from the livers of Sprague-Dawley rats treated with different genotoxic and nongenotoxic compounds in a 5 day repeat dose in vivo study. To assess the potential of these two genes as acute markers of carcinogenesis, we investigated their modulation during a long-term nongenotoxic study in the rat using a classic initiation-promotion regime. Clofibric acid (CLO), which belongs to the broad class of chemicals known as peroxisome proliferators, was used as a nongenotoxic hepatocarcinogen. Male F344 rats were given a single nonnecrogenic injection of diethylnitrosamine (0 or 30 mg/kg) and fed a diet containing none or 5000 ppm CLO for up to 20 months. Necropsies of five rats per groups were performed at 18, 46, 102, 264, 377, 447 (control, DEN, and DEN + CLO rats), 524, and 608 days (for the CLO and control rats). Gross macroscopic and microscopic evaluation and gene expression profiling (on Affymetrix microarrays) were performed in peritumoral and tumoral liver tissues. Bioanalysis of the liver gene expression data revealed that TSC-22 was strongly down-regulated early in the study. Its underexpression was maintained throughout the study but disappeared upon CLO withdrawal. These modulations were confirmed by real-time polymerase chain reaction. However, CYP-R gene expression was not significantly altered in our study. Taken together, our results showed that TSC-22, but not CYP-R, has the potential to be an acute early molecular marker for nongenotoxic hepatocarcinogenesis in rodents.

Contrasting influences of Drosophila white/mini-white on ethanol sensitivity in two different behavioral assays.

PubMed

Chan, Robin F; Lewellyn, Lara; DeLoyht, Jacqueline M; Sennett, Kristyn; Coffman, Scarlett; Hewitt, Matthew; Bettinger, Jill C; Warrick, John M; Grotewiel, Mike

2014-06-01

The fruit fly Drosophila melanogaster has been used extensively to investigate genetic mechanisms of ethanol (EtOH)-related behaviors. Many past studies in flies, including studies from our laboratory, have manipulated gene expression using transposons carrying the genetic-phenotypic marker mini-white(mini-w), a derivative of the endogenous gene white(w). Whether the mini-w transgenic marker or the endogenous w gene influences behavioral responses to acute EtOH exposure in flies has not been systematically investigated. We manipulated mini-w and w expression via (i) transposons marked with mini-w, (ii) RNAi against mini-w and w, and (iii) a null allele of w. We assessed EtOH sensitivity and tolerance using a previously described eRING assay (based on climbing in the presence of EtOH) and an assay based on EtOH-induced sedation. In eRING assays, EtOH-induced impairment of climbing correlated inversely with expression of the mini-w marker from a series of transposon insertions. Additionally, flies harboring a null allele of w or flies with RNAi-mediated knockdown of mini-w were significantly more sensitive to EtOH in eRING assays than controls expressing endogenous w or the mini-w marker. In contrast, EtOH sensitivity and rapid tolerance measured in the EtOH sedation assay were not affected by decreased expression of mini-w or endogenous w in flies. EtOH sensitivity measured in the eRING assay is noticeably influenced by w and mini-w, making eRING problematic for studies on EtOH-related behavior in Drosophila using transgenes marked with mini-w. In contrast, the EtOH sensitivity assay described here is a suitable behavioral paradigm for studies on EtOH sensitivity and rapid tolerance in Drosophila including those that use widely available transgenes marked with mini-w. Copyright © 2014 by the Research Society on Alcoholism.

Expression Plasmids for Use in Candida glabrata

PubMed Central

Zordan, Rebecca E.; Ren, Yuxia; Pan, Shih-Jung; Rotondo, Giuseppe; Peñas, Alejandro De Las; Iluore, Joseph; Cormack, Brendan P.

2013-01-01

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata. PMID:23934995

In situ expression of heat-shock proteins and 3-nitrotyrosine in brains of young rats exposed to a WiFi signal in utero and in early life.

PubMed

Aït-Aïssa, Saliha; de Gannes, Florence Poulletier; Taxile, Murielle; Billaudel, Bernard; Hurtier, Annabelle; Haro, Emmanuelle; Ruffié, Gilles; Athané, Axel; Veyret, Bernard; Lagroye, Isabelle

2013-06-01

The bioeffects of exposure to Wireless High-Fidelity (WiFi) signals on the developing nervous systems of young rodents was investigated by assessing the in vivo and in situ expression levels of three stress markers: 3-Nitrotyrosine (3-NT), an oxidative stress marker and two heat-shock proteins (Hsp25 and Hsp70). These biomarkers were measured in the brains of young rats exposed to a 2450 MHz WiFi signal by immunohistochemistry. Pregnant rats were first exposed or sham exposed to WiFi from day 6 to day 21 of gestation. In addition three newborns per litter were further exposed up to 5 weeks old. Daily 2-h exposures were performed blind in a reverberation chamber and whole-body specific absorption rate levels were 0, 0.08, 0.4 and 4 W/kg. 3-NT and stress protein expression was assayed in different areas of the hippocampus and cortex. No significant difference was observed among exposed and sham-exposed groups. These results suggest that repeated exposure to WiFi during gestation and early life has no deleterious effects on the brains of young rats.

The embryonic genes Dkk3, Hoxd8, Hoxd9 and Tbx1 identify muscle types in a diet-independent and fiber-type unrelated way.

PubMed

de Wilde, Janneke; Hulshof, Martijn F M; Boekschoten, Mark V; de Groot, Philip; Smit, Egbert; Mariman, Edwin C M

2010-03-15

The mouse skeletal muscle is composed of four distinct fiber types that differ in contractile function, number of mitochondria and metabolism. Every muscle type has a specific composition and distribution of the four fiber types. To find novel genes involved in specifying muscle types, we used microarray analysis to compare the gastrocnemius with the quadriceps from mice fed a low fat diet (LFD) or high fat diet (HFD) for 8 weeks. Additional qPCR analysis were performed in the gastrocnemius, quadriceps and soleus muscle from mice fed an LFD or HFD for 20 weeks. In mice fed the 8-week LFD 162 genes were differentially expressed in the gastrocnemius vs. the quadriceps. Genes with the strongest differences in expression were markers for oxidative fiber types (e.g. Tnni1) and genes which are known to be involved in embryogenesis (Dkk3, Hoxd8,Hoxd9 and Tbx1). Also Dkk2, Hoxa5, Hoxa10, Hoxc9, Hoxc10, Hoxc6 and Tbx15 were detectably, but not differentially expressed in adult muscle tissue. Expression of differentially expressed genes was not influenced by an 8-week or 20-week HFD. Comparing gastrocnemius, quadriceps and soleus, expression of Hoxd8 and Hoxd9 was not related with expression of markers for the four different fiber types. We found that the expression of both Hoxd8 and Hoxd9 was much higher in the gastrocnemius than in the quadriceps or soleus, whereas the expression of Dkk3 was high in quadriceps, but low in both gastrocnemius and soleus. Finally, expression of Tbx1 was high in quadriceps, intermediate in soleus and low in gastrocnemius. We found that genes from the Dkk family, Hox family and Tbx family are detectably expressed in adult mouse muscle. Interestingly, expression of Dkk3, Hoxd8, Hoxd9 and Tbx1 was highly different between gastrocnemius, quadriceps and soleus. In fact, every muscle type showed a unique combination of expression of these four genes which was not influenced by diet. Altogether, we conclude that genes important for embryogenesis identify mouse muscle types in a diet-independent and fiber type-unrelated manner.

Behavior of Human Bone Marrow-Derived Mesenchymal Stem Cells on Various Titanium-Based Coatings

PubMed Central

Qu, Chengjuan; Kaitainen, Salla; Kröger, Heikki; Lappalainen, Reijo; Lammi, Mikko J.

2016-01-01

The chemical composition and texture of titanium coatings can influence the growth characteristics of the adhered cells. An enhanced proliferation of the human mesenchymal stem cells (hMSCs) would be beneficial. The present study was aimed to investigate whether titanium deposited at different atmospheres would affect the cell growth properties, cellular morphology, and expression of surface markers of hMSCs. Titanium-based coatings were deposited on silicon wafers under oxygen, nitrogen, or argon atmospheres by ultra-short pulsed laser deposition using two different gas pressures followed by heating at 400 °C for 2 h. The characteristics of the coated surfaces were determined via contact angle, zeta potential, and scanning electron microscopy (SEM) techniques. Human MSCs were cultivated on differently coated silicon wafers for 48 h. Subsequently, the cell proliferation rates were analyzed with an MTT assay. The phenotype of hMSCs was checked via immunocytochemical stainings of MSC-associated markers CD73, CD90, and CD105, and the adhesion, spreading, and morphology of hMSCs on coated materials via SEM. The cell proliferation rates of the hMSCs were similar on all coated silicon wafers. The hMSCs retained the MSC phenotype by expressing MSC-associated markers and fibroblast-like morphology with cellular projections. Furthermore, no significant differences could be found in the size of the cells when cultured on all various coated surfaces. In conclusion, despite certain differences in the contact angles and the zeta potentials of various titanium-based coatings, no single coating markedly improved the growth characteristics of hMSCs. PMID:28773947

Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies

PubMed Central

2011-01-01

Micro-RNAs (miRs) are important regulators of mRNA and protein expression; the ability of miR expression profilings to distinguish different cancer types and classify their sub-types has been well-described. They also represent a novel biological entity with potential value as tumour biomarkers, which can improve diagnosis, prognosis, and monitoring of treatment response for human cancers. This endeavour has been greatly facilitated by the stability of miRs in formalin-fixed paraffin-embedded (FFPE) tissues, and their detection in circulation. This review will summarize some of the key dysregulated miRs described to date in human epithelial malignancies, and their potential value as molecular bio-markers in FFPE tissues and blood samples. There remain many challenges in this domain, however, with the evolution of different platforms, the complexities of normalizing miR profiling data, and the importance of evaluating sufficiently-powered training and validation cohorts. Nonetheless, well-conducted miR profiling studies should contribute important insights into the molecular aberrations driving human cancer development and progression. PMID:22128797

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.

Expression of interest: transcriptomics and the designation of conservation units.

PubMed

Hansen, Michael M

2010-05-01

An important task within conservation genetics consists in defining intraspecific conservation units. Most conceptual frameworks involve two steps: (i) identifying demographically independent units, and (ii) evaluating their degree of adaptive divergence. Whereas a plethora of methods are available for delineating genetic population structure, assessment of functional genetic divergence remains a challenge. In this issue, Tymchuk et al. (2010) study Atlantic salmon (Salmo salar) populations using both microsatellite markers and analysis of global gene expression. They show that important gene expression differences exist that can be interpreted in the context of different ecological conditions experienced by the populations, along with the populations' histories. This demonstrates an important potential role of transcriptomics for designating conservation units.

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

PubMed Central

Varghese, Divya S.; Parween, Shama; Ardah, Mustafa T.; Emerald, Bright Starling

2017-01-01

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results. PMID:29147115

Current molecular profile of juvenile nasopharyngeal angiofibroma: First comprehensive study from India.

PubMed

Pandey, Praveen; Mishra, Anupam; Tripathi, Ashoak Mani; Verma, Veerendra; Trivedi, Ritu; Singh, Hitendra Prakash; Kumar, Sunil; Patel, Brijesh; Singh, Vinay; Pandey, Shivani; Pandey, Amita; Mishra, Subhash Chandra

2017-03-01

An attempt is made to analyze the molecular behavior of juvenile nasopharyngeal angiofibroma (JNA). Case Series METHODS: Quantification of mRNAs expression was undertaken through real-time polymerase chain reaction in JNA (9-24) samples for VEGF-A, basic fibroblast growth factor (b-FGF), platelet-derived growth factor PDGF-A, KIT proto-oncogene receptor tyrosine kinase (c-Kit), Avian myelomatosis viral oncogene homolog (c-Myc), Harvey rat sarcoma viral oncogene homolog (H-Ras), tumor suppressor gene TP53, and androgen receptor and interleukin 6 (IL-6). The β-catenin expression was evaluated by western blot in 16 samples. Nasal polyp was taken as control. A significantly increased (P < 0.01) expression of c-myc, VEGFA, bFGF, PDGFA, c-kit, and TP53 was seen, along with enhanced expression of β-catenin. A massive enhancement of H-Ras expression was seen for the first time. Androgen receptor expression was no different, whereas IL-6 despite showing upregulation trend was not significant. The enhanced expressions of various markers suggest their potential role in JNA. Although the biological significance of c-kit, c-myc, and one of the novel markers H-Ras has yet to be defined, their significant expression may have a therapeutic importance. NA. Laryngoscope, 127:E100-E106, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

USGS Publications Warehouse

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Immunohistochemical localization of cystic fibrosis transmembrane regulator and clara cell secretory protein in taste receptor cells of rat circumvallate papillae.

PubMed

Merigo, Flavia; Benati, Donatella; Galiè, Mirco; Crescimanno, Caterina; Osculati, Francesco; Sbarbati, Andrea

2008-03-01

Taste receptor cells (TRCs) are the sensory cells of taste transduction and are organized into taste buds embedded in the epithelium of the tongue, palate, pharynx, and larynx. Several studies have demonstrated that TRCs involved in sweet as well as bitter and umami responses express alpha-gustducin, an alpha-subunit of the G-protein complex. It has been further demonstrated that this typical taste protein is a potent marker of chemosensory cells located in several tissues, including gastric and pancreatic mucosa and the respiratory apparatus. We recently observed that alpha-gustducin and phospholipase C beta 2-immunoreactive cells were colocalized in the airways with cystic fibrosis transmembrane regulator (CFTR) and Clara cell-specific secretory protein of 10 (CC10) and 26 kDa (CC26). This finding suggests that TRCs might themselves express secretory markers. To test this hypothesis, we investigated the expression of CFTR, CC10, and CC26 in rat circumvallate papillae using reverse transcriptase-polymerase chain reaction analysis, immunohistochemistry, and confocal laser microscopy. The results showed that secretory markers such as CFTR, CC10, and CC26 are present in taste cells of rat circumvallate papillae, and their immunoreactivity is expressed, to a different extent, in subsets of taste cells that express alpha-gustducin. The presence of CFTR, CC10, and CC26 in taste bud cells and their coexpression pattern with alpha-gustducin confirms and extends our previous findings in airway epithelium, lending further credence to the notion that chemoreception and secretion may be related processes.

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.

Molecular Markers in Patients with Chronic Wounds to Guide Surgical Debridement

PubMed Central

Brem, Harold; Stojadinovic, Olivera; Diegelmann, Robert F; Entero, Hyacinth; Lee, Brian; Pastar, Irena; Golinko, Michael; Rosenberg, Harvey; Tomic-Canic, Marjana

2007-01-01

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients’ wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hyperkeratotic epidermis; dermal fibrosis; increased procollagen synthesis). Fibroblasts deriving from this location exhibit impaired migration in comparison to the cells from adjacent nonulcerated biopsies, which exhibit normalization of morphology and normal migration capacity. The nonhealing edges have a specific, identifiable, and reproducible gene expression profile. The adjacent nonulcerated biopsies have their own distinctive reproducible gene expression profile, signifying that particular wound areas can be identified by gene expression profiling. We conclude that chronic ulcers contain distinct subpopulations of cells with different capacity to heal and that gene expression profiling can be utilized to identify them. In the future, molecular markers will be developed to identify the nonimpaired tissue, thereby making surgical debridement more accurate and more efficacious. PMID:17515955

Histogenesis of pure and combined Merkel cell carcinomas: An immunohistochemical study of 14 cases.

PubMed

Narisawa, Yutaka; Koba, Shinichi; Inoue, Takuya; Nagase, Kotaro

2015-05-01

The histogenesis of Merkel cell carcinoma (MCC) has remained unresolved. Moreover, one of the questions is whether pure MCC and combined MCC represent the same histogenesis and entity. The existence of combined MCC suggests that MCC likely arise from pluripotent stem cells. Merkel cells (MC) localize within the bulge area, which is populated by hair follicle stem cells. We used hair follicle stem cell markers to investigate whether MCC share certain characteristics of these stem cells. Fourteen MCC specimens were examined histologically and immunohistochemically. There were six pure MCC and eight combined MCC. In six combined MCC, both MCC components and squamous components at least focally shared the expression of one or more of cytokeratin (CK)15, CK19 and CD200, which are hair follicle stem cell markers. On the other hand, four cases of pure MCC showed partially distinct CK19 expression, but did not show CK15 and/or CD200 expression. There was a distinct difference between pure MCC and combined MCC on the expression of hair follicle stem cell markers. The normal skin expressed CK15, CK19 and CD200 in the bulge area, whereas CK15 and CD200 were absent in the MC-rich glabrous skin and touch domes. The results led us to hypothesize that combined MCC originate from the hair follicle stem cells. We postulate that combined MCC undergo multidirectional differentiation into squamous, glandular, mesenchymal and Merkel cells. Further investigation is warranted to confirm the histogenesis of pure MCC and combined MCC. © 2015 Japanese Dermatological Association.

Substance-specific importance of EGFR for vascular smooth muscle cells motility in primary culture.

PubMed

Schreier, Barbara; Schwerdt, Gerald; Heise, Christian; Bethmann, Daniel; Rabe, Sindy; Mildenberger, Sigrid; Gekle, Michael

2016-07-01

Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations. In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed. VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

PubMed Central

Yamamoto, K; Oda, Y; Haseda, A; Fujito, S; Mikami, T; Onodera, Y

2014-01-01

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03–336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ∼12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes. PMID:24169648

Differences in microRNA expression during tumor development in the transition and peripheral zones of the prostate

PubMed Central

2013-01-01

Background The prostate is divided into three glandular zones, the peripheral zone (PZ), the transition zone (TZ), and the central zone. Most prostate tumors arise in the peripheral zone (70-75%) and in the transition zone (20-25%) while only 10% arise in the central zone. The aim of this study was to investigate if differences in miRNA expression could be a possible explanation for the difference in propensity of tumors in the zones of the prostate. Methods Patients with prostate cancer were included in the study if they had a tumor with Gleason grade 3 in the PZ, the TZ, or both (n=16). Normal prostate tissue was collected from men undergoing cystoprostatectomy (n=20). The expression of 667 unique miRNAs was investigated using TaqMan low density arrays for miRNAs. Student’s t-test was used in order to identify differentially expressed miRNAs, followed by hierarchical clustering and principal component analysis (PCA) to study the separation of the tissues. The ADtree algorithm was used to identify markers for classification of tissues and a cross-validation procedure was used to test the generality of the identified miRNA-based classifiers. Results The t-tests revealed that the major differences in miRNA expression are found between normal and malignant tissues. Hierarchical clustering and PCA based on differentially expressed miRNAs between normal and malignant tissues showed perfect separation between samples, while the corresponding analyses based on differentially expressed miRNAs between the two zones showed several misplaced samples. A classification and cross-validation procedure confirmed these results and several potential miRNA markers were identified. Conclusions The results of this study indicate that the major differences in the transcription program are those arising during tumor development, rather than during normal tissue development. In addition, tumors arising in the TZ have more unique differentially expressed miRNAs compared to the PZ. The results also indicate that separate miRNA expression signatures for diagnosis might be needed for tumors arising in the different zones. MicroRNA signatures that are specific for PZ and TZ tumors could also lead to more accurate prognoses, since tumors arising in the PZ tend to be more aggressive than tumors arising in the TZ. PMID:23890084

Epithelial to mesenchymal transition in human endocrine islet cells

PubMed Central

Moreno-Amador, José Luis; Téllez, Noèlia; Marin, Sandra; Aloy-Reverté, Caterina; Semino, Carlos; Nacher, Montserrat

2018-01-01

Background β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. Objective To investigate whether EMT takes place in the endocrine non-β cells of human islets. Methodology Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. Results Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. Conclusions In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype. PMID:29360826

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

PubMed Central

Rodrigues‐Pinto, Ricardo; Berry, Andrew; Piper‐Hanley, Karen; Hanley, Neil; Richardson, Stephen M.

2016-01-01

ABSTRACT In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016. PMID:26910849

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord-specific markers during early human intervertebral disc development.

PubMed

Rodrigues-Pinto, Ricardo; Berry, Andrew; Piper-Hanley, Karen; Hanley, Neil; Richardson, Stephen M; Hoyland, Judith A

2016-08-01

In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte-like cells. Although animal studies indicate that notochord-derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5-18 weeks post-conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E-cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co-expressed by sclerotomal cells. CD90, Tie2, and E-cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord-specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327-1340, 2016. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc.

Newly available antibodies with practical applications in surgical pathology.

PubMed

Chan, John K C

2013-12-01

Selected antibodies that have become available in recent years and have applications in diagnostic pathology are discussed. They include antibodies that are organ-related, provide information on cellular differentiation or histogenetic type, have predictive value in tumors, and highlight infective agents. PAX8 (paired box gene 8) is a marker expressed in the lower female genital tract, thyroid, and kidney and their tumors. Napsin A is expressed in the lung and kidney and is an alternative marker for pulmonary adenocarcinoma. Arginase A is a sensitive and specific marker for liver tumors. ERG (Ets-related gene) is an excellent marker for endothelium and vascular tumors as well as prostatic cancer (about 50% of cases). SOX10 (SRY-related HMG box) is expressed predominantly in melanocytic and Schwann cells and the corresponding tumors. DOG1 (discovered on GIST 1) is an excellent marker for gastrointestinal stromal tumor (GIST) and acinic cell carcinoma. OCT3/4 is a pan-germ cell tumor marker, except yolk sac tumor. SALL4 is positive in various types of germ cell tumors, including yolk sac tumor. MUC4 (mucin-related antigen 4) is a sensitive and specific marker for low-grade fibromyxoid sarcoma. Langerin is a specific marker for Langerhans cells and their tumors. SOX11 is a sensitive marker for mantle cell lymphoma. New generation antibodies against anaplastic lymphoma kinase (ALK) are required to reliably demonstrate ALK gene translocation in pulmonary carcinomas. Lack of expression of succinate dehydrogenase B is seen in paragangliomas of the hereditary form and in the pediatric type of GIST. Antibodies against Trepenoma pallidum can facilitate the diagnosis of syphilis, whereas those against SV40 (simian virus 40) are helpful for diagnosis of BK virus infection and progressive multifocal leukoencephalopathy.

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.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture

PubMed Central

Eildermann, K.; Gromoll, J.; Behr, R.

2012-01-01

BACKGROUND Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells. PMID:22442249

Characterization of EST-derived and non-EST simple sequence repeats in an F₁ hybrid population of Vitis vinifera L.

PubMed

Kayesh, E; Bilkish, N; Liu, G S; Chen, W; Leng, X P; Fang, J G

2014-03-31

Among different classes of molecular markers, expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping in F1 hybrid populations of Vitis vinifera L. Recently, because of the availability of an enormous amount of data for ESTs in the public domain, the emphasis has shifted from genomic SSRs to EST-SSRs, which belong to transcribed regions of the genome and may have a role in gene expression or function. The objective of this study was to assess the polymorphisms among 94 F1 hybrids from "Early Rose" and "Red Globe" using 25 EST-derived and 25 non-EST SSR markers. A total collection of 362,375 grape ESTs that were retrieved from the National Center for Biotechnology Information (NCBI) and 2522 EST-SSR sequences were identified. From them, 205 primer pairs were randomly selected, including 176 pairs that were EST-derived and 29 non-EST SSR primer pairs, for polymerase chain reaction amplification. A total of 131 alleles were amplified using 50 pairs of primers; 78 alleles were amplified using EST-derived SSR primers and 53 were from non-EST SSR primers. At most, 6 and 5 alleles were amplified by EST-derived and non-EST SSR primers, respectively. The EST-derived SSR markers showed a maximum polymorphic information content (PIC) value of 1 and a minimum of 0.33 while non-EST SSR markers had maximum and minimum PIC values of 1 and 0.25, respectively. The average PIC value was 0.56 for EST-derived SSR markers and 0.45 for non-EST SSR markers.

Transgenic mice that accept Luciferase- or GFP-expressing syngeneic tumor cells at high efficiencies.

PubMed

Aoyama, Naoki; Miyoshi, Hiroyuki; Miyachi, Hitoshi; Sonoshita, Masahiro; Okabe, Masaru; Taketo, Makoto Mark

2018-05-11

Jellyfish green fluorescent protein (GFP) and firefly luciferase can serve as versatile tracking markers for identification and quantification of transplanted cancer cells in vivo. However, immune reactions against these markers can hamper the formation of syngraft tumors and metastasis that follows. Here, we report two transgenic (Tg) mouse lines that express nonfunctional mutant marker proteins, namely modified firefly luciferase (Luc2) or enhanced GFP (EGFP). These mice, named as Tg-mLuc2 and Tg-mEGFP, turned out to be immunologically tolerant to the respective tracking markers and thus efficiently accepted syngeneic cancer cells expressing the active forms of the markers. We then injected intrarectally the F 1 hybrid Tg mice (BALB/c × C57BL/6J) with Colon-26 (C26) colon cancer cells that originated from a BALB/c mouse. Even when C26 cells expressed active Luc2 or EGFP, they formed primary tumors in the Tg mice with only 10 4 cells per mouse compared with more than 10 6 cells required in the nontransgenic BALB/c hosts. Furthermore, we detected metastatic foci of C26 cells in the liver and lungs of the Tg mice by tracking the specific reporter activities. These results show the usefulness of the Tg mouse lines as recipients for transplantation experiments with the non-self tracking marker-expressing cells. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial.

PubMed

Yung, Sofie Shuk Fei; Lee, Vivian Chi Yan; Chiu, Philip Chi Ngong; Li, Hang Wun Raymond; Ng, Ernest Hung Yu; Yeung, William Shu Biu; Ho, Pak Chung

2016-04-01

To evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. We performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. We excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17 ± 2.67 (mean ± SD) in the letrozole group and 9.01 ± 2.82 in the placebo group (p=0.36). We did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. We did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first trimester. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking Spatial and Temporal Changes in Microbial Metabolic Potential and Gene Expression Patterns Across Geochemical Gradients at Axial Seamount

NASA Astrophysics Data System (ADS)

Fortunato, C. S.; Butterfield, D. A.; Larson, B.; Algar, C. K.; Huber, J. A.

2016-12-01

Microbial communities living both near and within the subseafloor are important players in the biogeochemical cycling of the deep ocean. To better understand the metabolic and gene expression patterns of these understudied communities, we collected low-temperature diffuse fluids for metagenomic, metatranscriptomic, and geochemical analyses from Axial Seamount, an active submarine volcano off the coast of Oregon, USA in 2013-2015. In April of 2015 Axial Seamount erupted along its north rift, five months before the 2015 samples were collected. This study thus provides both spatial and temporal analysis of subseafloor microbial communities pre and post eruption. The time series for this study focused on three vents at the south end of the caldera: Anemone, Marker 33, and Marker 113. Chemistry data shows that at each vent there are different geochemical conditions and thus a potentially different microbial metabolic profile. Anemone has the most oxidizing conditions and the highest abundance and expression of sulfur oxidation genes, attributed to both SUP05 and Epsilonproteobacteria. The most reducing conditions were observed at Marker 113, the site with the lowest oxygen concentration and where methanogenesis was the dominant metabolism, with 18.5% of all annotated transcripts attributed to methanogenesis. Although individual vents were metabolically distinct, there was very little variation in the overall taxonomic and metabolic profiles of each vent across years, even after the 2015 eruption. A diffuse fluid sample taken from the North Rift Zone post eruption showed similar community taxonomy to both Anemone and Marker 33; analyses of the metabolic potential and gene expression at this site is ongoing and will act as a comparison between the communities of the time series vents and those that were closer to the eruption site. Together, these chemical and `omic datasets reveal a dynamic microbial community at each vent, taxonomically diverse and involved in a wide array of biogeochemical transformations. Results are being used to model the functional dynamics and fluxes of vent communities to more closely link microbiological productivity at hydrothermal systems to deep-sea biogeochemical processes and will be also used to inform future projects using instrumentation on the cabled array at Axial Seamount.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

PubMed

Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

2018-06-01

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

Differential marker expression by cultures rich in mesenchymal stem cells

PubMed Central

2013-01-01

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

Differences in E-Cadherin and Syndecan-1 Expression in Different Types of Ameloblastomas

PubMed Central

López-Verdín, Sandra; Pereira-Prado, Vanesa

2018-01-01

Ameloblastomas are a group of benign, locally aggressive, recurrent tumors characterized by their slow and infiltrative growth. E-Cadherin and syndecan-1 are cell adhesion molecules related to the behavior of various tumors, including ameloblastomas. Ninety-nine ameloblastoma samples were studied; the expression of E-cadherin and syndecan-1 were evaluated by immunohistochemistry. E-Cadherin and epithelial syndecan-1 were more highly expressed in intraluminal/luminal unicystic ameloblastoma than in mural unicystic ameloblastoma and solid/multicystic ameloblastoma, whereas the stromal expression of syndecan-1 was higher in mural unicystic ameloblastoma and solid/multicystic ameloblastoma. Synchronicity was observed between E-cadherin and epithelial syndecan-1; the expression was correlated with intensity in all cases. There was a strong association between expression and tumor size and recurrence. The evaluation of the expression of E-cadherin and syndecan-1 are important for determining the potential aggressiveness of ameloblastoma variants. Future studies are required to understand how the expression of these markers is related to tumor aggressiveness.

Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context

PubMed Central

Geffroy, Benjamin; Guilbaud, Florian; Amilhat, Elsa; Beaulaton, Laurent; Vignon, Matthias; Huchet, Emmanuel; Rives, Jacques; Bobe, Julien; Fostier, Alexis; Guiguen, Yann; Bardonnet, Agnès

2016-01-01

Environmental sex determination (ESD) has been detected in a range of vertebrate reptile and fish species. Eels are characterized by an ESD that occurs relatively late, since sex cannot be histologically determined before individuals reach 28 cm. Because several eel species are at risk of extinction, assessing sex at the earliest stage is a crucial management issue. Based on preliminary results of RNA sequencing, we targeted genes susceptible to be differentially expressed between ovaries and testis at different stages of development. Using qPCR, we detected testis-specific expressions of dmrt1, amh, gsdf and pre-miR202 and ovary-specific expressions were obtained for zar1, zp3 and foxn5. We showed that gene expressions in the gonad of intersexual eels were quite similar to those of males, supporting the idea that intersexual eels represent a transitional stage towards testicular differentiation. To assess whether these genes would be effective early molecular markers, we sampled juvenile eels in two locations with highly skewed sex ratios. The combined expression of six of these genes allowed the discrimination of groups according to their potential future sex and thus this appears to be a useful tool to estimate sex ratios of undifferentiated juvenile eels. PMID:27658729

Porous titanium and Ti-35Nb alloy: effects on gene expression of osteoblastic cells derived from human alveolar bone.

PubMed

do Prado, Renata Falchete; Rabêlo, Sylvia Bicalho; de Andrade, Dennia Perez; Nascimento, Rodrigo Dias; Henriques, Vinicius André Rodrigues; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves; de Vasconcellos, Luana Marotta Reis

2015-11-01

Tests on titanium alloys that possess low elastic modulus, corrosion resistance and minimal potential toxicity are ongoing. This study aimed to evaluate the behavior of human osteoblastic cells cultured on dense and porous Titanium (Ti) samples comparing to dense and porous Ti-35 Niobium (Ti-35Nb) samples, using gene expression analysis. Scanning electronic microscopy confirmed surface porosity and pore interconnectivity and X-ray diffraction showed titanium beta-phase stabilization in Ti-35Nb alloy. There were no differences in expression of transforming growth factor-β, integrin-β1, alkaline phosphatase, osteopontin, macrophage colony stimulating factor, prostaglandin E synthase, and apolipoprotein E regarding the type of alloy, porosity and experimental period. The experimental period was a significant factor for the markers: bone sialoprotein II and interleukin 6, with expression increasing over time. Porosity diminished Runt-related transcription factor-2 (Runx-2) expression. Cells adhering to the Ti-35Nb alloy showed statistically similar expression to those adhering to commercially pure Ti grade II, for all the markers tested. In conclusion, the molecular mechanisms of interaction between human osteoblasts and the Ti-35Nb alloy follow the principal routes of osseointegration of commercially pure Ti grade II. Porosity impaired the route of transcription factor Runx-2.

Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context

NASA Astrophysics Data System (ADS)

Geffroy, Benjamin; Guilbaud, Florian; Amilhat, Elsa; Beaulaton, Laurent; Vignon, Matthias; Huchet, Emmanuel; Rives, Jacques; Bobe, Julien; Fostier, Alexis; Guiguen, Yann; Bardonnet, Agnès

2016-09-01

Environmental sex determination (ESD) has been detected in a range of vertebrate reptile and fish species. Eels are characterized by an ESD that occurs relatively late, since sex cannot be histologically determined before individuals reach 28 cm. Because several eel species are at risk of extinction, assessing sex at the earliest stage is a crucial management issue. Based on preliminary results of RNA sequencing, we targeted genes susceptible to be differentially expressed between ovaries and testis at different stages of development. Using qPCR, we detected testis-specific expressions of dmrt1, amh, gsdf and pre-miR202 and ovary-specific expressions were obtained for zar1, zp3 and foxn5. We showed that gene expressions in the gonad of intersexual eels were quite similar to those of males, supporting the idea that intersexual eels represent a transitional stage towards testicular differentiation. To assess whether these genes would be effective early molecular markers, we sampled juvenile eels in two locations with highly skewed sex ratios. The combined expression of six of these genes allowed the discrimination of groups according to their potential future sex and thus this appears to be a useful tool to estimate sex ratios of undifferentiated juvenile eels.

DDAH2 mRNA expression is inversely associated with some cardiovascular risk-related features in healthy young adults.

PubMed

Puchau, Blanca; Hermsdorff, Helen Hermana M; Zulet, M Angeles; Martínez, J Alfredo

2009-01-01

The purpose of this study was to evaluate whether the mRNA expression profiles of three genes (PRMT1, DDAH2 and NOS3) are related to ADMA metabolism and signalling, and the potential relationships with anthropometrical, biochemical, lifestyle and inflammatory indicators in healthy young adults. An emphasis on the putative effect of different mRNA expression on cardiovascular risk-related features was paid. Anthropometrical measurements as well as lifestyle features were analyzed in 120 healthy young adults. Fasting blood samples were collected for the measurement of glucose and lipid profiles as well as the concentrations of selected inflammatory markers. Profiles of mRNA expression were assessed for PRMT1, DDAH2 and NOS3 genes from peripheral blood mononuclear cells. Regarding inflammatory biomarkers, DDAH2 was inversely associated with IL-6 and TNF-alpha. Moreover, subjects in the highest quintile of DDAH2 mRNA expression showed a reduced risk to have higher values of waist circumference, and to be more prone to show higher values of HDL-c. Interestingly, DDAH2 gene expression seemed to be related with some anthropometrical, biochemical, lifestyle and inflammatory indicators linked to cardiovascular risk in apparently healthy young adults, emerging as a potential disease marker.

Assessing stemness and proliferation properties of the newly established colon cancer 'stem' cell line, CSC480 and novel approaches to identify dormant cancer cells.

PubMed

Alowaidi, Faisal; Hashimi, Saeed Mujahid; Alqurashi, Naif; Alhulais, Reem; Ivanovski, Saso; Bellette, Bernadette; Meedenyia, Adrian; Lam, Alfred; Wood, Stephen

2018-06-01

To date two questions that remain unanswered regarding cancer are the following: i) how is it initiated, and ii) what is the role that cancer stem cells (CSCs) play in the disease process? Understanding the biology of CSCs and how they are generated is pivotal for the development of successful treatment regimens. To date, the lack of a representative cell model has prevented the successful identification and eradication of CSCs in vivo. The current methods of CSC identification are dependent on the protocol used to generate these cells, which has introduced variation and made the identification process more complicated. Furthermore, the list of possible markers is increasing in complexity. This is further confounded by the fact that there is insufficient information to determine whether the cells these markers detect are truly self‑renewing stem cells or, instead, progenitor cells. In the present study, we investigated a novel cell line model, CSC480, which can be employed to assess CSC markers and for testing novel therapeutic regimens. CSC480 cells have been revealed to express markers of CSCs such as CD44, ALDH1 and Sox2, that have lower expression in the SW480 cell line. CSC480 cells also expressed higher levels of the cancer resistance marker, ABCG2 and had higher proliferative and growth capacity than SW480 cells. In the present study, we also evaluated a novel approach to identify different cell types present in heterogeneous cancer cell populations according to their proliferative ability using the proliferation marker 5‑ethynyl‑2'‑deoxyuridine (EdU). Furthermore, using EdU, we identified dormant cells with a modified label‑retaining cell (LRC) protocol. Through this novel LRC method, we assessed newly discovered markers of stemness to ascertain their capability to identify quiescent from dividing CSCs. In conclusion, the CSC480 cell line was an important model to be used in unravelling the underlying mechanisms that control fast‑dividing and partially self‑renewing stem cells (SCs) that may give rise to cancer.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

PubMed

Patil, Pradeep B; Begum, Setara; Joshi, Meghnad; Kleman, Marika I; Olausson, Michael; Sumitran-Holgersson, Suchitra

2014-06-01

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma.

PubMed

Shimoda, Yuki; Ubukata, Yasunari; Handa, Tadashi; Yokobori, Takehiko; Watanabe, Takayoshi; Gantumur, Dolgormaa; Hagiwara, Kei; Yamanaka, Takahiro; Tsukagoshi, Mariko; Igarashi, Takamichi; Watanabe, Akira; Kubo, Norio; Araki, Kenichiro; Harimoto, Norifumi; Katayama, Ayaka; Hikino, Toshiaki; Sano, Takaaki; Ogata, Kyoichi; Kuwano, Hiroyuki; Shirabe, Ken; Oyama, Tetsunari

2018-05-25

Hepatocellular carcinoma (HCC) is one of the major causes of tumor death; thus, the identification of markers related to its diagnosis and prognosis is critical. Previous studies have revealed that epithelial-to-mesenchymal transition (EMT) is involved in tumor invasion and metastasis, and the forkhead box protein C2 (FOXC2) has been shown to promote tumor cell proliferation, invasion, and EMT. In the present study, we examined the clinicopathological significance of FOXC2 and EMT-related markers in clinical HCC specimens and identified factors related to the diagnosis and prognosis of HCC. The expression of FOXC2 and EMT-related markers was evaluated by immunohistochemistry in 84 cases of hepatocellular carcinoma. A high expression of FOXC2 was observed in 26 of 84 cases, and expression was significantly correlated with background liver cirrhosis, poor tumor differentiation, high serum AFP, and elevated cell proliferation markers. In addition, this high expression was related to the induction of the Cadherin switch and vimentin expression and was an independent predictor for poor prognosis. The high expression of FOXC2 in HCC is correlated with tumor malignancy and poor prognosis, suggesting that FOXC2 may be an important prognostic factor for HCC.

Alveolar epithelial cells in idiopathic pulmonary fibrosis display upregulation of TRAIL, DR4 and DR5 expression with simultaneous preferential over-expression of pro-apoptotic marker p53.

PubMed

Akram, Khondoker M; Lomas, Nicola J; Forsyth, Nicholas R; Spiteri, Monica A

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease of unknown aetiology with no current cure. The pathogenesis of IPF remains unclear but repeated alveolar epithelial cell (AEC) injuries and subsequent apoptosis are believed to be among the initiating/ongoing triggers. However, the precise mechanism of apoptotic induction is hitherto elusive. In this study, we investigated expression of a panel of pro-apoptotic and cell cycle regulatory proteins in 21 IPF and 19 control lung tissue samples. We reveal significant upregulation of the apoptosis-inducing ligand TRAIL and its cognate receptors DR4 and DR5 in AEC within active lesions of IPF lungs. This upregulation was accompanied by pro-apoptotic protein p53 overexpression. In contrast, myofibroblasts within the fibroblastic foci of IPF lungs exhibited high TRAIL, DR4 and DR5 expression but negligible p53 expression. Similarly, p53 expression was absent or negligible in IPF and control alveolar macrophages and lymphocytes. No significant differences in TRAIL expression were noted in these cell types between IPF and control lungs. However, DR4 and DR5 upregulation was detected in IPF alveolar macrophages and lymphocytes. The marker of cellular senescence p21(WAF1) was upregulated within affected AEC in IPF lungs. Cell cycle regulatory proteins Cyclin D1 and SOCS3 were significantly enhanced in AEC within the remodelled fibrotic areas of IPF lungs but expression was negligible in myofibroblasts. Taken together these findings suggest that, within the remodelled fibrotic areas of IPF, AEC can display markers associated with proliferation, senescence, and apoptotosis, where TRAIL could drive the apoptotic response. Clear understanding of disease processes and identification of therapeutic targets will direct us to develop effective therapies for IPF.

Matrix metalloproteinase-9, a potential biological marker in invasive pituitary adenomas.

PubMed

Gong, Jian; Zhao, Yunge; Abdel-Fattah, Rana; Amos, Samson; Xiao, Aizhen; Lopes, M Beatriz S; Hussaini, Isa M; Laws, Edward R

2008-01-01

We analyzed MMP-9 expression using mRNA and protein level determinations and explored the possibility that matrix metalloproteinase-9 (MMP-9) is a potential biological marker of pituitary adenoma invasiveness and whether MMP-9 could be used to discriminate the extent of invasiveness among different hormonal subtypes, tumor sizes, growth characteristics, and primary versus recurrent tumors. 73 pituitary tumor specimens were snap frozen in liquid nitrogen immediately after surgical resection. RNA and protein were extracted. MMP-9 mRNA transcripts were analyzed by quantitative RT-PCR. MMP-9 protein activity was analyzed by gelatin zymography and validated by western blot analysis. Immunohistochemistry was performed to identify the presence and localization of MMP-9 in pituitary adenomas. Statistical differences between results were determined using Student's t-test or one way ANOVA. Comparing different hormonal subtypes of noninvasive and invasive pituitary tumors, MMP-9 mRNA expression was significantly increased in the majority of invasive adenomas. Considering the protein levels, our data also showed a significant increase in MMP-9 activity in the majority of invasive adenomas and these differences were confirmed by western blot analysis and immunohistochemistry. In addition, consistent differences in MMP-9 expression levels were found according to tumor subtype, tumor size, tumor extension and primary versus redo-surgery. MMP-9 expression can consistently distinguish invasive pituitary tumors from noninvasive pituitary tumors and would reflect the extent of invasiveness in pituitary tumors according to tumor subtype, size, tumor extension, primary and redo surgery, even at early stages of invasiveness. MMP-9 may be considered a potential biomarker to determine and predict the invasive nature of pituitary tumors.

Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

PubMed Central

Neubauer, Elisa; Wirtz, Ralph M.; Kaemmerer, Daniel; Athelogou, Maria; Schmidt, Lydia; Sänger, Jörg; Lupp, Amelie

2016-01-01

The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In this regard, we compared the expression of three proliferation markers, Ki-67, Topoisomerase II alpha (TOP2A), and RacGAP1, in a series of tumor samples from 104 BP-NEN patients (24 TC, 21 AC, 52 SCLC, 7 LCNEC) using different evaluation methods (immunohistochemistry (IHC): Average evaluation, Hotspot evaluation, digital image analysis; RT-qPCR). The results indicated that all three markers had increased protein and mRNA expression with poorer differentiation and correlated well with each other, as well as with grading, staging, and poor survival. Compared with Ki-67 and TOP2A, RacGAP1 allowed for a clearer prognostic statement. The cut-off limits obtained for Ki-67-Average (IHC) were TC-AC 1.5, AC-SCLC 19, and AC-LCNEC 23.5. The Hotspot evaluation generated equal to higher, the digital image analysis generally lower between-entity cut-off limits. All three markers enabled a clear-cut differentiation between the BP-NEN entities, and all methods evaluated were suitable for marker assessment. However, to define optimal cut-off limits, the Ki-67 evaluation methods should be standardized. RacGAP1 appeared to be a new marker with great potential. PMID:27259241

Different expression of FoxM1 in human benign and malignant pleural effusion.

PubMed

Tang, Zhonghao; Li, Hongqing; Zhu, Huili; Bai, Chunxue

2015-01-01

The aims of this study were as follows: to analyze the forkhead box M1 (FoxM1) expression in benign and malignant pleural effusion by reverse transcription-polymerase chain reaction assay (RT-PCR); to explore the role of FoxM1 in formation and progress in malignant pleural effusion, and whether there is significant difference in expression level of FoxM1 between benign and malignant pleural effusion; to seek a gene marker diagnostically useful to identify benign and malignant pleural effusion in diagnosis and treatment of pleural effusion; and to collect expression level data of FoxM1 in 23 malignant pleural effusion samples (17 adenocarcinoma samples, four squamous carcinoma samples and two small cell lung carcinoma samples) and 15 benign pleural effusion samples (11 inflammatory pleural effusions, two transudates, two tuberculous pleural effusions) by RT-PCR. Among all 38 samples, average FoxM1 expression level of benign pleural effusions is (235.09 ± 59.99), while malignant pleural effusions (828.77 ± 109.76). Among 23 malignant samples, average FoxM1 expression level is (529.27 ± 75.85) in samples without cytological diagnostic evidence, while (1,218.12 ± 167.21) in samples with cytological diagnostic evidence. Differences of FoxM1 expression level between benign pleural effusions and malignant ones have statistical significance. There is an area of 0.881 under the receiver-operating characteristic curve, which verifies the accuracy of using FoxM1 expression level as diagnostic index to identify benign and malignant pleural effusions. According to our study, diagnostic sensitivity and specificity for FoxM1 expression level at 418.1 were 82.6 and 86.7 %, respectively, while 47.8 and 100 %, respectively, at 768.7. FoxM1 expression level in malignant pleural effusions is significantly higher than in benign ones. This study provides a new approach in clinical diagnosis, with FoxM1 as a specific molecule marker to identify benign and malignant pleural effusions. FoxM1 expression level could provide evidence for diagnosis and treatment of malignant pleural effusions and lung cancer.

Long non-coding RNA BANCR regulates cancer stem cell markers in papillary thyroid cancer via the RAF/MEK/ERK signaling pathway.

PubMed

Wang, Yuanyuan; Lin, Xiangde; Fu, Xinghao; Yan, Wei; Lin, Fusheng; Kuang, Penghao; Luo, Yezhe; Lin, Ende; Hong, Xiaoquan; Wu, Guoyang

2018-06-18

Thyroid cancer is one of the most common malignant tumors of the endocrine system. Among all thyroid cancers, papillary thyroid carcinoma (PTC) is the most common type. The BRAF-activated non-coding RNA (BANCR) is a 693-bp nucleotide transcript which was first identified in melanoma. However, the role of BANCR in the development of thyroid cancer remains unclear. Therefore, the present study investigated the potential involvement of BANCR in the development of thyroid cancer in vitro using patient tissue samples and a panel of thyroid cancer cell lines, and in vivo using a xenograft mouse model. We observed that BANCR was expressed at a higher level in human thyroid tumor tissues than that noted in the adjacent normal tissues. The expression level of BANCR differed between cultured thyroid cancer cell lines; BANCR expression was lower in the BCPAP cell line than that observed in the CAL-62, WRO and FTC-133 cell lines. Western blot analysis and flow cytometry revealed that overexpression of BANCR in the BCPAP cell line resulted in increased expression of the cancer stem cell markers, LGR5 and EpCAM. Single-clone formation experiments showed that upregulated expression of BANCR in the BCPAP cell line promoted an increase in the number of clones formed. Similarly, in microsphere formation experiments, overexpression of BANCR resulted in increased number and size of microspheres compared with the control cell line. Western blotting experiments showed that BANCR overexpression in BCPAP upregulated the expression of phosphorylated c-Raf, MEK1/2 and ERK1/2. Inhibition of c-Raf via U0126 decreased the expression of LGR5 and EpCAM, as well as phosphorylated levels of c-Raf, MEK1/2 and ERK1/2 in the BCPAP cells, compared to levels in the DMSO controls. In the xenograft mouse model, BANCR overexpression in the thyroid cancer cells significantly increased tumor growth. Taken together, these results suggest that BANCR plays a role in PTC development by regulating the expression of cancer stem cell markers LGR5 and EpCAM via the c-Raf/MEK/ERK signaling pathway. Therefore, BANCR may be used as a novel prognostic marker for PTC.

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

PubMed

Riquelme, Paloma; Wundt, Judith; Hutchinson, James A; Brulport, Marc; Jun, Yu; Sotnikova, Anna; Girreser, Ulrich; Braun, Felix; Gövert, Felix; Soria, Bernat; Nüssler, Andreas; Clement, Bernd; Hengstler, Jan G; Fändrich, Fred

2009-03-01

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

Keratin K15 as a Biomarker of Epidermal Stem Cells

PubMed Central

Bose, Amrita; Teh, Muy-Teck; Mackenzie, Ian C.; Waseem, Ahmad

2013-01-01

Keratin 15 (K15) is type I keratin protein co-expressed with the K5/K14 pair present in the basal keratinocytes of all stratified epithelia. Although it is a minor component of the cytoskeleton with a variable expression pattern, nonetheless its expression has been reported as a stem cell marker in the bulge of hair follicles. Conversely, suprabasal expression of K15 has also been reported in both normal and diseased tissues, which is inconsistent with its role as a stem cell marker. Our recently published work has given evidence of the molecular pathways that seem to control the expression of K15 in undifferentiated and differentiated cells. In this article, we have critically reviewed the published work to establish the reliability of K15 as an epidermal stem cell marker. PMID:24071939

Zebrafish grainyhead-like1 is a common marker of different non-keratinocyte epidermal cell lineages, which segregate from each other in a Foxi3-dependent manner

PubMed Central

JÄNICKE, MARTINA; RENISCH, BJÖRN; HAMMERSCHMIDT, MATTHIAS

2012-01-01

Grainyhead/CP2 transcription factor family members are widely conserved among the animal kingdom and have been implicated in different developmental processes. Thus far, nothing has been known about their roles in zebrafish. Here we identify seven zebrafish grainyhead-like (grhl) / cp2 genes, with focus on grhl1, which is expressed in the periderm and in epidermal ionocyte progenitors, but downregulated when ionocytes differentiate. In addition, expression was detected in other “non-keratinocyte” cell types of the epidermis, such as pvalb8-expressing cells, which according to our lineage tracing experiments are derived from the same pool of progenitor cells like keratinocytes and ionocytes. Antisense morpholino oligonucleotide-based loss-of-function analysis revealed that grhl1 is dispensable for the development and function of all investigated epidermal cell types, but required as a negative regulator of its own transcription during ionocyte differentiation. Knockdown of the transcription factor Foxi3a, which is expressed in a subset of the grhl1 population, caused a loss of ionocytes and a corresponding increase in the number of pvalb8-expressing cells, while leaving the number of grhl1-positive cells unaltered. We propose that grhl1 is a novel common marker of all or most “non-keratinocyte” epidermal progenitors, and that the sub-functionalisation of these cells is regulated by differential positive and negative effects of Foxi3 factors. PMID:19757382

Prospective comparison of molecular signatures in urothelial cancer of the bladder and the upper urinary tract--is there evidence for discordant biology?

PubMed

Krabbe, Laura-Maria; Lotan, Yair; Bagrodia, Aditya; Gayed, Bishoy A; Darwish, Oussama M; Youssef, Ramy F; Bolenz, Christian; Sagalowsky, Arthur I; Raj, Ganesh V; Shariat, Shahrokh F; Kapur, Payal; Margulis, Vitaly

2014-04-01

Upper tract urothelial carcinoma is rare and less well studied than bladder cancer. It remains questionable if findings in bladder cancer can safely be extrapolated to upper tract urothelial carcinoma. We prospectively evaluate molecular profiles of upper tract urothelial carcinoma and bladder cancer using a cell cycle biomarker panel. Immunohistochemical staining for p21, p27, p53, cyclin E and Ki-67 was prospectively performed for 96 patients with upper tract urothelial carcinoma and 159 patients with bladder cancer with nonmetastatic high grade urothelial carcinoma treated with extirpative surgery. Data were compared between the groups according to pathological stage. Primary outcome was assessment of differences in marker expression. Secondary outcome was difference in survival according to marker status. During a median followup of 22.0 months 31.2% of patients with upper tract urothelial carcinoma and 28.3% of patients with bladder cancer had disease recurrence, and 20.8% and 27.7% died of upper tract urothelial carcinoma and bladder cancer, respectively. The number of altered markers was not significantly different between the study groups. Overall 34 patients (35.4%) with upper tract urothelial carcinoma and 62 (39.0%) with bladder cancer had an unfavorable marker score (more than 2 markers altered). There were no significant differences between upper tract urothelial carcinoma and bladder cancer in the alteration status of markers, the number of altered markers and biomarker score when substratified by pathological stage. There were no significant differences in survival outcomes between patients with upper tract urothelial carcinoma and those with bladder cancer according to the number of altered markers and biomarker score. Our results demonstrate the molecular similarity of upper tract urothelial carcinoma and bladder cancer in terms of cell cycle and proliferative tissue markers. These findings have important implications and support the further extrapolation of treatment paradigms established in bladder cancer to upper tract urothelial carcinoma. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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.

Aberrant expression of cancer stem cell markers (CD44, CD90, and CD133) contributes to disease progression and reduced survival in hepatoblastoma patients: 4-year survival data.

PubMed

Bahnassy, Abeer A; Fawzy, Mohamed; El-Wakil, Mohamed; Zekri, Abdel-Rahman N; Abdel-Sayed, Ahmed; Sheta, Marwa

2015-03-01

Hepatoblastoma (HB) is an embryonal tumor of the liver in children. Prognosis and response to treatment in HB are highly variable. Cancer stem cells (CSCs) constitute a population of cells, which contribute to the development and progression of many tumors. However, their role in HB is not well defined yet. We assessed the prognostic and predictive values of some CSC markers in HB patients. Protein and messenger RNA expressions of the CSC markers CD133, CD90, and CD44 were assessed in 43 HB patients and 20 normal hepatic tissues using immunohistochemistry and quantitative real-time polymerase chain reaction. The expression levels of these markers were correlated to standard prognostic factors, patients' response to treatment, overall survival (OS), and disease-free survival (DFS). CD44, CD90, and CD133 proteins were detected in 48.8%, 32.6%, and 48.8% compared with 46.5%, 41.7%, and 58.1% RNA, respectively (concordance, 77.8%-96%). None of the normal tissue samples was positive for any of the markers. Significant correlations were reported between α-fetoprotein and both CD44 and CD133 (P = 0.02) as well as between tumor types CD90 and CD133 (P = 0.009). Reduced OS correlated with CD44, CD90, and CD133 expressions (P < 0.001), advanced stage (P < 0.001), response to treatment (P < 0.001), and total excision of the tumor. Reduced DFS correlated with CD44 and CD133 expressions (P < 0.001) only. In conclusion, CD133, CD44, and CD90 could be used as prognostic and predictive markers in HB. High expression of these markers is significantly associated with poor response to treatment and reduced survival. Moreover, complete surgical resection and systemic chemotherapy are essential to achieve good response and prolonged survival, especially in early stage patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

PubMed Central

Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van IJcken, Wilfred

2007-01-01

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Electronic supplementary material The online version of this article (doi:10.1007/s00414-007-0182-6) contains supplementary material, which is available to authorized users. PMID:17579879

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.

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

DTIC Science & Technology

2008-12-01

transduction of dendritic cells (DCs) is inefficient because of the lack of the primary Ad receptor, CAR. CD40 is a surface marker expressed by DCs that...ligands or antibodies that can bind to the cell surface markers expressed by DCs. The tumor antigen or peptides are linked to the ligands...thus pose the risk of insertional mutagenesis and oncogenesis. The various cell- surface markers that have been exploited for targeting DCs have

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.

T- and NK-cell populations with regulatory phenotype and markers of apoptosis in circulating lymphocytes of patients with CIN3 or microcarcinoma of the cervix: evidence for potential mechanisms of immune suppression.

PubMed

Kurmyshkina, Olga V; Kovchur, Pavel I; Schegoleva, Ludmila V; Volkova, Tatyana O

2017-01-01

Processes and mechanisms responsible for systemic immune suppression in early-stage cervical cancer remain substantially underinvestigated. In this work, we focused on studying the frequencies of circulating regulatory T (CD4 and CD8 Tregs) and NK (NKregs) cells in parallel with assessment of apoptotic markers expression in T cells from patients with preinvasive and microinvasive cervical cancer, with the aim to determine whether up-regulation of apoptosis-associated markers in Т lymphocytes accompanies cervical cancer development and correlates with the change in percentages of regulatory cell populations at systemic level during the initial stages of invasive cervical cancer progression. Fourty two women with histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3, including carcinoma in situ) or cervical cancer (stage IA) and 30 healthy women (control) were enrolled in the study. Peripheral blood samples were taken immediately before surgery or any treatment and immediately subjected to multicolor flow cytometry. Analysis of a combination of CD4/CD8, CD25, CD127, and FoxP3 markers revealed a statistically significant increase in the frequencies of Tregs within both the CD4 and CD8 subsets of circulating lymphocytes in patients with CIN3 and stage IA cancer. In contrast, lower numbers of NKregs (defined as CD16 dim/neg CD56 bright subpopulation) and increased CD56 dim /CD56 bright NK ratio were found in patients compared to controls, with the percentage of CD16 bright CD56 dim cells (major subtype of circulating NKs) showing no difference. Patients also exhibited an increased expression of CD95 in total peripheral blood T lymphocytes, along with increased level of Annexin V binding to CD95-positive cells, suggesting higher susceptibility of T cells to apoptosis and potential involvement of CD95-dependent pathway in early-stage cervical cancer. Differential analysis of CD4 and CD8 T cells revealed different trends in the change of CD95 expression, confirming that this change likely has different functional significance for these two subsets. A search for correlations between the phenotypic parameters analyzed in this study was performed to demonstrate that women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of Treg markers in circulating lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on T cells, and apoptosis, compared to the control group. The results of this study suggest that a diversity of immune regulatory mechanisms that provide support for initial stages of invasive growth in cervical cancer patients includes systemic changes in the ratios between the principal regulatory and effector lymphocyte populations both within adaptive and innate immunity.

Predictive markers of chemoresistance in advanced stages epithelial ovarian carcinoma.

PubMed

Bonneau, Claire; Rouzier, Roman; Geyl, Caroline; Cortez, Annie; Castela, Mathieu; Lis, Raphael; Daraï, Emile; Touboul, Cyril

2015-01-01

DNA repair mechanisms, environment-mediated drug resistance and cancer initiating cells (CIC) are three major research concepts that can explain the chemoresistance of epithelial ovarian cancer (EOC). The objective was to test if changes in the expression of potential markers associated with drug resistance before and after chemotherapy would correlate with platinum resistance, defined as a recurrence within the first year after chemotherapy cessation, and with survival, in advanced EOC. We included 32 patients with stage IIIC-IV EOC who underwent laparoscopy to evaluate the extent of carcinomatosis, neoadjuvant chemotherapy (carboplatin/taxol) and interval surgery. Biopsies taken during the initial laparoscopies and interval surgeries were evaluated using immunohistochemistry for the expression of 7 proteins: CD117, CD44 and ALDH1 to evaluate CIC; IL-6, IL-8 and BMP2 to evaluate environment-mediated drug resistance; and ERCC1 to evaluate DNA repair. Expression measurements were correlated with platin resistance and survival. The markers' relevance was confirmed in vitro using chemoresistance tests and flow cytometric measurements of the proportion of CD44+ cells. 17 patients were chemoresistant and 15 patients were chemosensitive. We observed increases in CD44, IL-6 and ERCC1 expression and stable ALDH1, CD117, IL-8, and BMP2 expression. Reduced expression of cancer initiating cell markers and increased expression of environment-mediated drug resistance markers were associated with poor prognosis. We also demonstrated that CD44+ cells had survival advantages in vitro. Changes in CD44 and IL-8 expression on tumor cells appeared to correlate with overall survival and should be further tested as predictors of chemoresistance using larger cohort. Copyright © 2014 Elsevier Inc. All rights reserved.

Developmental expression of Toll‑like receptors in the guinea pig lung.

PubMed

Ma, Lingjie; Yang, Jiali; Yang, Li; Shi, Juan; Xue, Jing; Li, Yong; Liu, Xiaoming

2017-03-01

The guinea pig is a useful model for investigating infectious and non‑infectious lung diseases due to the sensitivity of its respiratory system and susceptibility to infectious agents. Toll‑like receptors (TLRs) are important components of the innate immune response and are critical for lung immune function. In the present study, the differentiation of epithelial cells in the guinea pig lung was examined during gestation by studying anatomic morphology and the major epithelial cell types using cell type‑specific markers. The developmental expression of all 9 TLRs and the TLR signaling adaptors myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor associated factor 6 (TRAF‑6) were investigated by reverse transcription‑quantitative polymerase chain reaction and western blotting analysis. The formation of lung lobes in guinea pigs was observed at 45 days of gestation (dGA), along with the expression of the basal cell marker keratin 14 and the alveolar type II cell marker pro‑surfactant protein. However, the cube cell marker secretoglobin family1A member 1 and ciliated cell marker b‑tubulin IV were only detected in the lungs from 52 dGA onward. The expression levels of all TLRs, MyD88 and TRAF‑6 were determined in lung tissues harvested from embryos, newborn, postnatal and adult animals. The expression levels of all TLR signaling components displayed similar dynamic expression patterns with gestation age and postnatal maturation time, except for TLR‑4 and TLR‑7. mRNA expression levels of TLR components were significantly increased in the lungs at 45 and 52 dGA, compared with later developmental stages. These results suggest that TLR expression in the guinea pig lung is developmentally regulated, enhancing the understanding of lung biology in guinea pig models.

Immunohistochemical comparative analysis of GFAP, MAP - 2, NOGO - A, OLIG - 2 and WT - 1 expression in WHO 2016 classified neuroepithelial tumours and their prognostic value.

PubMed

Schwab, David Emanuel; Lepski, Guilherme; Borchers, Christian; Trautmann, Katrin; Paulsen, Frank; Schittenhelm, Jens

2018-01-01

Immunohistochemistry is routinely used in differential diagnosis of tumours of the central nervous system (CNS). The latest 2016 WHO 2016 revision now includes molecular data such as IDH mutation and 1p/19q codeletion thus restructuring glioma classification. Direct comparative information between commonly used immunohistochemical markers for glial tumours GFAP, MAP - 2, NOGO - A, OLIG - 2 and WT - 1 concerning quality and quantity of expression and their relation to the new molecular markers are lacking. We therefore compared the immunohistochemical staining results of all five antibodies in 34 oligodendrogliomas, 106 ependymomas and 423 astrocytic tumours. GFAP expression was reduced in cases with higher WHO grade, oligodendroglial differentiation and in IDH wildtype diffuse astrocytomas. By contrast MAP - 2 expression was significantly increased in diffuse astrocytomas with IDH mutation, while NOGO - A expression was not associated with any molecular marker. WT - 1 expression was significantly decreased in tumours with IDH mutation and ATRX loss. OLIG - 2 was increased in IDH-mutant grade II astrocytomas and in cases with higher proliferation rate. In univariate survival analysis high WT - 1 expression was significantly associated with worse outcome in diffuse astrocytic tumours (log rank p < 0.0001; n = 211; median time: 280 days vs 562 days). None of the markers was prognostic in multivariate survival analysis. Among the evaluated markers MAP - 2, OLIG - 2 and WT - 1 showed the best potential to separate between glioma entities and can be recommended for a standardized immunohistochemical panel. Copyright © 2017 Elsevier GmbH. All rights reserved.

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.

A Transition Zone Showing Highly Discontinuous or Alternating Levels of Stem Cell and Proliferation Markers Characterizes the Development of PTEN-Haploinsufficient Colorectal Cancer.

PubMed

Arvai, Kevin J; Hsu, Ya-Hsuan; Lee, Lobin A; Jones, Dan

2015-01-01

Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC. We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases. In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This bottleneck-like zone is usually followed by the emergence of invasive tumors with intact PTEN expression but dysregulated TP53 and uniformly high proliferation rates.

Hodgkin lymphoma: A complex metabolic ecosystem with glycolytic reprogramming of the tumor microenvironment.

PubMed

Mikkilineni, Lekha; Whitaker-Menezes, Diana; Domingo-Vidal, Marina; Sprandio, John; Avena, Paola; Cotzia, Paolo; Dulau-Florea, Alina; Gong, Jerald; Uppal, Guldeep; Zhan, Tingting; Leiby, Benjamin; Lin, Zhao; Pro, Barbara; Sotgia, Federica; Lisanti, Michael P; Martinez-Outschoorn, Ubaldo

2017-06-01

Twenty percent of patients with classical Hodgkin Lymphoma (cHL) have aggressive disease defined as relapsed or refractory disease to initial therapy. At present we cannot identify these patients pre-treatment. The microenvironment is very important in cHL because non-cancer cells constitute the majority of the cells in these tumors. Non-cancer intra-tumoral cells, such as tumor-associated macrophages (TAMs) have been shown to promote tumor growth in cHL via crosstalk with the cancer cells. Metabolic heterogeneity is defined as high mitochondrial metabolism in some tumor cells and glycolysis in others. We hypothesized that there are metabolic differences between cancer cells and non-cancer tumor cells, such as TAMs and tumor-infiltrating lymphocytes in cHL and that greater metabolic differences between cancer cells and TAMs are associated with poor outcomes. A case-control study was conducted with 22 tissue samples of cHL at diagnosis from a single institution. The case samples were from 11 patients with aggressive cHL who had relapsed after standard treatment with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or were refractory to this treatment. The control samples were from 11 patients with cHL who achieved a remission and never relapsed after ABVD. Reactive non-cancerous lymph nodes from four subjects served as additional controls. Samples were stained by immunohistochemistry for three metabolic markers: translocase of the outer mitochondrial membrane 20 (TOMM20), monocarboxylate transporter 1 (MCT1), and monocarboxylate transporter 4 (MCT4). TOMM20 is a marker of mitochondrial oxidative phosphorylation (OXPHOS) metabolism. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is a marker of OXPHOS. Monocarboxylate transporter 4 (MCT4) is the main lactate exporter out of cells and is a marker of glycolysis. The immunoreactivity for TOMM20, MCT1, and MCT4 was scored based on staining intensity and percentage of positive cells, as follows: 0 for no detectable staining in > 50% of cells; 1+ for faint to moderate staining in > 50% of cells, and 2+ for high or strong staining in > 50% of cells. TOMM20, MCT1, and MCT4 expression was significantly different in Hodgkin and Reed Sternberg (HRS) cells, which are the cancerous cells in cHL compared with TAMs and tumor-associated lymphocytes. HRS have high expression of TOMM20 and MCT1, while TAMs have absent expression of TOMM20 and MCT1 in all but two cases. Tumor-infiltrating lymphocytes have low TOMM20 expression and absent MCT1 expression. Conversely, high MCT4 expression was found in TAMs, but absent in HRS cells in all but one case. Tumor-infiltrating lymphocytes had absent MCT4 expression. Reactive lymph nodes in contrast to cHL tumors had low TOMM20, MCT1, and MCT4 expression in lymphocytes and macrophages. High TOMM20 and MCT1 expression in cancer cells with high MCT4 expression in TAMs is a signature of high metabolic heterogeneity between cancer cells and the tumor microenvironment. A high metabolic heterogeneity signature was associated with relapsed or refractory cHL with a hazard ratio of 5.87 (1.16-29.71; two-sided P < .05) compared with the low metabolic heterogeneity signature. Aggressive cHL exhibits features of metabolic heterogeneity with high mitochondrial metabolism in cancer cells and high glycolysis in TAMs, which is not seen in reactive lymph nodes. Future studies will need to confirm the value of these markers as prognostic and predictive biomarkers in clinical practice. Treatment intensity may be tailored in the future to the metabolic profile of the tumor microenvironment and drugs that target metabolic heterogeneity may be valuable in this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Using genetic markers to orient the edges in quantitative trait networks: the NEO software.

PubMed

Aten, Jason E; Fuller, Tova F; Lusis, Aldons J; Horvath, Steve

2008-04-15

Systems genetic studies have been used to identify genetic loci that affect transcript abundances and clinical traits such as body weight. The pairwise correlations between gene expression traits and/or clinical traits can be used to define undirected trait networks. Several authors have argued that genetic markers (e.g expression quantitative trait loci, eQTLs) can serve as causal anchors for orienting the edges of a trait network. The availability of hundreds of thousands of genetic markers poses new challenges: how to relate (anchor) traits to multiple genetic markers, how to score the genetic evidence in favor of an edge orientation, and how to weigh the information from multiple markers. We develop and implement Network Edge Orienting (NEO) methods and software that address the challenges of inferring unconfounded and directed gene networks from microarray-derived gene expression data by integrating mRNA levels with genetic marker data and Structural Equation Model (SEM) comparisons. The NEO software implements several manual and automatic methods for incorporating genetic information to anchor traits. The networks are oriented by considering each edge separately, thus reducing error propagation. To summarize the genetic evidence in favor of a given edge orientation, we propose Local SEM-based Edge Orienting (LEO) scores that compare the fit of several competing causal graphs. SEM fitting indices allow the user to assess local and overall model fit. The NEO software allows the user to carry out a robustness analysis with regard to genetic marker selection. We demonstrate the utility of NEO by recovering known causal relationships in the sterol homeostasis pathway using liver gene expression data from an F2 mouse cross. Further, we use NEO to study the relationship between a disease gene and a biologically important gene co-expression module in liver tissue. The NEO software can be used to orient the edges of gene co-expression networks or quantitative trait networks if the edges can be anchored to genetic marker data. R software tutorials, data, and supplementary material can be downloaded from: http://www.genetics.ucla.edu/labs/horvath/aten/NEO.

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål).

PubMed

Jing, S; Liu, B; Peng, L; Peng, X; Zhu, L; Fu, Q; He, G

2012-02-01

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens Stål) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.

Study of Extrinsic Apoptotic Pathway in Oral Pemphigus Vulgaris Using TNFR 1 and FasL Immunohistochemical Markers and TUNEL Technique

PubMed Central

Deyhimi, Parviz; Alishahi, Batoul

2018-01-01

Statement of the Problem: Pemphigus vulgaris is characterized by intraepithelial vesicles, but pathogenesis of vesicle formation in this disease has not been substantiated yet. Purpose: The present study investigate extrinsic apoptotic pathway in oral pemphigus vulgaris using TUNEL and important immunohistochemical markers of extrinsic pathway, TNFR1 and FasL. Materials and Method: In the present cross sectional study, 25 oral pemphigus vulgaris samples and 6 normal oral mucosa were analyzed for the presence of apoptosis by TUNEL and the staining of TNFR1 and FasL in basal and parabasal layers around vesicle, vesicle floor, vesicle roof and acantholytic cells. The staining expression and intensity were measured and the obtained data were analyzed by Wilcoxon and Mann-Whitney tests. Results: There was no or faint staining of TUNEL, FasL and TNFR1 in normal oral mucosa. In addition, there was no significant difference between the staining of TUNEL technique in different layers. The staining of TNFR1 marker was very high in all regions. FasL marker was not positive in the basal and parabasal layers around vesicle in 92% of samples but showed a varied and different staining in vesicle region. There was a significant difference between the each two markers in all layers (p <0.001). Conclusion: Apoptosis is probably is a preceding phenomenon to acantholysis in pemphigus vulgaris. It appears that the apoptosis occurs mostly by extrinsic pathway using proapototic mediators TNFR1 and FasL. PMID:29854887

Transcription factor GATA-4 is a marker of anaplasia in adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

PubMed

Peterson, R A; Kiupel, M; Bielinska, M; Kiiveri, S; Heikinheimo, M; Capen, C C; Wilson, D B

2004-07-01

Adrenocortical neoplasms are a common cause of morbidity in neutered ferrets. Recently we showed that gonadectomized DBA/2J mice develop adrenocortical tumors that express transcription factor GATA-4. Therefore, we screened archival specimens of adrenocortical neoplasms from neutered ferrets to determine whether GATA-4 could be used as a tumor marker in this species. Nuclear immunoreactivity for GATA-4 was evident in 19/22 (86%) of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation. Normal adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice, inhibin-alpha and luteinizing hormone receptor, were coexpressed with GATA-4 in some of the ferret tumors. No GATA-4 expression was observed in three cases of nodular hyperplasia, but patches of anaplastic cells expressing GATA-4 were evident in 7/14 (50%) of tumors classified as adenomas. We conclude that GATA-4 can function as a marker of anaplasia in ferret adrenocortical tumors.

Insulin-like growth factor II messenger RNA-binding protein-3 is an independent prognostic factor in uterine leiomyosarcoma.

PubMed

Yasutake, Nobuko; Ohishi, Yoshihiro; Taguchi, Kenichi; Hiraki, Yuka; Oya, Masafumi; Oshiro, Yumi; Mine, Mari; Iwasaki, Takeshi; Yamamoto, Hidetaka; Kohashi, Kenichi; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

2018-04-01

The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis. © 2017 John Wiley & Sons Ltd.

Association of BRAFV600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers

PubMed Central

Aragon Han, Patricia; Kim, Hyun-seok; Cho, Soonweng; Fazeli, Roghayeh; Najafian, Alireza; Khawaja, Hunain; McAlexander, Melissa; Dy, Benzon; Sorensen, Meredith; Aronova, Anna; Sebo, Thomas J.; Giordano, Thomas J.; Fahey, Thomas J.; Thompson, Geoffrey B.; Gauger, Paul G.; Somervell, Helina; Bishop, Justin A.; Eshleman, James R.; Schneider, Eric B.; Witwer, Kenneth W.; Umbricht, Christopher B.

2016-01-01

Background: Studies have demonstrated an association of the BRAFV600E mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAFV600E mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM). Methods: Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAFV600E mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC. Results: Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAFV600E almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAFV600E mutation to predict CLNM independently. While BRAFV600E was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAFV600E was not significantly associated with CLNM. Conclusion: In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAFV600E mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers. PMID:26950846

The Prognostic Role of Cancer Stem Cell Markers for Long-term Outcome After Resection of Colonic Liver Metastases.

PubMed

Spelt, Lidewij; Sasor, Agata; Ansari, Daniel; Hilmersson, Katarzyna Said; Andersson, Roland

2018-01-01

To assess the expression of cancer stem cell (CSC) markers CD44, CD133 and CD24 in colon cancer liver metastases and analyse their predictive value for overall survival (OS) and disease-free survival (DFS) after liver resection. Patients operated on for colon cancer liver metastases were included. CSC marker expression was determined through immunohistochemistry analysis. OS and DFS were compared between marker-positive and marker-negative patients. Multivariate analysis was performed to select predictive variables for OS and DFS. CD133-positive patients had a worse DFS than CD133-negative patients, with a median DFS of 12 and 25 months (p=0.051). Multivariate analysis selected CD133 expression as a significant predictor for DFS. CD44 and CD24 were not found to predict OS or DFS. CD133 expression in colonic liver metastases is a negative prognostic factor for DFS after liver resection. In the future, CD133 could be used as a biomarker for risk stratification, and possibly for developing novel targeted therapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

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

Kumar, S.; Cole, R.; Chiappelli, F.

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase inmore » mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.« less

Isolation of Small SSEA-4-Positive Putative Stem Cells from the Ovarian Surface Epithelium of Adult Human Ovaries by Two Different Methods

PubMed Central

Virant-Klun, Irma; Skutella, Thomas; Hren, Matjaz; Gruden, Kristina; Cvjeticanin, Branko; Vogler, Andrej; Sinkovec, Jasna

2013-01-01

The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched. PMID:23509763