Resveratrol regulates microglia M1/M2 polarization via PGC-1α in conditions of neuroinflammatory injury.

PubMed

Yang, Xiaodong; Xu, Shaoqing; Qian, Yiwei; Xiao, Qin

2017-08-01

Microglia are the primary cells that exert immune function in the central nervous system (CNS), and accumulating evidence suggests that microglia act as key players in the initiation of neurodegenerative diseases. It is now well recognized that microglia have functional plasticity and dual phenotypes, proinflammatory M1 and anti-inflammatory M2 phenotypes. Inhibiting the M1 phenotype while stimulating the M2 phenotype has been suggested as a potential therapeutic approach for the treatment of neuroinflammation-related diseases. Resveratrol has been demonstrated to exert anti-inflammatory effects by suppressing M1 microglia activation. However, the role of resveratrol in regulating microglia polarization and the molecular mechanisms involved have not been fully clarified. In this study, we tested whether resveratrol could suppress microglia activation by promoting microglia polarization toward the M2 phenotype via PGC-1α by measuring M1 and M2 markers in vitro and in vivo. Our study demonstrated that resveratrol reduced inflammatory damage and promoted microglia polarization to the M2 phenotype in LPS-induced neuroinflammation. In addition, resveratrol ameliorated LPS-induced sickness behavior in mice. The promoting effects of resveratrol on M2 polarization were attenuated by knocking down PGC-1α. PGC-1α not only suppressed LPS-evoked M1 marker expression by inhibition of NF-κB activity but also increased M2 marker expression by coactivation of the STAT6 and STAT3 pathways. We propose that overexpression PGC-1α by resveratrol could be a potential therapeutic approach to suppress neuroinflammation by regulating microglia polarization. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamic, M2-like remodeling phenotypes of CD11c+ adipose tissue macrophages during high-fat diet--induced obesity in mice.

PubMed

Shaul, Merav E; Bennett, Grace; Strissel, Katherine J; Greenberg, Andrew S; Obin, Martin S

2010-05-01

To identify, localize, and determine M1/M2 polarization of epidydimal adipose tissue (eAT) macrophages (Phis) during high-fat diet (HFD)-induced obesity. Male C57BL/6 mice were fed an HFD (60% fat kcal) or low-fat diet (LFD) (10% fat kcal) for 8 or 12 weeks. eATMPhis (F4/80(+) cells) were characterized by in vivo fluorescent labeling, immunohistochemistry, fluorescence-activated cell sorting, and quantitative PCR. Recruited interstitial macrophage galactose-type C-type lectin (MGL)1(+)/CD11c(-) and crown-like structure-associated MGL1(-)/CD11c(+) and MGL1(med)/CD11c(+) eATMPhis were identified after 8 weeks of HFD. MGL1(med)/CD11c(+) cells comprised approximately 65% of CD11c(+) eATMPhis. CD11c(+) eATMPhis expressed a mixed M1/M2 profile, with some M1 transcripts upregulated (IL-12p40 and IL-1beta), others downregulated (iNOS, caspase-1, MCP-1, and CD86), and multiple M2 and matrix remodeling transcripts upregulated (arginase-1, IL-1Ra, MMP-12, ADAM8, VEGF, and Clec-7a). At HFD week 12, each eATMPhi subtype displayed an enhanced M2 phenotype as compared with HFD week 8. CD11c(+) subtypes downregulated IL-1beta and genes mediating antigen presentation (I-a, CD80) and upregulated the M2 hallmark Ym-1 and genes promoting oxidative metabolism (PGC-1alpha) and adipogenesis (MMP-2). MGL1(med)/CD11c(+) eATMPhis upregulated additional M2 genes (IL-13, SPHK1, CD163, LYVE-1, and PPAR-alpha). MGL1(med)/CD11c(+) ATMPhis expressing elevated PGC-1alpha, PPAR-alpha, and Ym-1 transcripts were selectively enriched in eAT of obese mice fed pioglitazone for 6 days, confirming the M2 features of the MGL1(med)/CD11c(+) eATMPhi transcriptional profile and implicating PPAR activation in its elicitation. These results 1) redefine the phenotypic potential of CD11c(+) eATMPhis and 2) suggest previously unappreciated phenotypic and functional commonality between murine and human ATMPhis in the development of obesity and its complications.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

PubMed Central

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field. PMID:26317353

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

PubMed

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

PubMed

Winchester, Lee J; Veeranki, Sudhakar; Givvimani, Srikanth; Tyagi, Suresh C

2015-07-01

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes.

PubMed

Wu, N; Qin, H; Wang, M; Bian, Y; Dong, B; Sun, G; Zhao, W; Chang, G; Xu, Q; Chen, G

2017-04-01

1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks. 2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head. 3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes. 4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers. 5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

Mice Lacking Alternatively Activated (M2) Macrophages Show Impairments in Restorative Sleep after Sleep Loss and in Cold Environment.

PubMed

Massie, Ashley; Boland, Erin; Kapás, Levente; Szentirmai, Éva

2018-06-05

The relationship between sleep, metabolism and immune functions has been described, but the cellular components of the interaction are incompletely identified. We previously reported that systemic macrophage depletion results in sleep impairment after sleep loss and in cold environment. These findings point to the role of macrophage-derived signals in maintaining normal sleep. Macrophages exist either in resting form, classically activated, pro-inflammatory (M1) or alternatively activated, anti-inflammatory (M2) phenotypes. In the present study we determined the contribution of M2 macrophages to sleep signaling by using IL-4 receptor α-chain-deficient [IL-4Rα knockout (KO)] mice, which are unable to produce M2 macrophages. Sleep deprivation induced robust increases in non-rapid-eye-movement sleep (NREMS) and slow-wave activity in wild-type (WT) animals. NREMS rebound after sleep deprivation was ~50% less in IL-4Rα KO mice. Cold exposure induced reductions in rapid-eye-movement sleep (REMS) and NREMS in both WT and KO mice. These differences were augmented in IL-4Rα KO mice, which lost ~100% more NREMS and ~25% more REMS compared to WTs. Our finding that M2 macrophage-deficient mice have the same sleep phenotype as mice with global macrophage depletion reconfirms the significance of macrophages in sleep regulation and suggests that the main contributors are the alternatively activated M2 cells.

Acute Penicillium marneffei infection stimulates host M1/M2a macrophages polarization in BALB/C mice.

PubMed

Dai, Xiaoying; Mao, Congzheng; Lan, Xiuwan; Chen, Huan; Li, Meihua; Bai, Jing; Deng, Jingmin; Liang, Qiuli; Zhang, Jianquan; Zhong, Xiaoning; Liang, Yi; Fan, Jiangtao; Luo, Honglin; He, Zhiyi

2017-08-18

Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to

Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

PubMed

Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

2017-08-01

M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive

Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages

PubMed Central

2014-01-01

Introduction The mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI. Methods MSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate. Results In vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL

TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection

PubMed Central

Allman, Windy R.; Dey, Ranadhir; Liu, Lunhua; Siddiqui, Shafiuddin; Coleman, Adam S.; Bhattacharya, Parna; Yano, Masahide; Uslu, Kadriye; Takeda, Kazuyo; Nakhasi, Hira L.; Akkoyunlu, Mustafa

2015-01-01

The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated. PMID:26170307

SIRT1/Adenosine Monophosphate-Activated Protein Kinase α Signaling Enhances Macrophage Polarization to an Anti-inflammatory Phenotype in Rheumatoid Arthritis

PubMed Central

Park, So Youn; Lee, Sung Won; Lee, Sang Yeob; Hong, Ki Whan; Bae, Sun Sik; Kim, Koanhoi; Kim, Chi Dae

2017-01-01

Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) α/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, FcεRII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon γ-mediated NF-κB activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNFα and IL-1β expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPKα signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA. PMID:28966618

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Li, Yiwei; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of Epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein. PMID:21503965

Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish.

PubMed

Gros-Louis, Francois; Kriz, Jasna; Kabashi, Edor; McDearmid, Jonathan; Millecamps, Stéphanie; Urushitani, Makoto; Lin, Li; Dion, Patrick; Zhu, Qinzhang; Drapeau, Pierre; Julien, Jean-Pierre; Rouleau, Guy A

2008-09-01

Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

Effect of Cocoa Polyphenolic Extract on Macrophage Polarization from Proinflammatory M1 to Anti-Inflammatory M2 State

PubMed Central

Dugo, Laura; Belluomo, Maria Giovanna; Fanali, Chiara; Russo, Marina; Cacciola, Francesco

2017-01-01

Polyphenols-rich cocoa has many beneficial effects on human health, such as anti-inflammatory effects. Macrophages function as control switches of the immune system, maintaining the balance between pro- and anti-inflammatory activities. We investigated the hypothesis that cocoa polyphenol extract may affect macrophage proinflammatory phenotype M1 by favoring an alternative M2 anti-inflammatory state on macrophages deriving from THP-1 cells. Chemical composition, total phenolic content, and antioxidant capacity of cocoa polyphenols extracted from roasted cocoa beans were determined. THP-1 cells were activated with both lipopolysaccharides and interferon-γ for M1 or with IL-4 for M2 switch, and specific cytokines were quantified. Cellular metabolism, through mitochondrial oxygen consumption, and ATP levels were evaluated. Here, we will show that cocoa polyphenolic extract attenuated in vitro inflammation decreasing M1 macrophage response as demonstrated by a significantly lowered secretion of proinflammatory cytokines. Moreover, treatment of M1 macrophages with cocoa polyphenols influences macrophage metabolism by promoting oxidative pathways, thus leading to a significant increase in O2 consumption by mitochondrial complexes as well as a higher production of ATP through oxidative phosphorylation. In conclusion, cocoa polyphenolic extract suppresses inflammation mediated by M1 phenotype and influences macrophage metabolism by promoting oxidative pathways and M2 polarization of active macrophages. PMID:28744339

Activation of mTORC1 in Collecting Ducts Causes Hyperkalemia

PubMed Central

Chen, Zhenguo; Dong, Heling; Jia, Chunhong; Song, Qiancheng; Chen, Juan; Zhang, Yue; Lai, Pinglin; Fan, Xiaorong; Zhou, Xuan; Liu, Miao; Lin, Jun; Yang, Cuilan; Li, Ming; Gao, Tianming

2014-01-01

Mutation of TSC (encoding tuberous sclerosis complex protein) and activation of mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of several renal diseases, such as diabetic nephropathy and polycystic kidney disease. However, the role of mTOR in renal potassium excretion and hyperkalemia is not known. We showed that mice with collecting-duct (CD)–specific ablation of TSC1 (CDTsc1KO) had greater mTOR complex 1 (mTORC1) activation in the CD and demonstrated features of pseudohypoaldosteronism, including hyperkalemia, hyperaldosteronism, and metabolic acidosis. mTORC1 activation caused endoplasmic reticulum stress, columnar cell lesions, and dedifferentiation of CD cells with loss of aquaporin-2 and epithelial-mesenchymal transition-like phenotypes. Of note, mTORC1 activation also reduced the expression of serum- and glucocorticoid-inducible kinase 1, a crucial regulator of potassium homeostasis in the kidney, and decreased the expression and/or activity of epithelial sodium channel-α, renal outer medullary potassium channel, and Na+, K+-ATPase in the CD, which probably contributed to the aldosterone resistance and hyperkalemia in these mice. Rapamycin restored these phenotypic changes. Overall, this study identifies a novel function of mTORC1 in regulating potassium homeostasis and demonstrates that loss of TSC1 and activation of mTORC1 results in dedifferentiation and dysfunction of the CD and causes hyperkalemia. The CDTsc1KO mice provide a novel model for hyperkalemia induced exclusively by dysfunction of the CD. PMID:24203997

Activation of mTORC1 in collecting ducts causes hyperkalemia.

PubMed

Chen, Zhenguo; Dong, Heling; Jia, Chunhong; Song, Qiancheng; Chen, Juan; Zhang, Yue; Lai, Pinglin; Fan, Xiaorong; Zhou, Xuan; Liu, Miao; Lin, Jun; Yang, Cuilan; Li, Ming; Gao, Tianming; Bai, Xiaochun

2014-03-01

Mutation of TSC (encoding tuberous sclerosis complex protein) and activation of mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of several renal diseases, such as diabetic nephropathy and polycystic kidney disease. However, the role of mTOR in renal potassium excretion and hyperkalemia is not known. We showed that mice with collecting-duct (CD)-specific ablation of TSC1 (CDTsc1KO) had greater mTOR complex 1 (mTORC1) activation in the CD and demonstrated features of pseudohypoaldosteronism, including hyperkalemia, hyperaldosteronism, and metabolic acidosis. mTORC1 activation caused endoplasmic reticulum stress, columnar cell lesions, and dedifferentiation of CD cells with loss of aquaporin-2 and epithelial-mesenchymal transition-like phenotypes. Of note, mTORC1 activation also reduced the expression of serum- and glucocorticoid-inducible kinase 1, a crucial regulator of potassium homeostasis in the kidney, and decreased the expression and/or activity of epithelial sodium channel-α, renal outer medullary potassium channel, and Na(+), K(+)-ATPase in the CD, which probably contributed to the aldosterone resistance and hyperkalemia in these mice. Rapamycin restored these phenotypic changes. Overall, this study identifies a novel function of mTORC1 in regulating potassium homeostasis and demonstrates that loss of TSC1 and activation of mTORC1 results in dedifferentiation and dysfunction of the CD and causes hyperkalemia. The CDTsc1KO mice provide a novel model for hyperkalemia induced exclusively by dysfunction of the CD.

Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia.

PubMed

Bertolino, Alessandro; Fazio, Leonardo; Caforio, Grazia; Blasi, Giuseppe; Rampino, Antonio; Romano, Raffaella; Di Giorgio, Annabella; Taurisano, Paolo; Papp, Audrey; Pinsonneault, Julia; Wang, Danxin; Nardini, Marcello; Popolizio, Teresa; Sadee, Wolfgang

2009-02-01

Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case-control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density.

Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia

PubMed Central

Fazio, Leonardo; Caforio, Grazia; Blasi, Giuseppe; Rampino, Antonio; Romano, Raffaella; Di Giorgio, Annabella; Taurisano, Paolo; Papp, Audrey; Pinsonneault, Julia; Wang, Danxin; Nardini, Marcello; Popolizio, Teresa; Sadee, Wolfgang

2009-01-01

Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case–control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density. PMID:18829695

Comparison of CYP1A2 and NAT2 Phenotypes between Black and White Smokers

PubMed Central

Muscat, Joshua E.; Pittman, Brian; Kleinman, Wayne; Lazarus, Philip; Stellman, Steven D.; Richie, John P.

2008-01-01

The lower incidence rate of transitional cell carcinoma of the urinary bladder in blacks than in whites may be due to racial differences in the catalytic activity of enzymes that metabolize carcinogenic arylamines in tobacco smoke. To examine this, we compared cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 activities (NAT2) in black and white smokers using urinary caffeine metabolites as a probe for enzyme activity in a community-based study of 165 black and 183 white cigarette smokers. The paraxanthine (1,7-dimethylxanthine, 17X)/caffeine (trimethylxanthine, 137X) ratio or [17X + 1,7-dimethyluric acid (17U)]/137X ratio was used as an indicator of CYP1A2 activity. The 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine (1X) ratio indicated NAT2 activity. The odds ratio for the slow NAT2 phenotype associated with black race was 0.4; 95% confidence intervals 0.2–0.7. The putative combined low risk phenotype (slow CYP1A2/rapid NAT2) was more common in blacks than in whites (25% vs. 15%, P<0.02). There were no significant racial differences in slow and rapid CYP1A2 phenotypes, and in the combined slow NAT2/rapid CYP1A2 phenotype. Age, education, cigarette smoking amount, body mass index, GSTM1 and GSTM3 genotypes were unrelated to CYP1A2 and NAT2 activity. Intake of cruciferous vegetables (primarily broccoli), red meat, carrots, grapefruit and onions predicted CYP1A2 activity either for all subjects or in race-specific analyses. Carrot and grapefruit consumption was related to NAT2 activity. Collectively, these results indicated that phenotypic differences in NAT2 alone or in combination with CYP1A2 might help explain the higher incidence rates of transitional cell bladder cancer in whites. PMID:18703023

Pseudomonas aeruginosa infection alters the macrophage phenotype switching process during wound healing in diabetic mice.

PubMed

Chen, Sinuo; Li, Renren; Cheng, Chun; Xu, Jing-Ying; Jin, Caixia; Gao, Furong; Wang, Juan; Zhang, Jieping; Zhang, Jingfa; Wang, Hong; Lu, Lixia; Xu, Guo-Tong; Tian, Haibin

2018-03-07

Macrophages play critical roles in wound healing process. They switch from "classically activated" (M1) phenotype in the early inflammatory phase to "alternatively activated" (M2) phenotype in the later healing phase. However, the dynamic process of macrophage phenotype switching in diabetic wounds burdened with bacteria is unclear. In this report, Pseudomonas aeruginosa, frequently detected in diabetic foot ulcers, was inoculated into cutaneous wounds of db/db diabetic mice to mimic bacterium-infected diabetic wound healing. We observed that P. aeruginosa infection impaired diabetic wound healing and quickly promoted the expression of pro-inflammatory genes (M1 macrophage markers) tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β) and il-6 in wounds. The expression of markers of M2 macrophages, including il-10, arginase-1, and ym1 were also upregulated. In addition, similar gene expression patterns were observed in macrophages isolated directly from wounds. Immunostaining showed that P. aeruginosa infection increased both the ratios of M1 and M2 macrophages in wounds compared with that in control groups, which was further confirmed by in vitro culturing macrophages with P. aeruginosa and skin fibroblast conditioned medium. However, the ratios of the expression levels of pro-inflammatory genes to anti-inflammatory gene il-10 was increased markedly in P. aeruginosa infected wounds and macrophages compared with that in control groups, and P. aeruginosa prolonged the presence of M1 macrophages in the wounds. These data demonstrated that P. aeruginosa in diabetic wounds activates a mixed M1/M2 macrophage phenotype with an excessive activation of M1 phenotype or relatively inadequate activation of M2 phenotype. © 2018 International Federation for Cell Biology.

Association of the hypertriglyceridemic waist phenotype and type 2 diabetes mellitus among adults in China.

PubMed

Ren, Yongcheng; Zhang, Ming; Zhao, Jingzhi; Wang, Chongjian; Luo, Xinping; Zhang, Jiatong; Zhu, Tian; Li, Xi; Yin, Lei; Pang, Chao; Feng, Tianping; Wang, Bingyuan; Zhang, Lu; Li, Linlin; Yang, Xiangyu; Zhang, Hongyan; Hu, Dongsheng

2016-09-01

To clarify the association of the hypertriglyceridemic waist phenotype and type 2 diabetes mellitus among adults in China. In the present case-control study, we included 1,685 patients with type 2 diabetes mellitus and 7,141 normal glucose-tolerant controls from the Henan Province of China in 2011. Elevated waist circumference (GW) was defined as ≥90 cm for men and ≥80 cm for women. Hypertriglyceridemia (HT) was defined as >1.7 m mol/L triglycerides (TG) level. The association of hypertriglyceridemic waist phenotype and type 2 diabetes mellitus was investigated by sex, body mass index, physical activity, and family history of diabetes. Cases and controls differed in age, waist circumference (WC), weight, TG level, fasting glucose, body mass index, smoking status, diabetic family history, physical activity and hypertriglyceridemic waist phenotype (P < 0.05), but not alcohol drinking (P = 0.63). In the overall sample, as compared with the phenotype of normal TG level and normal WC (NTNW), normal TG level/enlarged WC (NTGW), elevated TG level/normal WC (HTNW) and elevated TG level/enlarged WC (HTGW) were associated with type 2 diabetes mellitus (odds ratio 4.14, 2.42 and 6.23, respectively). Only HTGW was consistently associated with risk of type 2 diabetes mellitus, with or without adjustment. The strongest relationship between HTGW and type 2 diabetes mellitus was for subjects with body mass index <24.0 kg/m(2) (odds ratio 6.54, 95% confidence interval 4.22-10.14) after adjustment for cofounding variables. HTGW was stably and significantly associated with risk of type 2 diabetes mellitus in adult Chinese. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Inhibition of AGEs/RAGE/Rho/ROCK pathway suppresses non-specific neuroinflammation by regulating BV2 microglial M1/M2 polarization through the NF-κB pathway.

PubMed

Chen, Jingkao; Sun, Zhaowei; Jin, Minghua; Tu, Yalin; Wang, Shengnan; Yang, Xiaohong; Chen, Qiuhe; Zhang, Xiao; Han, Yifan; Pi, Rongbiao

2017-04-15

The microglia-mediated neuroinflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). Advanced glycation end products (AGEs)/receptor for advanced glycation end products (RAGE) or Rho/Rho kinase (ROCK) are both involved in the development of non-specific inflammation. However, there are few reports about their effects on neuroinflammation. Here, we explored the mechanism of AGEs/RAGE/Rho/ROCK pathway underlying the non-specific inflammation and microglial polarization in BV2 cells. AGEs could activate ROCK pathway in a concentration-dependent manner. ROCK inhibitor fasudil and RAGE-specific blocker FPS-ZM1 significantly inhibited AGEs-mediated activation of BV2 cells and induction of reactive oxygen species (ROS). FPS-ZM1 and fasudil exerted their anti-inflammatory effects by downregulating inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), NLRP3 and nuclear translocation of nuclear factor kappa B (NF-κB) p65. In addition, AGEs induced both M1 (CD16/32, M1 marker) and M2 (CD206, M2 marker) phenotype in BV2 cells. Fasudil and FPS-ZM1 led to a decreased M1 and increased M2 phenotype. Together, these results indicate that the AGEs/RAGE/Rho/ROCK pathway in BV2 cells could intensify the non-specific inflammation of AD, which will provide novel strategies for the development of anti-AD drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype.

PubMed

Rosciszewski, Gerardo; Cadena, Vanesa; Murta, Veronica; Lukin, Jeronimo; Villarreal, Alejandro; Roger, Thierry; Ramos, Alberto Javier

2018-05-01

Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1β and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

PubMed

Melo, Sônia C; Santos, Regineide X; Melgaço, Ana C; Pereira, Alanna C F; Pungartnik, Cristina; Brendel, Martin

2015-06-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

PubMed Central

Melo, Sônia C.; Santos, Regineide X.; Melgaço, Ana C.; Pereira, Alanna C. F.; Pungartnik, Cristina; Brendel, Martin

2015-01-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae. PMID:26039235

Detection of an endogenous urinary biomarker associated with CYP2D6 activity using global metabolomics.

PubMed

Tay-Sontheimer, Jessica; Shireman, Laura M; Beyer, Richard P; Senn, Taurence; Witten, Daniela; Pearce, Robin E; Gaedigk, Andrea; Gana Fomban, Cletus L; Lutz, Justin D; Isoherranen, Nina; Thummel, Kenneth E; Fiehn, Oliver; Leeder, J Steven; Lin, Yvonne S

2014-12-01

We sought to discover endogenous urinary biomarkers of human CYP2D6 activity. Healthy pediatric subjects (n = 189) were phenotyped using dextromethorphan and randomized for candidate biomarker selection and validation. Global urinary metabolomics was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Candidate biomarkers were tested in adults receiving fluoxetine, a CYP2D6 inhibitor. A biomarker, M1 (m/z 444.3102) was correlated with CYP2D6 activity in both the pediatric training and validation sets. Poor metabolizers had undetectable levels of M1, whereas it was present in subjects with other phenotypes. In adult subjects, a 9.56-fold decrease in M1 abundance was observed during CYP2D6 inhibition. Identification and validation of M1 may provide a noninvasive means of CYP2D6 phenotyping.

Niacin and olive oil promote skewing to the M2 phenotype in bone marrow-derived macrophages of mice with metabolic syndrome.

PubMed

Montserrat-de la Paz, Sergio; Naranjo, Maria C; Lopez, Sergio; Abia, Rocio; Muriana, Francisco J G; Bermudez, Beatriz

2016-05-18

Metabolic syndrome (MetS) is associated with obesity, dyslipemia, type 2 diabetes and chronic low-grade inflammation. The aim of this study was to determine the role of high-fat low-cholesterol diets (HFLCDs) rich in SFAs (HFLCD-SFAs), MUFAs (HFLCD-MUFAs) or MUFAs plus omega-3 long-chain PUFAs (HFLCD-PUFAs) on polarisation and inflammatory potential in bone marrow-derived macrophages (BMDMs) from niacin (NA)-treated Lep(ob/ob)LDLR(-/-) mice. Animals fed with HFLCD-SFAs had increased weight and serum triglycerides, and their BMDMs accumulated triglycerides over the animals fed with HFLCD-MUFAs or -PUFAs. Furthermore, BMDMs from animals fed with HFLCD-SFAs were polarised towards the M1 phenotype with functional competence to produce pro-inflammatory cytokines, whereas BMDMs from animals fed with HFLCD-MUFAs or -PUFAs were skewed to the anti-inflammatory M2 phenotype. These findings open opportunities for developing novel nutritional strategies with olive oil as the most important dietary source of MUFAs (notably oleic acid) to prevent development and progression of metabolic complications in the NA-treated MetS.

Treatment in vitro with PPARα and PPARγ ligands drives M1-to-M2 polarization of macrophages from T. cruzi-infected mice.

PubMed

Penas, Federico; Mirkin, Gerardo A; Vera, Marcela; Cevey, Ágata; González, Cintia D; Gómez, Marisa I; Sales, María Elena; Goren, Nora B

2015-05-01

Trypanosoma cruzi, the etiological agent of Chagas' disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1β and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-β. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas' disease deserves further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

A multifamily study on the relationship between CYP2C19 genotype and s-mephenytoin oxidation phenotype.

PubMed

Brøsen, K; de Morais, S M; Meyer, U A; Goldstein, J A

1995-10-01

It has recently been shown that the most common mutation (named m1) in both Caucasian and Japanese poor metabolizers (PM) of S-mephenytoin is a single base pair mutation (G-->A) in exon 5 of the CYP2C19 gene. In Japanese, a second defective allele of CYP2C19 named m2 consists of a G-->A mutation in exon 4. In the present study, we have investigated the inheritance of the CYP2C19 wild type allele (wt) and the two defective alleles (m1 and m2) in families of 11 Danish PM probands. The study was carried out for two principal reasons. First, we wanted to confirm the autosomal recessive inheritance of the defective alleles, and second, we wanted to examine the specificity and sensitivity of the CYP2C19 genotyping test. Individuals were phenotyped by measuring the ratio of S/R mephenytoin excreted in the urine after administration of mephenytoin, and genotyping was carried out by a PCR-based DNA amplification procedure. The genotypes of nine of the 11 probands were consistent with their phenotypes. Eight were homozygous m1/m1, and one was heterozygous m1/m2. The genotypes of two putative PM probands (wt/m1) were not consistent with their phenotypes. On the basis of extended phenotyping (additional late urine collections (24-36 h) and acidification of urine), one of these could probably be reclassified as an extensive metabolizer (EM) while the other was considered to be a true PM. This suggests the presence of an additional unknown mutant allele in the latter. Seven of the 41 phenotyped relatives in the 11 families were phenotyped as PMs, and with the exception of the father of family 10, their genotypes (m1/m1) were consistent with their phenotypes. Extended phenotyping (acidification of urine) suggested that the father of family 10 in fact is an EM and hence that his genotype (wt/m1) is concordant with his phenotype. Thus, the specificity of genotyping tests for PM was 100%, while the sensitivity was 15/16 or 94%. Our study provides unequivocal evidence for autosomal

Hyperoxia exacerbates postnatal inflammation-induced lung injury in neonatal BRP-39 null mutant mice promoting the M1 macrophage phenotype.

PubMed

Syed, Mansoor A; Bhandari, Vineet

2013-01-01

Hyperoxia exposure to developing lungs-critical in the pathogenesis of bronchopulmonary dysplasia-may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39(-/-) mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39(-/-) mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.

Phenotypic dysregulation of microglial activation in young offspring rats with maternal sleep deprivation-induced cognitive impairment

PubMed Central

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

2015-01-01

Despite the potential adverse effects of maternal sleep deprivation (MSD) on physiological and behavioral aspects of offspring, the mechanisms remain poorly understood. The present study was intended to investigate the roles of microglia on neurodevelopment and cognition in young offspring rats with prenatal sleep deprivation. Pregnant Wistar rats received 72 h sleep deprivation in the last trimester of gestation, and their prepuberty male offspring were given the intraperitoneal injection with or without minocycline. The results showed the number of Iba1+ microglia increased, that of hippocampal neurogenesis decreased, and the hippocampus-dependent spatial learning and memory were impaired in MSD offspring. The classical microglial activation markers (M1 phenotype) IL-1β, IL-6, TNF-α, CD68 and iNOS were increased, while the alternative microglial activation markers (M2 phenotype) Arg1, Ym1, IL-4, IL-10 and CD206 were reduced in hippocampus of MSD offspring. After minocycline administration, the MSD offspring showed improvement in MWM behaviors and increase in BrdU+/DCX+ cells. Minocycline reduced Iba1+ cells, suppressed the production of pro-inflammatory molecules, and reversed the reduction of M2 microglial markers in the MSD prepuberty offspring. These results indicate that dysregulation in microglial pro- and anti-inflammatory activation is involved in MSD-induced inhibition of neurogenesis and impairment of spatial learning and memory. PMID:25830666

Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

PubMed

Tanner, Julie-Anne; Prasad, Bhagwat; Claw, Katrina G; Stapleton, Patricia; Chaudhry, Amarjit; Schuetz, Erin G; Thummel, Kenneth E; Tyndale, Rachel F

2017-01-01

Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine. Copyright © 2016 by The American Society for Pharmacology and

Disorders with similar clinical phenotypes reveal underlying genetic interaction: SATB2 acts as an activator of the UPF3B gene

PubMed Central

Leoyklang, Petcharat; Suphapeetiporn, Kanya; Srichomthong, Chalurmpon; Tongkobpetch, Siraprapa; Fietze, Stefanie; Dorward, Heidi; Cullinane, Andrew R.; Gahl, William A.; Huizing, Marjan; Shotelersuk, Vorasuk

2014-01-01

Two syndromic cognitive impairment disorders have very similar craniofacial dysmorphisms. One is caused by mutations of SATB2, a transcription regulator, and the other by heterozygous mutations leading to premature stop codons in UPF3B, encoding a member of the nonsense-mediated mRNA decay complex. Here we demonstrate that the products of these two causative genes function in the same pathway. We show that the SATB2 nonsense mutation in our patient leads to a truncated protein that localizes to the nucleus, forms a dimer with wild-type SATB2 and interferes with its normal activity. This suggests that the SATB2 nonsense mutation has a dominant negative effect. The patient’s leukocytes had significantly decreased UPF3B mRNA compared to controls. This effect was replicated both in vitro, where siRNA knockdown of SATB2 in HEK293 cells resulted in decreased UPF3B expression, and in vivo, where embryonic tissue of Satb2 knock-out mice showed significantly decreased Upf3b expression. Furthermore, chromatin immunoprecipitation demonstrates that SATB2 binds to the UPF3B promoter, and a luciferase reporter assay confirmed that SATB2 expression significantly activates gene transcription using the UPF3B promoter. These findings indicate that SATB2 acts as an activator UPF3B expression through binding to its promoter. This study emphasizes the value of recognizing disorders with similar clinical phenotypes to explore underlying mechanisms of genetic interaction. PMID:23925499

Delayed pituitary adenylate cyclase-activating polypeptide delivery after brain stroke improves functional recovery by inducing m2 microglia/macrophage polarization.

PubMed

Brifault, Coralie; Gras, Marjorie; Liot, Donovan; May, Victor; Vaudry, David; Wurtz, Olivier

2015-02-01

Until now, except thrombolysis, the therapeutical strategies targeting the acute phase of cerebral ischemia have been proven ineffective, and no approach is available to attenuate the delayed cell death mechanisms and the resulting functional deficits in the late phase. Then, we investigated whether a targeted and delayed delivery of pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide known to exert neuroprotective activities, may dampen delayed pathophysiological processes improving functional recovery. Three days after permanent focal ischemia, PACAP-producing stem cells were transplanted intracerebro ventricularly in nonimmunosuppressed mice. At 7 and 14 days post ischemia, the effects of this stem cell-based targeted delivery of PACAP on functional recovery, volume lesions, and inflammatory processes were analyzed. The delivery of PACAP in the vicinity of the infarct zone 3 days post stroke promotes fast, stable, and efficient functional recovery. This was correlated with a modulation of the postischemic inflammatory response. Transcriptomic and Ingenuity Pathway Analysis-based bioinformatic analyses identified several gene networks, functions, and key transcriptional factors, such as nuclear factor-κB, C/EBP-β, and Notch/RBP-J as PACAP's potential targets. Such PACAP-dependent immunomodulation was further confirmed by morphometric and phenotypic analyses of microglial cells showing increased number of Arginase-1(+) cells in mice treated with PACAP-expressing cells specifically, demonstrating the redirection of the microglial response toward a neuroprotective M2 phenotype. Our results demonstrated that immunomodulatory strategies capable of redirecting the microglial response toward a neuroprotective M2 phenotype in the late phase of brain ischemia could represent attractive options for stroke treatment in a new and unexploited therapeutical window. © 2014 American Heart Association, Inc.

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and 'Schizophrenia-Like Behaviors' in Mice.

PubMed

Vitucci, Daniela; Di Giorgio, Annabella; Napolitano, Francesco; Pelosi, Barbara; Blasi, Giuseppe; Errico, Francesco; Attrotto, Maria Teresa; Gelao, Barbara; Fazio, Leonardo; Taurisano, Paolo; Di Maio, Anna; Marsili, Valentina; Pasqualetti, Massimo; Bertolino, Alessandro; Usiello, Alessandro

2016-02-01

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia.

Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis.

PubMed

Mahmood, Dler Faieeq Darweesh; Abderrazak, Amna; Couchie, Dominique; Lunov, Oleg; Diderot, Vimala; Syrovets, Tatiana; Slimane, Mohamed-Naceur; Gosselet, Fabien; Simmet, Thomas; Rouis, Mustapha; El Hadri, Khadija

2013-07-01

Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases. Copyright © 2013 Wiley Periodicals, Inc.

Viral Activation of MK2-hsp27-p115RhoGEF-RhoA Signaling Axis Causes Cytoskeletal Rearrangements, P-body Disruption and ARE-mRNA Stabilization

PubMed Central

Corcoran, Jennifer A.; Johnston, Benjamin P.; McCormick, Craig

2015-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers, including the endothelial cell (EC) neoplasm Kaposi's sarcoma (KS). KSHV-infected ECs secrete abundant host-derived pro-inflammatory molecules and angiogenic factors that contribute to tumorigenesis. The precise contributions of viral gene products to this secretory phenotype remain to be elucidated, but there is emerging evidence for post-transcriptional regulation. The Kaposin B (KapB) protein is thought to contribute to the secretory phenotype in infected cells by binding and activating the stress-responsive kinase MK2, thereby selectively blocking decay of AU-rich mRNAs (ARE-mRNAs) encoding pro-inflammatory cytokines and angiogenic factors. Processing bodies (PBs) are cytoplasmic ribonucleoprotein foci in which ARE-mRNAs normally undergo rapid 5′ to 3′ decay. Here, we demonstrate that PB dispersion is a feature of latent KSHV infection, which is dependent on kaposin protein expression. KapB is sufficient to disperse PBs, and KapB-mediated ARE-mRNA stabilization could be partially reversed by treatments that restore PBs. Using a combination of genetic and chemical approaches we provide evidence that KapB-mediated PB dispersion is dependent on activation of a non-canonical Rho-GTPase signaling axis involving MK2, hsp27, p115RhoGEF and RhoA. PB dispersion in latently infected cells is likewise dependent on p115RhoGEF. In addition to PB dispersion, KapB-mediated RhoA activation in primary ECs caused actin stress fiber formation, increased cell motility and angiogenesis; these effects were dependent on the activity of the RhoA substrate kinases ROCK1/2. By contrast, KapB-mediated PB dispersion occurred in a ROCK1/2-independent manner. Taken together, these observations position KapB as a key contributor to viral reprogramming of ECs, capable of eliciting many of the phenotypes characteristic of KS tumor cells, and strongly contributing to the post

To Genotype or Phenotype for Personalized Medicine? CYP450 Drug Metabolizing Enzyme Genotype-Phenotype Concordance and Discordance in the Ecuadorian Population.

PubMed

De Andrés, Fernando; Terán, Santiago; Hernández, Francisco; Terán, Enrique; LLerena, Adrián

2016-12-01

Genetic variations within the cytochrome P450 (CYP450) superfamily of drug metabolizing enzymes confer substantial person-to-person and between-population differences in pharmacokinetics, and by extension, highly variable clinical effects of medicines. In this context, "personalized medicine," "precision medicine," and "stratified medicine" are related concepts attributed to what is essentially targeted therapeutics and companion diagnostics, aimed at improving safety and effectiveness of health interventions. We report here, to the best of our knowledge, the first comparative clinical pharmacogenomics study, in an Ecuadorian population sample, of five key CYP450s involved in drug metabolism: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In 139 unrelated, medication-free, and healthy Ecuadorian subjects, we measured the phenotypic activity of these drug metabolism pathways using the CEIBA multiplexed phenotyping cocktail. The subjects were genotyped for each CYP450 enzyme gene as well. Notably, based on the CYP450 metabolic phenotypes estimated by the genotype data, 0.75% and 3.10% of the subjects were genotypic poor metabolizers (gPMs) for CYP2C19 and CYP2D6, respectively. Additionally, on the other extreme, genotype-estimated ultrarapid metabolizer (gUMs) phenotype was represented by 15.79% of CYP2C19, and 5.43% of CYP2D6. There was, however, considerable discordance between directly measured phenotypes (mPMs and mUMs) and the above genotype-estimated enzyme phenotypes. For example, among individuals genotypically carrying enhanced activity alleles (gUMs), many showed a lower actual drug metabolism capacity than expected by their genotypes, even lower than individuals with reduced or no activity alleles. In conclusion, for personalized medicine in the Ecuadorian population, we recommend CYP450 multiplexed phenotyping, or genotyping and phenotyping in tandem, rather than CYP450 genotypic tests alone. Additionally, we recommend, in consideration of equity, ethical

IgE-Api m 4 Is Useful for Identifying a Particular Phenotype of Bee Venom Allergy.

PubMed

Ruiz, B; Serrano, P; Moreno, C

Different clinical behaviors have been identified in patients allergic to bee venom. Compound-resolved diagnosis could be an appropriate tool for investigating these differences. The aims of this study were to analyze whether specific IgE to Api m 4 (sIgE-Api m 4) can identify a particular kind of bee venom allergy and to describe response to bee venom immunotherapy (bVIT). Prospective study of 31 patients allergic to bee venom who were assigned to phenotype group A (sIgE-Api m 4 <0.98 kU/L), treated with native aqueous (NA) extract, or phenotype group B (sIgE-Api m 4 ≥0.98 kU/L), treated with purified aqueous (PA) extract. Sex, age, cardiovascular risk, severity of preceding sting reaction, exposure to beekeeping, and immunological data (intradermal test, sIgE/sIgG4-Apis-nApi m 1, and sIgE-rApi m 2-Api m 4 were analyzed. Systemic reactions (SRs) during bVIT build-up were analyzed. Immunological and sting challenge outcomes were evaluated in each group after 1 and 2 years of bVIT. Phenotype B patients had more severe reactions (P=.049) and higher skin sensitivity (P=.011), baseline sIgE-Apis (P=.0004), sIgE-nApi m 1 (P=.0004), and sIgG4-Apis (P=.027) than phenotype A patients. Furthermore, 41% of patients in group B experienced SRs during the build-up phase with NA; the sting challenge success rate in this group was 82%. There were no significant reductions in serial intradermal test results, but an intense reduction in sIgE-nApi m 1 (P=.013) and sIgE-Api m 4 (P=.004) was observed after the first year of bVIT. Use of IgE-Api m 4 as the only discrimination criterion demonstrated differences in bee venom allergy. Further investigation with larger populations is necessary.

Hyperoxia Exacerbates Postnatal Inflammation-Induced Lung Injury in Neonatal BRP-39 Null Mutant Mice Promoting the M1 Macrophage Phenotype

PubMed Central

Syed, Mansoor A.

2013-01-01

Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury. PMID:24347826

Polymorphisms of CYP1A2 and CYP2A6 activity: phenotypes and the effect of age and sex in a Nigerian population.

PubMed

Adehin, Ayorinde; Bolaji, Oluseye O

2015-09-01

CYP1A2 and CYP2A6 are polymorphic enzymes that metabolise several compounds of clinical importance. This study investigated the prevalent phenotypes of these enzymes and the influence of age and sex on enzyme activity in a Nigerian population. Caffeine (110 mg) was administered to each of 129 healthy, unrelated subjects (85 males and 44 females) who were non-smokers. Urine voided within 7 h after caffeine administration was collected for a high performance liquid chromatographic assay of caffeine (137X), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X). CYP1A2 activity was measured as a ratio of (17U+17X) to 137X, while 17U/17X served as marker for CYP2A6. Transformed data were analysed and the influences of age and sex on activity were also determined. Distribution of CYP1A2 activity in the population was bimodal with a mean±SD of 0.82±0.41, while that of CYP2A6 was trimodal with a mean±SD activity of 0.27±0.42 of the log-transformed urinary molar ratio of metabolites. The influences of age and sex on enzyme activity for both CYP1A2 and CYP2A6 were not significant (p>0.05). The study established the prevalence of polymorphism in phenotypes of CYP1A2 and CYP2A6 activity in the Nigerian population, but no influence of age and sex on enzyme activity was observed in this population.

Novel SOX2 mutations and genotype-phenotype correlation in anophthalmia and microphthalmia.

PubMed

Schneider, Adele; Bardakjian, Tanya; Reis, Linda M; Tyler, Rebecca C; Semina, Elena V

2009-12-01

SOX2 represents a High Mobility Group domain containing transcription factor that is essential for normal development in vertebrates. Mutations in SOX2 are known to result in a spectrum of severe ocular phenotypes in humans, also typically associated with other systemic defects. Ocular phenotypes include anophthalmia/microphthalmia (A/M), optic nerve hypoplasia, ocular coloboma and other eye anomalies. We screened 51 unrelated individuals with A/M and identified SOX2 mutations in the coding region of the gene in 10 individuals. Seven of the identified mutations are novel alterations, while the remaining three individuals carry the previously reported recurrent 20-nucleotide deletion in SOX2, c.70del20. Among the SOX2-positive cases, seven patients had bilateral A/M and mutations resulting in premature termination of the normal protein sequence (7/38; 18% of all bilateral cases), one patient had bilateral A/M associated with a single amino acid insertion (1/38; 3% of bilateral cases), and the final two patients demonstrated unilateral A/M associated with missense mutations (2/13; 15% of all unilateral cases). These findings and review of previously reported cases suggest a potential genotype/phenotype correlation for SOX2 mutations with missense changes generally leading to less severe ocular defects. In addition, we report a new familial case of affected siblings with maternal mosaicism for the identified SOX2 mutation, which further underscores the importance of parental testing to provide accurate genetic counseling to families.

Novel SOX2 Mutations and Genotype-Phenotype Correlation in Anophthalmia and Microphthalmia

PubMed Central

Schneider, Adele; Bardakjian, Tanya; Reis, Linda M.; Tyler, Rebecca C.; Semina, Elena V.

2009-01-01

SOX2 represents a High Mobility Group domain containing transcription factor that is essential for normal development in vertebrates. Mutations in SOX2 are known to result in a spectrum of severe ocular phenotypes in humans, also typically associated with other systemic defects. Ocular phenotypes include anophthalmia/microphthalmia (A/M), optic nerve hypoplasia, ocular coloboma and other eye anomalies. We screened 51 unrelated individuals with A/M and indentified SOX2 mutations in the coding region of the gene in 10 individuals. Seven of the identified mutations are novel alterations, while the remaining three individuals carry the previously reported recurrent 20-nucleotide deletion in SOX2, c.70del20. Among the SOX2-positive cases, seven patients had bilateral A/M and mutations resulting in premature termination of the normal protein sequence (7/38; 18% of all bilateral cases), one patient had bilateral A/M associated with a single amino acid insertion (1/38; 3% of bilateral cases), and the final two patients demonstrated unilateral A/M associated with missense mutations (2/13; 15% of all unilateral cases). These findings and review of previously reported cases suggest a potential genotype/phenotype correlation for SOX2 mutations with missense changes generally leading to less severe ocular defects. In addition, we report a new familial case of affected siblings with maternal mosaicism for the identified SOX2 mutation, which further underscores the importance of parental testing to provide accurate genetic counseling to families. PMID:19921648

Murine Lupus Susceptibility Locus Sle2 Activates DNA-Reactive B Cells through Two Sub-Loci with Distinct Phenotypes

PubMed Central

Zeumer, Leilani; Sang, Allison; Niu, Haitao; Morel, Laurence

2010-01-01

The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B cell differentiation which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b, and Sle2c. Sle2 also enhances the breach of B cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that play a critical role in B cell tolerance. PMID:21270826

Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages

PubMed Central

Canton, Johnathan; Khezri, Rojyar; Glogauer, Michael; Grinstein, Sergio

2014-01-01

Macrophages respond to changes in environmental stimuli by assuming distinct functional phenotypes, a phenomenon referred to as macrophage polarization. We generated classically (M1) and alternatively (M2) polarized macrophages—two extremes of the polarization spectrum—to compare the properties of their phagosomes. Specifically, we analyzed the regulation of the luminal pH after particle engulfment. The phagosomes of M1 macrophages had a similar buffering power and proton (equivalent) leakage permeability but significantly reduced proton-pumping activity compared with M2 phagosomes. As a result, only the latter underwent a rapid and profound acidification. By contrast, M1 phagosomes displayed alkaline pH oscillations, which were caused by proton consumption upon dismutation of superoxide, followed by activation of a voltage- and Zn2+-sensitive permeation pathway, likely HV1 channels. The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes. The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify. Thus, in M1 cells, elimination of pathogens through deployment of the microbicidal NADPH oxidase is given priority at the expense of delayed acidification. By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively. PMID:25165138

Hepatic Oval Cells Have the Side Population Phenotype Defined by Expression of ATP-Binding Cassette Transporter ABCG2/BCRP1

PubMed Central

Shimano, Koichi; Satake, Makoto; Okaya, Atsuhito; Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Sakagami, Masafumi; Terada, Nobuyuki; Tsujimura, Tohru

2003-01-01

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver. PMID:12819005

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

PubMed

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

PubMed Central

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation. PMID:26584640

Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

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

Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

1995-09-01

The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had amore » genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.« less

Tachykinin activation of human alveolar macrophages in tobacco smoke and sarcoidosis: a phenotypical and functional study.

PubMed

Brunelleschi, S; Guidotto, S; Viano, I; Fantozzi, R; Pozzi, E; Ghio, P; Albera, C

1996-10-01

Substance P (SP) and neurokinin A (NKA), which exert bronchoconstrictor effects on human airways, are known to interact with inflammatory and immune cells, including monocyte macrophages. We have evaluated the effects of SP, NKA and the NK2 selective agonist [beta-Ala8]-NKA(4-10) on alveolar macrophages (AM) isolated from 4 healthy smokers and 4 non-smoker active pulmonary sarcoid patients. An accumulation of activated mononuclear phagocytes, as well as elevated angiotensin-converting enzyme (ACE) activity, has been evidenced in both clinical conditions. The phenotype of AMs in the studied subjects was characterized by an elevated expression of CD68+, HLA-DR+ and CD14+, CD14+ being significantly less in sarcoidosis as compared to smokers. SP, NKA and the NK2 selective agonist evoked superoxide anion (O2-) production in AMs obtained from sarcoid patients or healthy smokers. While SP acted in a non-dose-dependent manner in both conditions, NKA and [beta-Ala8]-NKA(4-10) evoked a dose-dependent respiratory burst (ED50 = 0.25 and 0.26 nM, respectively) in smokers, but not in sarcoidosis. The more marked phenotypical expression correlated well with the ability of NK2 receptors to activate AMs in smoker subjects.

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and ‘Schizophrenia-Like Behaviors' in Mice

PubMed Central

Vitucci, Daniela; Di Giorgio, Annabella; Napolitano, Francesco; Pelosi, Barbara; Blasi, Giuseppe; Errico, Francesco; Attrotto, Maria Teresa; Gelao, Barbara; Fazio, Leonardo; Taurisano, Paolo; Di Maio, Anna; Marsili, Valentina; Pasqualetti, Massimo; Bertolino, Alessandro; Usiello, Alessandro

2016-01-01

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia. PMID:26228524

Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro

PubMed Central

Chhor, Vibol; Le Charpentier, Tifenn; Lebon, Sophie; Oré, Marie-Virgine; Celador, Idoia Lara; Josserand, Julien; Degos, Vincent; Jacotot, Etienne; Hagberg, Henrik; Sävman, Karin; Mallard, Carina; Gressens, Pierre; Fleiss, Bobbi

2013-01-01

Microglia mediate multiple facets of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype is an appealing neurotherapeutic strategy but a comprehensive study of classical and more novel microglial phenotypic markers in vitro is lacking. The aim of this study was to outline the temporal expression of a battery of phenotype markers from polarised microglia to generate an in vitro tool for screening the immunomodulatory potential of novel compounds. We characterised expression of thirty-one macrophage/microglial phenotype markers in primary microglia over time (4, 12, 36, and 72 h), using RT-qPCR or multiplex protein assay. Firstly, we selected Interleukin-4 (IL-4) and lipopolysaccharide (LPS) as the strongest M1–M2 polarising stimuli, from six stimuli tested. At each time point, markers useful to identify that microglia were M1 included iNOS, Cox-2 and IL-6 and a loss of M2a markers. Markers useful for quantifying M2b-immunomodulatory microglia included, increased IL-1RA and SOCS3 and for M2a-repair and regeneration, included increased arginase-1, and a loss of the M1 and M2b markers were discriminatory. Additional markers were regulated at fewer time points, but are still likely important to monitor when assessing the immunomodulatory potential of novel therapies. Further, to facilitate identification of how novel immunomodulatory treatments alter the functional affects of microglia, we characterised how the soluble products from polarised microglia affected the type and rate of neuronal death; M1/2b induced increasing and M2a-induced decreasing neuronal loss. We also assessed any effects of prior activation state, to provide a way to identify how a novel compound may alter phenotype depending on the stage of injury/insult progression. We identified generally that a prior M1/2b reduced the ability of microglia to switch to

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.).

PubMed

Huang, Jian; Tang, Ding; Shen, Yi; Qin, Baoxiang; Hong, Lilan; You, Aiqing; Li, Ming; Wang, Xin; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2010-01-01

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2beta-hydroxylation. In the present study, we isolated a CaMV 35S-enhancer activation tagged mutant, H032. This mutant exhibited a dominant dwarf and GA-deficient phenotype, with a final stature that was less than half of its wild-type counterpart. The endogenous bioactive GAs are markedly decreased in the H032 mutant, and application of bioactive GAs (GA(3) or GA(4)) can reverse the dwarf phenotype. The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR. An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant, which might be due to the enhancer role of the CaMV 35S promoter. RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene. BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases, which is a novel type of GA2ox that uses C20-GAs (GA(12) and/or GA(53)) as the substrates. Interestingly, we found that a GA biosynthesis inhibitor, paclobutrazol, positively regulated the OsGA2ox6 gene. Unlike the over-expression of OsGA2ox1, which led to a high rate of seed abortion, the H032 mutant retained normal flowering and seed production. These results indicate that OsGA2ox6 mainly affects plant stature, and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

m-Trifluoromethyl-diphenyl Diselenide Regulates Prefrontal Cortical MOR and KOR Protein Levels and Abolishes the Phenotype Induced by Repeated Forced Swim Stress in Mice.

PubMed

Rosa, Suzan Gonçalves; Pesarico, Ana Paula; Martini, Franciele; Nogueira, Cristina Wayne

2018-04-05

The present study aimed to investigate the m-trifluoromethyl-diphenyl diselenide [(m-CF 3 -PhSe) 2 ] effects on prefrontal cortical MOR and KOR protein levels and phenotype induced by repeated forced swim stress (FSS) in mice. Adult Swiss mice were subjected to repeated FSS sessions, and after that, they performed the spontaneous locomotor/exploratory activity, tail suspension, and splash tests. (m-CF 3 -PhSe) 2 (0.1 to 5 mg/kg) was administered to mice 30 min before the first FSS session and 30 min before the subsequent repeated FSS. (m-CF 3 -PhSe) 2 abolished the phenotype induced by repeated FSS in mice. In addition, a single FSS session increased μ but reduced δ-opioid receptor contents, without changing the κ content. Mice subjected to repeated FSS had an increase in the μ content when compared to those of naïve group or subjected to single FSS. Repeated FSS induced an increase of δ-opioid receptor content compared to those mice subjected to single FSS. However, the δ-opioid receptor contents were lower than those found in the naïve group. The mice subjected to repeated FSS showed an increase in the κ-opioid receptor content when compared to that of the naïve mice. (m-CF 3 -PhSe) 2 regulated the protein contents of μ and κ receptors in mice subjected to repeated FSS. These findings demonstrate that (m-CF 3 -PhSe) 2 was effective to abolish the phenotype induced by FSS, which was accompanied by changes in the contents of cortical μ- and κ-opioid receptors.

IL-33, IL-25, and TSLP induce a distinct phenotypic and activation profile in human type 2 innate lymphoid cells.

PubMed

Camelo, Ana; Rosignoli, Guglielmo; Ohne, Yoichiro; Stewart, Ross A; Overed-Sayer, Catherine; Sleeman, Matthew A; May, Richard D

2017-04-11

Innate lymphoid cells (ILCs) represent a distinct branch of the lymphoid lineage composed of 3 major subpopulations: ILC1, ILC2, and ILC3. ILCs are mainly described as tissue-resident cells but can be detected at low levels in human blood. However, unlike mouse ILCs, there is still no consistent methodology to purify and culture these cells that enables in-depth analysis of their intrinsic biology. Here, we describe defined culture conditions for ILC2s, which allowed us to dissect the roles of interleukin 2 (IL-2), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) individually, or in combination, in modulating ILC2 phenotype and function. We show that TSLP is important for ILC2 survival, while ILC2 activation is more dependent on IL-33, especially when in combination with IL-2 or TSLP. We found that activation of ILC2s by IL-33 and TSLP dramatically upregulated their surface expression of c-Kit and downregulated expression of the canonical markers IL-7Rα and CRTH2. IL-2 further amplified ILC2 production of IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor but also induced a more natural killer (NK)-like phenotype in ILC2, with upregulation of granzyme B production by these cells. Furthermore, ILC2 plasticity was observed in serum-free SFEM II media in response to IL-33, IL-25, and TSLP stimulation and independently of IL-12 and IL-1β. This is the first comprehensive report of an in vitro culture system for human ILC2s, without the use of feeder layers, which additionally evaluates the impact of IL-25, IL-33, and TSLP alone or in combination on ILC2 surface phenotype and activation status.

Hypoxia inducible factor 1 links fast-patterned muscle activity and fast muscle phenotype in rats.

PubMed

Lunde, Ida G; Anton, Siobhan L; Bruusgaard, Jo C; Rana, Zaheer A; Ellefsen, Stian; Gundersen, Kristian

2011-03-15

Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. PO2 can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle.When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1β than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles.When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

Delivery strategies to control inflammatory response: Modulating M1-M2 polarization in tissue engineering applications.

PubMed

Alvarez, Mario Moisés; Liu, Julie C; Trujillo-de Santiago, Grissel; Cha, Byung-Hyun; Vishwakarma, Ajaykumar; Ghaemmaghami, Amir M; Khademhosseini, Ali

2016-10-28

Macrophages are key players in many physiological scenarios including tissue homeostasis. In response to injury, typically the balance between macrophage sub-populations shifts from an M1 phenotype (pro-inflammatory) to an M2 phenotype (anti-inflammatory). In tissue engineering scenarios, after implantation of any device, it is desirable to exercise control on this M1-M2 progression and to ensure a timely and smooth transition from the inflammatory to the healing stage. In this review, we briefly introduce the current state of knowledge regarding macrophage function and nomenclature. Next, we discuss the use of controlled release strategies to tune the balance between the M1 and M2 phenotypes in the context of tissue engineering applications. We discuss recent literature related to the release of anti-inflammatory molecules (including nucleic acids) and the sequential release of cytokines to promote a timely M1-M2 shift. In addition, we describe the use of macrophages as controlled release agents upon stimulation by physical and/or mechanical cues provided by scaffolds. Moreover, we discuss current and future applications of "smart" implantable scaffolds capable of controlling the cascade of biochemical events related to healing and vascularization. Finally, we provide our opinion on the current challenges and the future research directions to improve our understanding of the M1-M2 macrophage balance and properly exploit it in tissue engineering and regenerative medicine applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Delivery strategies to control inflammatory response: Modulating M1-M2 polarization in tissue engineering applications

PubMed Central

Alvarez, Mario Moisés; Liu, Julie C.; Santiago, Grissel Trujillo-de; Cha, Byung-Hyun; Vishwakarma, Ajaykumar; Ghaemmaghami, Amir; Khademhosseini, Ali

2016-01-01

Macrophages are key players in many physiological scenarios including tissue homeostasis. In response to injury, typically the balance between macrophage sub-populations shifts from an M1 phenotype (pro-inflammatory) to an M2 phenotype (anti-inflammatory). In tissue engineering scenarios, after implantation of any device, it is desirable to exercise control on this M1-M2 progression and to ensure a timely and smooth transition from the inflammatory to the healing stage. In this review, we briefly introduce the current state of knowledge regarding macrophage function and nomenclature. Next, we discuss the use of controlled release strategies to tune the balance between the M1 and M2 phenotypes in the context of tissue engineering applications. We discuss recent literature related to the release of anti-inflammatory molecules (including nucleic acids) and the sequential release of cytokines to promote a timely M1-M2 shift. In addition, we describe the use of macrophages as controlled release agents upon stimulation by physical and/or mechanical cues provided by scaffolds. Moreover, we discuss current and future applications of “smart” implantable scaffolds capable of controlling the cascade of biochemical events related to healing and vascularization. Finally, we provide our opinion on the current challenges and the future research directions to improve our understanding of the M1-M2 macrophage balance and properly exploit it in tissue engineering and regenerative medicine applications. PMID:26778695

Wnt signaling pathway involvement in genotypic and phenotypic variations in Waardenburg syndrome type 2 with MITF mutations.

PubMed

Wang, Xue-Ping; Liu, Ya-Lan; Mei, Ling-Yun; He, Chu-Feng; Niu, Zhi-Jie; Sun, Jie; Zhao, Yu-Lin; Feng, Yong; Zhang, Hua

2018-05-01

Mutation in the gene encoding microphthalmia-associated transcription factor (MITF) lead to Waardenburg syndrome 2 (WS2), an autosomal dominantly inherited syndrome with auditory-pigmentary abnormalities, which is clinically and genetically heterogeneous. Haploinsufficiency may be the underlying mechanism for WS2. However, the mechanisms explaining the genotypic and phenotypic variations in WS2 caused by MITF mutations are unclear. A previous study revealed that MITF interacts with LEF-1, an important factor in the Wnt signaling pathway, to regulate its own transcription through LEF-1-binding sites on the MITF promoter. In this study, four different WS2-associated MITF mutations (p.R217I, p.R217G, p.R255X, p.R217del) that are associated with highly variable clinical features were chosen. According to the results, LEF-1 can activate the expression of MITF on its own, but MITF proteins inhibited the activation. This inhibition weakens when the dosage of MITF is reduced. Except for p.R217I, p.R255X, p.R217G, and p.R217del lose the ability to activate TYR completely and do not inhibit the LEF-1-mediated activation of the MITF-M promoter, and the haploinsufficiency created by mutant MITF can be overcome; correspondingly, the mutants' associated phenotypes are less severe than that of p.R217I. The dominant negative of p.R217del made it have a second-most severe phenotype. This study's data imply that MITF has a negative feedback loop of regulation to stabilize MITF gene dosage that involves the Wnt signaling pathway and that the interaction of MITF mutants with this pathway drives the genotypic and phenotypic differences observed in Waardenburg syndrome type 2 associated with MITF mutations.

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor–activated macrophages

PubMed Central

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-01-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages. PMID:22675026

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

PubMed

López-Pelaéz, Marta; Fumagalli, Stefano; Sanz, Carlos; Herrero, Clara; Guerra, Susana; Fernandez, Margarita; Alemany, Susana

2012-08-01

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

PubMed

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Phenotypical expression of CYP2D6 in amerindians of tepehuano origin from Durango, Mexico.

PubMed

Lares-Asseff, Ismael; Sosa-Macías, Martha; Elizondo-Azuela, Guillermo; Flores-Pérez, Carmen; Flores-Pérez, Janett; Bradley-Alvarez, Francisco

2005-01-01

Cytochrome P4502D6 (CYP2D6) shows genetic polymorphism, which is clinically important in the metabolism of drugs and other xenobiotics. Dextrometorphan (DM) has been used as a test compound to evaluate the in vivo activity of CYP2D6. Phenotypical frequencies of CYP2D6 have been determined in some populations, but little is known about them in native populations. The object of this study was to characterize the phenotypical expression of CYP2D6 in Amerindian subjects of Tepehuano origin from the State of Durango, using DM as metabolic marker, as well as the effect of age, sex and nutritional status on this activity. Three hr after oral administration of a single 30 mg dose of DM, the plasma concentration of DM and its metabolite dextrophan (DX) were determined with HPLC in 55 Tepehuano subjects. All subjects were extensive metabolizers (metabolic ratio MR < 0.3). No correlation of age, sex or nutritional status was found with the DM/DX metabolic ratio. However, we found a monoexponential relationship between the metabolic ratio of DM and DX, and their concentrations respectively, which can have clinical applications, since metabolic ratio can be predicted from a known DM or DX concentration. Three hr after ingestion of DM, 18 individuals showed DM plasma concentrations of 5 to 10 ng/mL, 15 subjects of 11 to 20 ng/mL, 8 subjects of 21 to 50 ng/mL and 14 subjects >51 ng/mL pointing out that DM concentrations and MR must be determined to establish toxicity risk levels.

Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex.

PubMed

Zeng, Ling-Hui; Rensing, Nicholas R; Zhang, Bo; Gutmann, David H; Gambello, Michael J; Wong, Michael

2011-02-01

Tuberous Sclerosis Complex (TSC) is an autosomal dominant, multi-system disorder, typically involving severe neurological symptoms, such as epilepsy, cognitive deficits and autism. Two genes, TSC1 and TSC2, encoding the proteins hamartin and tuberin, respectively, have been identified as causing TSC. Although there is a substantial overlap in the clinical phenotype produced by TSC1 and TSC2 mutations, accumulating evidence indicates that TSC2 mutations cause more severe neurological manifestations than TSC1 mutations. In this study, the neurological phenotype of a novel mouse model involving conditional inactivation of the Tsc2 gene in glial-fibrillary acidic protein (GFAP)-positive cells (Tsc2(GFAP1)CKO mice) was characterized and compared with previously generated Tsc1(GFAP1)CKO mice. Similar to Tsc1(GFAP1)CKO mice, Tsc2(GFAP1)CKO mice exhibited epilepsy, premature death, progressive megencephaly, diffuse glial proliferation, dispersion of hippocampal pyramidal cells and decreased astrocyte glutamate transporter expression. However, Tsc2(GFAP1)CKO mice had an earlier onset and higher frequency of seizures, as well as significantly more severe histological abnormalities, compared with Tsc1(GFAP1)CKO mice. The differences between Tsc1(GFAP1)CKO and Tsc2(GFAP1)CKO mice were correlated with higher levels of mammalian target of rapamycin (mTOR) activation in Tsc2(GFAP1)CKO mice and were reversed by the mTOR inhibitor, rapamycin. These findings provide novel evidence in mouse models that Tsc2 mutations intrinsically cause a more severe neurological phenotype than Tsc1 mutations and suggest that the difference in phenotype may be related to the degree to which Tsc1 and Tsc2 inactivation causes abnormal mTOR activation.

Combination of EPA with Carotenoids and Polyphenol Synergistically Attenuated the Transformation of Microglia to M1 Phenotype Via Inhibition of NF-κB.

PubMed

Hadad, Nurit; Levy, Rachel

2017-09-01

Microglia activation toward the M1 phenotype has been reported to contribute to the neurodegenerative processes and cognition alterations due to the release of pro-inflammatory mediators and cytokines. The aim of the present research was to assess the effectiveness of free fatty acids omega-3 preparations: eicosapentaenoic acid (EPA) or/and docosahexaenoic acid (DHA), carotenoids and phenolics combinations, in inhibiting the release of inflammatory mediators from activated microglia. Preincubation of BV-2 microglia cells with each of the FFAs omega-3 preparations in a range of 0.03-2 μM together with Lyc-O-mato ® (0.1 μM), Carnosic acid (0.2 μM) with or without Lutein (0.2 μM), 1 h before addition of lipopolysaccharide (LPS) for 16 h caused a synergistic inhibition of nitric oxide (NO) production with a rank order of EPA > Ropufa (EPA/DHA 2/1) > Krill (EPA/DHA 1.23/1). The optimal inhibitory combinations of EPA (0.125 μM) with the phytonutrients caused a synergistic inhibition of prostaglandin E 2 (PGE 2 ) release, IL-6 secretion, superoxide and NO production and prevention of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) upregulation and elevated CD40 expression in microglia exposed to LPS or interferon-γ (IFN-γ), representing infection or inflammation, respectively. The presence of the combination caused a synergistic increase in the release of the anti-inflammatory cytokine IL-10. The inhibitory effects by the combinations of EPA with the phytonutrients were mediated by the inhibition of the redox-sensitive NF-κB activation and detected by its phosphorylated p-65 on serine 536 in microglia stimulated by either LPS or IFN-γ. In addition, phosphorylated CREB on serine 133 which was shown to be involved in the induction of iNOS was inhibited by the combinations in stimulated cells. In conclusion, the results suggest that low concentrations of EPA with the phytonutrients are very efficient in inhibiting the transformation of

M2 polarization enhances silica nanoparticle uptake by macrophages.

PubMed

Hoppstädter, Jessica; Seif, Michelle; Dembek, Anna; Cavelius, Christian; Huwer, Hanno; Kraegeloh, Annette; Kiemer, Alexandra K

2015-01-01

While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but

M2 polarization enhances silica nanoparticle uptake by macrophages

PubMed Central

Hoppstädter, Jessica; Seif, Michelle; Dembek, Anna; Cavelius, Christian; Huwer, Hanno; Kraegeloh, Annette; Kiemer, Alexandra K.

2015-01-01

While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but

Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.

PubMed

Burczyk, Martina; Burkhalter, Martin D; Blätte, Tamara; Matysik, Sabrina; Caron, Marc G; Barak, Lawrence S; Philipp, Melanie

2015-01-27

The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.

Active Learning Strategies for Phenotypic Profiling of High-Content Screens.

PubMed

Smith, Kevin; Horvath, Peter

2014-06-01

High-content screening is a powerful method to discover new drugs and carry out basic biological research. Increasingly, high-content screens have come to rely on supervised machine learning (SML) to perform automatic phenotypic classification as an essential step of the analysis. However, this comes at a cost, namely, the labeled examples required to train the predictive model. Classification performance increases with the number of labeled examples, and because labeling examples demands time from an expert, the training process represents a significant time investment. Active learning strategies attempt to overcome this bottleneck by presenting the most relevant examples to the annotator, thereby achieving high accuracy while minimizing the cost of obtaining labeled data. In this article, we investigate the impact of active learning on single-cell-based phenotype recognition, using data from three large-scale RNA interference high-content screens representing diverse phenotypic profiling problems. We consider several combinations of active learning strategies and popular SML methods. Our results show that active learning significantly reduces the time cost and can be used to reveal the same phenotypic targets identified using SML. We also identify combinations of active learning strategies and SML methods which perform better than others on the phenotypic profiling problems we studied. © 2014 Society for Laboratory Automation and Screening.

Open innovation for phenotypic drug discovery: The PD2 assay panel.

PubMed

Lee, Jonathan A; Chu, Shaoyou; Willard, Francis S; Cox, Karen L; Sells Galvin, Rachelle J; Peery, Robert B; Oliver, Sarah E; Oler, Jennifer; Meredith, Tamika D; Heidler, Steven A; Gough, Wendy H; Husain, Saba; Palkowitz, Alan D; Moxham, Christopher M

2011-07-01

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

IRF5 regulates lung macrophages M2 polarization during severe acute pancreatitis in vitro.

PubMed

Sun, Kang; He, Song-Bing; Qu, Jian-Guo; Dang, Sheng-Chun; Chen, Ji-Xiang; Gong, Ai-Hua; Xie, Rong; Zhang, Jian-Xin

2016-11-14

To investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP) in vitro . A mouse SAP model was established by intraperitoneal (ip) injections of 20 μg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR. SAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype in vitro . The expressions of M1 markers, including IRF5 (S + IRF5 siRNA vs S + PBS, 0.013 ± 0.01 vs 0.054 ± 0.047, P < 0.01), TNF-α (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.019 ± 0.018, P < 0.001), iNOS (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.026 ± 0.018, P < 0.001) and IL-12 (S + IRF5 siRNA vs S + PBS, 0.000005 ± 0.00004 vs 0.024 ± 0.016, P < 0.001), were decreased. In contrast, the expressions of M2 markers, including IL-10 (S + IRF5 siRNA vs S + PBS, 0.060 ± 0.055 vs 0.0230 ± 0.018, P < 0.01) and Arg-1 (S + IRF5 siRNA vs S + PBS, 0.910 ± 0.788 vs 0.0036 ± 0.0025, P < 0.001), were increased. IRF5 siRNA could reverse the lung macrophage polarization more effectively than IL-4. Treatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI.

IRF5 regulates lung macrophages M2 polarization during severe acute pancreatitis in vitro

PubMed Central

Sun, Kang; He, Song-Bing; Qu, Jian-Guo; Dang, Sheng-Chun; Chen, Ji-Xiang; Gong, Ai-Hua; Xie, Rong; Zhang, Jian-Xin

2016-01-01

AIM To investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP) in vitro. METHODS A mouse SAP model was established by intraperitoneal (ip) injections of 20 μg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR. RESULTS SAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype in vitro. The expressions of M1 markers, including IRF5 (S + IRF5 siRNA vs S + PBS, 0.013 ± 0.01 vs 0.054 ± 0.047, P < 0.01), TNF-α (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.019 ± 0.018, P < 0.001), iNOS (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.026 ± 0.018, P < 0.001) and IL-12 (S + IRF5 siRNA vs S + PBS, 0.000005 ± 0.00004 vs 0.024 ± 0.016, P < 0.001), were decreased. In contrast, the expressions of M2 markers, including IL-10 (S + IRF5 siRNA vs S + PBS, 0.060 ± 0.055 vs 0.0230 ± 0.018, P < 0.01) and Arg-1 (S + IRF5 siRNA vs S + PBS, 0.910 ± 0.788 vs 0.0036 ± 0.0025, P < 0.001), were increased. IRF5 siRNA could reverse the lung macrophage polarization more effectively than IL-4. CONCLUSION Treatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI. PMID:27895424

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides.

PubMed

Pannell, Maria; Labuz, Dominika; Celik, Melih Ö; Keye, Jacqueline; Batra, Arvind; Siegmund, Britta; Machelska, Halina

2016-10-07

During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 10 5 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and β-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the

[Inhibitory effects of pseudolaric acid B on inflammatory response and M1 phenotype polarization in RAW264.7 macrophages induced by lipopolysaccharide].

PubMed

Li, Yuxiu; Li, Tan; Ji, Wenjie; Li, Xiao; Ma, Yongqiang; Zhao, Jihong; Zhou, Xin; Li, Yuming

2016-05-01

To investigate the effects of pseudolaric acid B (PLAB) on the inflammatory response and M1 phenotype polarization in RAW264.7 cells induced by lipopolysaccharide (LPS) and the related mechanisms. The inflammatory model in vitro was made using RAW264.7 cells stimulated by LPS, and then was treated with 0.5 μmol/L PLAB and 1 μmol/L GW9662, a peroxisome proliferators-activated receptor γ (PPARγ) antagonist. The cell cycle was tested by flow cytometry. The mRNA expressions of PPARγ and M1 phenotype markers interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α) were measured by real-time PCR. The expression levels of signal molecules involved in nuclear factor-κB (NF-κB) signal pathway were detected by Western blotting. PLAB markedly decreased the expressions of IL-1β and TNF-α mRNAs induced by LPS and increased PPARγ mRNA level. Moreover, the expressions of NF-κB p65, pNF-κB p65, IKKα, IKKβ, pIKKα/β, IκBα and pIκBα decreased in PLAB-treated cells. Meanwhile, RAW264.7 cells were arrested in G0 and G2 phase after the treatment with PLAB. However, the effects of PLAB on RAW264.7 cells could be reversed by GW9662 obviously. PLAB could inhibit the inflammatory response and M1 phenotype polarization in RAW264.7 cells induced by LPS via modulating cell cycle and NF-κB/PPARγ signal pathway.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PubMed

Litviakov, Nikolai; Tsyganov, Matvey; Larionova, Irina; Ibragimova, Marina; Deryusheva, Irina; Kazantseva, Polina; Slonimskaya, Elena; Frolova, Irina; Choinzonov, Eugeniy; Cherdyntseva, Nadezhda; Kzhyshkowska, Julia

2018-05-04

High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39 + CCL18 - or YKL-39 - CCL18 + ) and only one patient had M2- macrophage phenotype (YKL-39 - CCL18 - ). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39 - CCL18 - ). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes

Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism.

PubMed

Hasan, Maroof; Gonugunta, Vijay K; Dobbs, Nicole; Ali, Aktar; Palchik, Guillermo; Calvaruso, Maria A; DeBerardinis, Ralph J; Yan, Nan

2017-01-24

Three-prime repair exonuclease 1 knockout (Trex1 -/- ) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-TANK-binding kinase-interferon regulatory factor 3 (cGAS-STING-TBK1-IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1 -/- mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1 -/- background, and many metabolic defects persist in Trex1 -/- Irf3 -/- cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1 -/- mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1 -/- cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

PubMed Central

Pal, Rituraj; Bajaj, Lakshya; Sharma, Jaiprakash; Palmieri, Michela; Di Ronza, Alberto; Lotfi, Parisa; Chaudhury, Arindam; Neilson, Joel; Sardiello, Marco; Rodney, George G.

2016-01-01

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD. PMID:26960433

Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes

NASA Astrophysics Data System (ADS)

Qie, Yaqing; Yuan, Hengfeng; von Roemeling, Christina A.; Chen, Yuanxin; Liu, Xiujie; Shih, Kevin D.; Knight, Joshua A.; Tun, Han W.; Wharen, Robert E.; Jiang, Wen; Kim, Betty Y. S.

2016-05-01

Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines.

ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

PubMed Central

Sammons, Morgan A.; Antons, Amanda K.; Bendjennat, Mourad; Udd, Bjarne; Krahe, Ralf; Link, Andrew J.

2010-01-01

Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5′UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype. PMID:20174632

mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice

PubMed Central

Inoki, Ken; Mori, Hiroyuki; Wang, Junying; Suzuki, Tsukasa; Hong, SungKi; Yoshida, Sei; Blattner, Simone M.; Ikenoue, Tsuneo; Rüegg, Markus A.; Hall, Michael N.; Kwiatkowski, David J.; Rastaldi, Maria P.; Huber, Tobias B.; Kretzler, Matthias; Holzman, Lawrence B.; Wiggins, Roger C.; Guan, Kun-Liang

2011-01-01

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition–like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN. PMID:21606597

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro

PubMed Central

Heinrich, Franziska; Lehmbecker, Annika; Raddatz, Barbara B.; Kegler, Kristel; Tipold, Andrea; Stein, Veronika M.; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

2017-01-01

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as “respiratory burst”, whereas M2-polarization was associated with processes such as “mitosis”. Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for

mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells.

PubMed

Eid, Walaa; Dauner, Kristin; Courtney, Kevin C; Gagnon, AnneMarie; Parks, Robin J; Sorisky, Alexander; Zha, Xiaohui

2017-07-25

mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5 -/- and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.

Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

PubMed Central

Aoki, Yutaka; Helzlsouer, Kathy J.; Strickland, Paul T.

2014-01-01

Context: Cholinesterase (ChE) specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking) with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose. PMID:24473115

Satb2-Independent Acquisition of the Cholinergic Sudomotor Phenotype in Rodents

PubMed Central

Schütz, Burkhard; Schaäfer, Martin K.-H.; Gördes, Markus; Eiden, Lee E.; Weihe, Eberhard

2014-01-01

Expression of Satb2 (Special AT-rich sequence-binding protein-2) elicits expression of the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in cultured rat sympathetic neurons exposed to soluble differentiation factors. Here, we determined whether or not Satb2 plays a similar role in cholinergic differentiation in vivo, by comparing the postnatal profile of Satb2 expression in the rodent stellate ganglion to that of VAChT and ChAT. Throughout postnatal development, VAChT and ChAT were found to be co-expressed in a numerically stable subpopulation of rat stellate ganglion neurons. Nerve fibers innervating rat forepaw sweat glands on P1 were VAChT immunoreactive, while ChAT was detectable at this target only after P5. The postnatal abundance of VAChT transcripts in the stellate ganglion was at maximum already on P1, whereas ChAT mRNA levels increased from low levels on P1 to reach maximum levels between P5 and P21. Satb2 mRNA was detected in cholinergic neurons in the stellate ganglion beginning with P8, thus coincident with the onset of unequivocal detection of ChAT immunoreactivity in forepaw sweat gland endings. Satb2 knockout mice exhibited no change in the P1 cholinergic VAChT/ChAT co-phenotype in stellate ganglion neurons. Thus, cholinergic phenotype maturation involves first, early target (sweat-gland)-independent expression and trafficking of VAChT, and later, potentially target- and Satb2-dependent elevation of ChAT mRNA and protein transport into sweat gland endings. In rat sudomotor neurons that, unlike mouse sudomotor neurons, co-express calcitonin gene-related peptide (CGRP), Satb2 may also be related to the establishment of species-specific neuropeptide co-phenotypes during postnatal development. PMID:25239161

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

PubMed Central

Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

2017-01-01

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought

Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

PubMed

Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

2015-01-01

Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.

Treatment targets for M2 microglia polarization in ischemic stroke.

PubMed

Wang, Ji; Xing, Hongyi; Wan, Lin; Jiang, Xingjun; Wang, Chen; Wu, Yan

2018-06-05

As the first line of defense in the nervous system, resident microglia are the predominant immune cells in the brain. In diseases of the central nervous system such as stroke, Alzheimer's disease, and Parkinson's disease, they often cause inflammation or phagocytosis; however, some studies have found that despite the current controversy over M1, M2 polarization could be beneficial. Ischemic stroke is the third most common cause of death in humans. Patients who survive an ischemic stroke might experience a clear decline in their quality of life, owing to conditions such as hemiplegic paralysis and aphasia. After stroke, the activated microglia become a double-edged sword, with distinct phenotypic changes to the deleterious M1 and neuroprotective M2 types. Therefore, methods for promoting the differentiation of microglia into the M2 polarized form to alleviate harmful reactions after stroke have become a topic of interest in recent years. Subsequently, the discovery of new drugs related to M2 polarization has enabled the realization of targeted therapies. In the present review, we discussed the neuroprotective effects of microglia M2 polarization and the potential mechanisms and drugs by which microglia can be transformed into the M2 polarized type after stroke. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Inhibition of NF-κB prevents the acidic bile-induced oncogenic mRNA phenotype, in human hypopharyngeal cells

PubMed Central

Vageli, Dimitra P.; Doukas, Sotirios G.; Sasaki, Clarence T.

2018-01-01

Bile-containing gastro-duodenal reflux has been clinically considered an independent risk factor in hypopharyngeal carcinogenesis. We recently showed that the chronic effect of acidic bile, at pH 4.0, selectively induces NF-κB activation and accelerates the transcriptional levels of genes, linked to head and neck cancer, in normal hypopharyngeal epithelial cells. Here, we hypothesize that NF-κB inhibition is capable of preventing the acidic bile-induced and cancer-related mRNA phenotype, in treated normal human hypopharyngeal cells. In this setting we used BAY 11-7082, a specific and well documented pharmacologic inhibitor of NF-κB, and we observed that BAY 11-7082 effectively inhibits the acidic bile-induced gene expression profiling of the NF-κB signaling pathway (down-regulation of 72 out of 84 analyzed genes). NF-κB inhibition significantly prevents the acidic bile-induced transcriptional activation of NF-κB transcriptional factors, RELA (p65) and c-REL, as well as genes related to and commonly found in established HNSCC cell lines. These include anti-apoptotic bcl-2, oncogenic STAT3, EGFR, ∆Np63, TNF-α and WNT5A, as well as cytokines IL-1β and IL-6. Our findings are consistent with our hypothesis demonstrating that NF-κB inhibition effectively prevents the acidic bile-induced cancer-related mRNA phenotype in normal human hypopharyngeal epithelial cells supporting an understanding that NF-κB may be a critical link between acidic bile and early preneoplastic events in this setting. PMID:29464041

PPAR Activation Induces M1 Macrophage Polarization via cPLA2-COX-2 Inhibition, Activating ROS Production against Leishmania mexicana

PubMed Central

Díaz-Gandarilla, J. A.; Osorio-Trujillo, C.; Hernández-Ramírez, V. I.; Talamás-Rohana, P.

2013-01-01

Defence against Leishmania depends upon Th1 inflammatory response and, a major problem in susceptible models, is the turnoff of the leishmanicidal activity of macrophages with IL-10, IL-4, and COX-2 upregulation, as well as immunosuppressive PGE2, all together inhibiting the respiratory burst. Peroxisome proliferator-activated receptors (PPAR) activation is responsible for macrophages polarization on Leishmania susceptible models where microbicide functions are deactivated. In this paper, we demonstrated that, at least for L. mexicana, PPAR activation, mainly PPARγ, induced macrophage activation through their polarization towards M1 profile with the increase of microbicide activity against intracellular pathogen L. mexicana. PPAR activation induced IL-10 downregulation, whereas the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 remained high. Moreover, PPAR agonists treatment induced the deactivation of cPLA2-COX-2-prostaglandins pathway together with an increase in TLR4 expression, all of whose criteria meet the M1 macrophage profile. Finally, parasite burden, in treated macrophages, was lower than that in infected nontreated macrophages, most probably associated with the increase of respiratory burst in these treated cells. Based on the above data, we conclude that PPAR agonists used in this work induces M1 macrophages polarization via inhibition of cPLA2 and the increase of aggressive microbicidal activity via reactive oxygen species (ROS) production. PMID:23555077

Uncoupling oxidative phosphorylation with 2,4-dinitrophenol promotes development of the adhesion phenotype.

PubMed

Shavell, Valerie I; Fletcher, Nicole M; Jiang, Zhong L; Saed, Ghassan M; Diamond, Michael P

2012-03-01

To determine the effect of uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP) on adhesion phenotype development. Prospective experimental study. Academic medical center. Women undergoing laparotomy for pelvic pain from whom normal peritoneum and adhesions were excised to create primary cultures of normal peritoneal and adhesion fibroblasts. Treatment of normal peritoneal and adhesion fibroblasts isolated from the same patient(s) with or without 0.2 mM DNP for 24 hours. Evaluation of adhesion phenotype markers type I collagen, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α. In agreement with prior findings, adhesion fibroblasts exhibited significantly higher basal levels of type I collagen, VEGF, and HIF-1α compared with normal peritoneal fibroblasts. Treatment of normal peritoneal fibroblasts with DNP resulted in significant increases in type I collagen (10.2 ± 1.4 vs. 18.4 ± 1.9 fg/μg RNA) and VEGF (8.2 ± 1.1 vs. 13.7 ± 0.4 fg/μg RNA) over baseline. HIF-1α levels did not increase when normal peritoneal fibroblasts were treated with DNP. The adhesion phenotype, which is normally expressed in response to hypoxia, is reproduced in a normoxic environment by uncoupling oxidative phosphorylation with DNP, as evidenced by an increase in type I collagen and VEGF. Acquisition of the adhesion phenotype was via a mechanism distinct from up-regulation of HIF-1α. These observations are consistent with the hypothesis that the adhesion phenotype represents a state of intracellular metabolic depletion. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The Role of Macrophage Phenotype in Vascularization of Tissue Engineering Scaffolds

PubMed Central

Spiller, Kara L.; Anfang, Rachel; Spiller, Krista J.; Ng, Johnathan; Nakazawa, Kenneth R.; Daulton, Jeffrey W.; Vunjak-Novakovic, Gordana

2014-01-01

Angiogenesis is crucial for the success of most tissue engineering strategies. The natural inflammatory response is a major regulator of vascularization, through the activity of different types of macrophages and the cytokines they secrete. Macrophages exist on a spectrum of diverse phenotypes, from “classically activated” M1 to “alternatively activated” M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are typically considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic, although these classifications are controversial. Here we show that in contrast to this traditional paradigm, primary human M1 macrophages secrete the highest levels of potent angiogenic stimulators including VEGF; M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant stabilizing pericytes, and also promote anastomosis of sprouting endothelial cells in vitro; and M2c macrophages secrete the highest levels of MMP9, an important protease involved in vascular remodeling. In a murine subcutaneous implantation model, porous collagen scaffolds were surrounded by a fibrous capsule, coincident with high expression of M2 macrophage markers, while scaffolds coated with the bacterial lipopolysaccharide were degraded by inflammatory macrophages, and glutaraldehyde-crosslinked scaffolds were infiltrated by substantial numbers of blood vessels accompanied by high levels of M1 and M2 macrophages. These results suggest that coordinated efforts by both M1 and M2 macrophages are required for angiogenesis and scaffold vascularization, which may explain some of the controversy over which phenotype is the angiogenic phenotype. PMID:24589361

Microglia M2A Polarization as Potential Link between Food Allergy and Autism Spectrum Disorders.

PubMed

Kalkman, Hans O; Feuerbach, Dominik

2017-12-09

Atopic diseases are frequently co-morbid with autism spectrum disorders (ASD). Allergic responses are associated with an activation of mast cells, innate lymphoid cells, and Th2 cells. These cells produce type-2 cytokines (IL4 and IL13), which stimulate microglia and macrophages to adopt a phenotype referred to as 'alternative activation' or 'M2A'. M2A-polarized macrophages and microglia play a physiological role in tissue repair by secreting growth factors such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1. In ASD there is evidence for increased type-2 cytokines, microglia activation, M2A polarization, and increased levels of growth factors. In neurons, these growth factors drive a signal transduction pathway that leads to activation of the enzyme mammalian Target of Rapamycin (mTOR), and thereby to the inhibition of autophagy. Activation of mTOR is an effect that is also common to several of the genetic forms of autism. In the central nervous system, redundant synapses are removed via an autophagic process. Activation of mTOR would diminish the pruning of redundant synapses, which in the context of ASD is likely to be undesired. Based on this line of reasoning, atopic diseases like food allergy, eczema or asthma would represent risk factors for autism spectrum disorders.

Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction.

PubMed

Biliczki, Peter; Girmatsion, Zenawit; Brandes, Ralf P; Harenkamp, Sabine; Pitard, Bruno; Charpentier, Flavien; Hébert, Terence E; Hohnloser, Stefan H; Baró, Isabelle; Nattel, Stanley; Ehrlich, Joachim R

2009-12-01

KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function. We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers. Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed. KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes. The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially

NOX2 drives M1-like microglial/macrophage activation and neurodegeneration following experimental traumatic brain injury

PubMed Central

Kumar, Alok; Barrett, James P.; Alvarez-Croda, Dulce-Mariely; Stoica, Bogdan A.; Faden, Alan I.; Loane, David J.

2016-01-01

Following traumatic brain injury (TBI), activation of microglia and peripherally derived inflammatory macrophages occurs in association with tissue damage. This neuroinflammatory response may have beneficial or detrimental effects on neuronal survival, depending on the functional polarization of these cells along a continuum from M1-like to M2-like activation states. The mechanisms that regulate M1-like and M2-like activation after TBI are not well understood, but appear in part to reflect the redox state of the lesion microenvironment. NADPH oxidase (NOX2) is a critical enzyme system that generates reactive oxygen species in microglia/macrophages. After TBI, NOX2 is strongly up-regulated in M1-like, but not in M2-like polarized cells. Therefore, we hypothesized that NOX2 drives M1-like neuroinflammation and contributes to neurodegeneration and loss of neurological function after TBI. In the present studies we inhibited NOX2 activity using NOX2-knockout mice or the selective peptide inhibitor gp91ds-tat. We show that NOX2 is highly up-regulated in infiltrating macrophages after injury, and that NOX2 deficiency reduces markers of M1-like activation, limits tissue loss and neurodegeneration, and improves motor recovery after moderate-level control cortical injury (CCI). NOX2 deficiency also promotes M2-like activation after CCI, through increased IL-4Rα signaling in infiltrating macrophages, suggesting that NOX2 acts as a critical switch between M1- and M2-like activation states after TBI. Administration of gp91ds-tat to wild-type CCI mice starting at 24 hours post-injury reduces deficits in cognitive function and increased M2-like activation in the hippocampus. Collectively, our data indicate that increased NOX2 activity after TBI drives M1-like activation that contributes to inflammatory-mediated neurodegeneration, and that inhibiting this pathway provides neuroprotection, in part by altering M1-/M2-like balance towards the M2-like neuroinflammatory response. PMID

Behavioral and Psychological Phenotyping of Physical Activity and Sedentary Behavior: Implications for Weight Management.

PubMed

Bryan, Angela D; Jakicic, John M; Hunter, Christine M; Evans, Mary E; Yanovski, Susan Z; Epstein, Leonard H

2017-10-01

Risk for obesity is determined by a complex mix of genetics and lifetime exposures at multiple levels, from the metabolic milieu to psychosocial and environmental influences. These phenotypic differences underlie the variability in risk for obesity and response to weight management interventions, including differences in physical activity and sedentary behavior. As part of a broader effort focused on behavioral and psychological phenotyping in obesity research, the National Institutes of Health convened a multidisciplinary workshop to explore the state of the science in behavioral and psychological phenotyping in humans to explain individual differences in physical activity, both as a risk factor for obesity development and in response to activity-enhancing interventions. Understanding the behavioral and psychological phenotypes that contribute to differences in physical activity and sedentary behavior could allow for improved treatment matching and inform new targets for tailored, innovative, and effective weight management interventions. This summary provides the rationale for identifying psychological and behavioral phenotypes relevant to physical activity and identifies opportunities for future research to better understand, define, measure, and validate putative phenotypic factors and characterize emerging phenotypes that are empirically associated with initiation of physical activity, response to intervention, and sustained changes in physical activity. © 2017 The Obesity Society.

Standards-Based Procedural Phenotyping: The Arden Syntax on i2b2.

PubMed

Mate, Sebastian; Castellanos, Ixchel; Ganslandt, Thomas; Prokosch, Hans-Ulrich; Kraus, Stefan

2017-01-01

Phenotyping, or the identification of patient cohorts, is a recurring challenge in medical informatics. While there are open source tools such as i2b2 that address this problem by providing user-friendly querying interfaces, these platforms lack semantic expressiveness to model complex phenotyping algorithms. The Arden Syntax provides procedural programming language construct, designed specifically for medical decision support and knowledge transfer. In this work, we investigate how language constructs of the Arden Syntax can be used for generic phenotyping. We implemented a prototypical tool to integrate i2b2 with an open source Arden execution environment. To demonstrate the applicability of our approach, we used the tool together with an Arden-based phenotyping algorithm to derive statistics about ICU-acquired hypernatremia. Finally, we discuss how the combination of i2b2's user-friendly cohort pre-selection and Arden's procedural expressiveness could benefit phenotyping.

[Mg2+]o/[Ca2+]o determines Ca2+ response at fertilization: tuning of adult phenotype?

PubMed

Ozil, Jean-Pierre; Sainte-Beuve, Thierry; Banrezes, Bernadette

2017-11-01

Alteration of the postnatal phenotype has sparked great concern about the developmental impact of culture media used at fertilization. However, the mechanisms and compounds involved are yet to be determined. Here, we used the Ca 2+ responses from mouse eggs fertilized by ICSI as a dynamic and quantitative marker to understand the role of compounds in egg functioning and establish possible correlations with adult phenotypes. We computed 134 Ca 2+ responses from the first to the last oscillation in media with specific formulations. Analyses demonstrate that eggs generated two times as many Ca 2+ oscillations in KSOM as in M16 media (18.8 ± 7.0 vs 9.2 ± 2.5). Moreover, the time increment of the delay between two consecutive oscillations, named TIbO, is the most sensitive coefficient characterizing the mechanism that paces Ca 2+ oscillations once the egg has been fertilized. Neither doubling external free Ca 2+ nor dispermic fertilization increased significantly the total number of Ca 2+ oscillations. In contrast, removing Mg 2+ from the M16 boosted Ca 2+ oscillations to 54.0 ± 35.2. Hence, [Mg 2+ ] o /[Ca 2+ ] o appears to determine the number, duration and frequency of the Ca 2+ oscillations. These changes were correlated with long-term effects. The rate of female's growth was impacted with the 'KSOM' females having only half the fat deposit of 'M16' females. Moreover, adult animals issued from M16 had significantly smaller brain weight vs 'KSOM' and 'control' animals. TIbO is a new Ca 2+ coefficient that gauges the very early functional impact of culture media. It offers the possibility of establishing correlations with postnatal consequences according to IVF medium formulation.Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/154/5/675/suppl/DC2. © 2017 Society for Reproduction and Fertility.

Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes

PubMed Central

Lauvrak, S U; Munthe, E; Kresse, S H; Stratford, E W; Namløs, H M; Meza-Zepeda, L A; Myklebost, O

2013-01-01

Background: Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations. Methods: To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling. Results: The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes—COL1A2, KYNU, ACTG2 and NPPB—were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease. Interpretation: The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is

MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

PubMed

Yang, Bo; Xu, Qiu-Yun; Guo, Chun-Yan; Huang, Jin-Wen; Wang, Shu-Mei; Li, Yong-Mei; Tu, Ying; He, Li; Bi, Zhi-Gang; Ji, Chao; Cheng, Bo

2017-02-21

Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

M-atrial natriuretic peptide and nitroglycerin in a canine model of experimental acute hypertensive heart failure: differential actions of 2 cGMP activating therapeutics.

PubMed

McKie, Paul M; Cataliotti, Alessandro; Ichiki, Tomoko; Sangaralingham, S Jeson; Chen, Horng H; Burnett, John C

2014-01-02

Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator. HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M-ANP (139 ± 4 to 118 ± 3 mm Hg, P<0.05) and nitroglycerin (137 ± 3 to 116 ± 4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M-ANP (12 ± 2 to 6 ± 2 mm Hg, P<0.05) and nitroglycerin (12 ± 1 to 6 ± 1 mm Hg, P<0.05). M-ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38 ± 4 to 53 ± 5 mL/min), renal blood flow (132 ± 18 to 236 ± 23 mL/min), and natriuresis (11 ± 4 to 689 ± 37 mEq/min) and also inhibited aldosterone activation (32 ± 3 to 23 ± 2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation. Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

PubMed Central

Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

2012-01-01

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

Differential S1P Receptor Profiles on M1- and M2-Polarized Macrophages Affect Macrophage Cytokine Production and Migration.

PubMed

Müller, Jan; von Bernstorff, Wolfram; Heidecke, Claus-Dieter; Schulze, Tobias

2017-01-01

Introduction . Macrophages are key players in complex biological processes. In response to environmental signals, macrophages undergo polarization towards a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that acts via 5 G-protein coupled receptors (S1P 1-5 ) in order to influence a broad spectrum of biological processes. This study assesses S1P receptor expression on macrophages before and after M1 and M2 polarization and performs a comparative analysis of S1P signalling in the two activational states of macrophages. Methods . Bone marrow derived macrophages (BMDM) from C57 BL/6 mice were cultured under either M1- or M2-polarizing conditions. S1P-receptor expression was determined by quantitative RT-PCR. Influence of S1P on macrophage activation, migration, phagocytosis, and cytokine secretion was assessed in vitro. Results . All 5 S1P receptor subclasses were expressed in macrophages. Culture under both M1- and M2-polarizing conditions led to significant downregulation of S1P 1 . In contrast, M1-polarized macrophages significantly downregulated S1P 4 . The expression of the remaining three S1P receptors did not change. S1P increased expression of iNOS under M2-polarizing conditions. Furthermore, S1P induced chemotaxis in M1 macrophages and changed cytokine production in M2 macrophages. Phagocytosis was not affected by S1P-signalling. Discussion . The expression of different specific S1P receptor profiles may provide a possibility to selectively influence M1- or M2-polarized macrophages.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation*

PubMed Central

Walker, Natalie M.; Belloli, Elizabeth A.; Stuckey, Linda; Chan, Kevin M.; Lin, Jules; Lynch, William; Chang, Andrew; Mazzoni, Serina M.; Fingar, Diane C.; Lama, Vibha N.

2016-01-01

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states. PMID:26755732

RIPK3/MLKL-Mediated Neuronal Necroptosis Modulates the M1/M2 Polarization of Microglia/Macrophages in the Ischemic Cortex

PubMed Central

Yang, Jiping; Zhao, Youyi; Zhang, Li; Fan, Hong; Qi, Chuchu; Zhang, Kun; Liu, Xinyu; Fei, Lin; Chen, Siwei; Wang, Mengmeng; Kuang, Fang; Wang, Yazhou; Wu, Shengxi

2018-01-01

Abstract Cell death and subsequent inflammation are 2 key pathological changes occurring in cerebral ischemia. Active microglia/macrophages play a double-edged role depending on the balance of their M1/M2 phenotypes. Necrosis is the predominant type of cell death following ischemia. However, how necrotic cells modulate the M1/M2 polarization of microglia/macrophages remains poorly investigated. Here, we reported that ischemia induces a rapid RIPK3/MLKL-mediated neuron-dominated necroptosis, a type of programmed necrosis. Ablating RIPK3 or MLKL could switch the activation of microglia/macrophages from M1 to the M2 type in the ischemic cortex. Conditioned medium of oxygen-glucose deprivation (OGD)-treated wild-type (WT) neurons induced M1 polarization, while that of RIPK3−/− neurons favored M2 polarization. OGD treatment induces proinflammatory IL-18 and TNFα in WT but not in RIPK3−/− neurons, which in turn upregulate anti-inflammatory IL-4 and IL-10. Furthermore, the expression of Myd88—a common downstream adaptor of toll-like receptors—is significantly upregulated in the microglia/macrophages of ischemic WT but not of RIPK3−/− or MLKL−/− cortices. Antagonizing the function of Myd88 could phenocopy the effects of RIPK3/MLKL-knockout on the polarization of microglia/macrophages and was neuroprotective. Our data revealed a novel role of necroptotic neurons in modulating the M1/M2 balance of microglia/macrophages in the ischemic cortex, possibly through Myd88 signaling. PMID:29746630

Three Hypothetical Inflammation Pathobiology Phenotypes and Pediatric Sepsis-Induced Multiple Organ Failure Outcome.

PubMed

Carcillo, Joseph A; Halstead, E Scott; Hall, Mark W; Nguyen, Trung C; Reeder, Ron; Aneja, Rajesh; Shakoory, Bita; Simon, Dennis

2017-06-01

We hypothesize that three inflammation pathobiology phenotypes are associated with increased inflammation, proclivity to develop features of macrophage activation syndrome, and multiple organ failure-related death in pediatric severe sepsis. Prospective cohort study comparing children with severe sepsis and any of three phenotypes: 1) immunoparalysis-associated multiple organ failure (whole blood ex vivo tumor necrosis factor response to endotoxin < 200 pg/mL), 2) thrombocytopenia-associated multiple organ failure (new onset thrombocytopenia with acute kidney injury and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 activity < 57%), and/or 3) sequential multiple organ failure with hepatobiliary dysfunction (respiratory distress followed by liver dysfunction with soluble Fas ligand > 200 pg/mL), to those without any of these phenotypes. Tertiary children's hospital PICU. One hundred consecutive severe sepsis admissions. Clinical data were recorded daily, and blood was collected twice weekly. Multiple organ failure developed in 75 cases and eight died. Multiple organ failure cases with any of the three inflammation phenotypes (n = 37) had higher inflammation (C-reactive protein, p = 0.009 and ferritin, p < 0.001) than multiple organ failure cases without any of these phenotypes (n = 38) or cases with only single organ failure (n = 25). Development of features of macrophage activation syndrome and death were more common among multiple organ failure cases with any of the phenotypes (macrophage activation syndrome: 10/37, 27%; death: 8/37, 22%) compared to multiple organ failure cases without any phenotype (macrophage activation syndrome: 1/38, 3%; p = 0.003 and death: 0/38, 0%; p = 0.002). Our approach to phenotype categorization remains hypothetical, and the phenotypes identified need to be confirmed in multicenter studies of pediatric multiple organ dysfunction syndrome.

Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

PubMed

Siemens, Nikolai; Kreikemeyer, Bernd

2013-01-01

Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS). In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i) which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii) is Ralp3 actively lost during evolution in these serotypes. We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

mNos2 deletion and human NOS2 replacement in Alzheimer disease models.

PubMed

Colton, Carol A; Wilson, Joan G; Everhart, Angela; Wilcock, Donna M; Puoliväli, Jukka; Heikkinen, Taneli; Oksman, Juho; Jääskeläinen, Olli; Lehtimäki, Kimmo; Laitinen, Teemu; Vartiainen, Nina; Vitek, Michael P

2014-08-01

Understanding the pathophysiologic mechanisms underlying Alzheimer disease relies on knowledge of disease onset and the sequence of development of brain pathologies. We present a comprehensive analysis of early and progressive changes in a mouse model that demonstrates a full spectrum of characteristic Alzheimer disease-like pathologies. This model demonstrates an altered immune redox state reminiscent of the human disease and capitalizes on data indicating critical differences between human and mouse immune responses, particularly in nitric oxide levels produced by immune activation of the NOS2 gene. Using the APPSwDI(+)/(+)mNos2(-/-) (CVN-AD) mouse strain, we show a sequence of pathologic events leading to neurodegeneration,which include pathologically hyperphosphorylated tau in the perforant pathway at 6 weeks of age progressing to insoluble tau, early appearance of β-amyloid peptides in perivascular deposits around blood vessels in brain regions known to be vulnerable to Alzheimer disease, and progression to damage and overt loss in select vulnerable neuronal populations in these regions. The role of species differences between hNOS2 and mNos2 was supported by generating mice in which the human NOS2 gene replaced mNos2. When crossed with CVN-AD mice, pathologic characteristics of this new strain (APPSwDI(+)/(-)/HuNOS2(tg+)/(+)/mNos2(-/-)) mimicked the pathologic phenotypes found in the CVN-AD strain.

Correlation of beta-catenin localization with cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in colorectal cancer.

PubMed

Kawasaki, Takako; Nosho, Katsuhiko; Ohnishi, Mutsuko; Suemoto, Yuko; Kirkner, Gregory J; Dehari, Reiko; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

2007-07-01

The WNT/beta-catenin (CTNNB1) pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2)/prostaglandin pathways have been suggested. The relationship between beta-catenin activation and microsatellite instability (MSI) in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between beta-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed beta-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear beta-catenin expressions (i.e., beta-catenin activation) and associated positively with membrane expression. The inverse relation between beta-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic beta-catenin expression (even after tumors were stratified by CIMP status), but did not correlate significantly with nuclear or membrane expression. In conclusion, beta-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic beta-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic beta-catenin in stabilizing PTGS2 (COX-2) mRNA.

Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

PubMed Central

2016-01-01

The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling

PubMed Central

Yang, Bo; Xu, Qiu-Yun; Guo, Chun-Yan; Huang, Jin-Wen; Wang, Shu-Mei; Li, Yong-Mei; Tu, Ying; He, Li; Bi, Zhi-Gang; Ji, Chao; Cheng, Bo

2017-01-01

Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling. PMID:28061443

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2

PubMed Central

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim

2015-01-01

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2−/− mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2−/− mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2−/− mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2−/− mice. Our results demonstrate that Eif4ebp2−/− mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. SIGNIFICANCE STATEMENT Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2.

PubMed

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-08-05

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2(-/-) mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2(-/-) mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2(-/-) mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2(-/-) mice. Our results demonstrate that Eif4ebp2(-/-) mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E

A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

PubMed Central

Kato, Satoshi; Tomita, Katsuro; Titus, Louisa; Boden, Scott D.

2011-01-01

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1−/− knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. PMID:21110071

A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

PubMed

Kato, Satoshi; Sangadala, Sreedhara; Tomita, Katsuro; Titus, Louisa; Boden, Scott D

2011-03-01

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β.

PubMed

Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Sha'ri, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

2017-12-01

The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

PubMed

Lee, Ok Ran; Cho, Hyung-Taeg

2012-12-01

Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

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

Reimers, Kerstin; Buchholz, Katja; Werchau, Hermann

2005-01-20

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cellsmore » revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.« less

The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2

PubMed Central

Chen, Bo; Xu, Ming; Zhang, Hui; Xu, Ming-zheng; Wang, Xu-jing; Tang, Qing-he

2015-01-01

In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK–mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer. PMID:26284306

The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2.

PubMed

Chen, Bo; Xu, Ming; Zhang, Hui; Xu, Ming-zheng; Wang, Xu-jing; Tang, Qing-he; Tang, Jian-ying

2015-10-01

In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells both in vitro and in vivo. We demonstrated that OSI-027 inhibited survival and growth of both primary and transformed (PANC-1 and MIA PaCa-2 lines) human pancreatic cancer cells. Meanwhile, OSI-027 induced caspase-dependent apoptotic death of the pancreatic cancer cells. On the other hand, caspase inhibitors alleviated cytotoxicity by OSI-027. At the molecular level, OSI-027 treatment blocked mTORC1 and mTORC2 activation simultaneously, without affecting ERK-mitogen-activated protein kinase activation. Importantly, OSI-027 activated cytoprotective autophagy in the above cancer cells. Whereas pharmacological blockage of autophagy or siRNA knockdown of Beclin-1 significantly enhanced the OSI-027-induced activity against pancreatic cancer cells. Specifically, a relatively low dose of OSI-027 sensitized gemcitabine-induced pancreatic cancer cell death in vitro. Further, administration of OSI-027 or together with gemcitabine dramatically inhibited PANC-1 xenograft growth in severe combined immunodeficiency mice, leading to significant mice survival improvement. In summary, the preclinical results of this study suggest that targeting mTORC1/2 synchronously by OSI-027 could be further investigated as a valuable treatment for pancreatic cancer.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

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

Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.

2012-08-26

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface,more » a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.« less

Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages

PubMed Central

Montana, Giovanna

2016-01-01

Substance P (SP) is a neuropeptide that mediates many physiological as well as inflammatory responses. Recently, SP has been implicated in the resolution of inflammation through induction of M2 macrophages phenotype. The shift between M1-like and M2-like, allowing the resolution of inflammatory processes, also takes place by means of hemeoxygenase-1 (HO-1). HO-1 is induced in response to oxidative stress and inflammatory stimuli and modulates the immune response through macrophages polarisation. SP induces HO-1 expression in human periodontal ligament (PDL), the latter potentially plays a role in cytoprotection. We demonstrated that SP promotes M2-like phenotype from resting as well as from M1 macrophages. Indeed, SP triggers the production of interleukine-10 (IL-10), interleukine-4 (IL-4) and arginase-1 (Arg1) without nitric oxide (NO) generation. In addition, SP increases HO-1 expression in a dose- and time-dependent manner. Here we report that SP, without affecting cell viability, significantly reduces the production of pro-inflammatory cytokines and enzymes, such as tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and ameliorates migration and phagocytic properties in LPS-stimulated RAW 264.7 cells. M2-like conversion required retention of NF-κB p65 into the cytoplasm and HO-1 induced expression. Silencing of the HO-1 mRNA expression reversed the induction of pro-inflammatory cytokines in RAW 264.7 stimulated by LPS and down-regulated anti-inflammatory hallmarks of M2 phenotype. In conclusion, our data show that SP treatment might be associated with anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression. PMID:27907187

Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

PubMed

Scherrer, A; Kruithof, E K; Grob, J P

1991-06-01

Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

PubMed Central

Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

2016-01-01

Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

Fibronectin type III domain containing 5 attenuates NLRP3 inflammasome activation and phenotypic transformation of adventitial fibroblasts in spontaneously hypertensive rats.

PubMed

Ling, Li; Chen, Dan; Tong, Ying; Zang, Ying-Hao; Ren, Xing-Sheng; Zhou, Hong; Qi, Xiao-Hong; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

2018-05-01

Phenotypic transformation of adventitial fibroblasts is important in the pathogenesis of hypertension. This study was designed to determine whether fibronectin type III domain containing 5 (FNDC5) alleviates the phenotypic transformation of adventitial fibroblasts in hypertension and the underlying mechanisms. Experiments were carried out in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and primary aortic adventitial fibroblasts. FNDC5 was downregulated and NLRP3 inflammasome was activated in aortic adventitia of SHR. FNDC5 overexpression attenuated adventitial fibroblasts phenotypic transformation, excessive synthesis and secretion of matrix components, NLRP3 inflammasome activation and inflammation in adventitial fibroblasts from SHR. Moreover, FNDC5 overexpression reduced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production in adventitial fibroblasts from SHR. Similarly, exogenous FNDC5 inhibited adventitial fibroblasts phenotypic transformation, expression of matrix components, NLRP3 inflammasome activation and NOX2 expression in adventitial fibroblasts from SHR. FNDC5 overexpression in rats attenuated phenotypic transformation, inflammation and reactive oxygen species (ROS) production in the aortic adventitia of SHR. Furthermore, FNDC5 overexpression reduced blood pressure and alleviated vascular remodeling in SHR. FNDC5 reduces NOX2-derived ROS production, NLRP3 inflammasome activation and phenotypic transformation in adventitial fibroblasts of SHR. FNDC5 plays a beneficial role in attenuating vascular inflammation, vascular remodeling and hypertension in SHR.

Variants of the ankyrin repeat domain 6 gene (ANKRD6) and muscle and physical activity phenotypes among European-derived American adults.

PubMed

Van Deveire, Katherine N; Scranton, Sarah K; Kostek, Mathew A; Angelopoulos, Theodore J; Clarkson, Priscilla M; Gordon, Paul M; Moyna, Niall M; Visich, Paul S; Zoeller, Robert F; Thompson, Paul D; Devaney, Joseph M; Gordish-Dressman, Heather; Hoffman, Eric P; Maresh, Carl M; Pescatello, Linda S

2012-07-01

Ankyrin repeat domain 6 (ANKRD6) is a ubiquitous protein that associates with early development in mammals and is highly expressed in the brain, spinal cord, and heart of humans. We examined the role of 8 ANKRD6 single-nucleotide polymorphisms (SNPs) on muscle performance and habitual physical activity (PA). Single-nucleotide polymorphisms were 545 T>A (rs9362667), 485 M>L (rs61736690), 233 T>M (rs2273238), 128 I>L (rs3748085), 631 P>L (rs61739327), 122 Q>E (rs16881983), 197805 G>A (rs9344950), and 710 L>X (NOVEL). This study consisted of 922 healthy, untrained, European-derived American men (n = 376, 23.6 ± 0.3 years, 25.0 ± 0.2 kg·m(-2)) and women (n = 546, 23.2 ± 0.2 years, 24.0 ± 0.2 kg·m(-2)). Muscle strength (maximum voluntary contraction [MVC] and 1 repetition maximum [1RM]) and size (cross-sectional area [CSA]) were assessed before and after 12 weeks of unilateral resistance training (RT). A subsample (n = 536, 23.4 ± 0.2 years, 24.6 ± 0.2 kg·m(-2)) completed the Paffenbarger Physical Activity Questionnaire. Associations among ANKRD6 genotypes and muscle phenotypes were tested with repeated measure analysis of covariance (ANCOVA) and PA phenotypes with multivariate ANCOVA, with age and body mass index as covariates. ANKRD6 122 Q>E was associated with increased baseline biceps CSA. ANKRD6 545 A>T and ANKRD6 710 L>X were associated with increased 1RM and MVC in response to RT, respectively. ANKRD6 631 P>L was associated with increased biceps CSA response to RT and time spent in moderate-intensity PA among the total sample and women. ANKRD6 genetic variants were associated with the muscle size and strength response to RT and habitual PA levels. Further research is needed to validate our results and explore mechanisms for the associations we observed.

Secondary hyperalgesia phenotypes exhibit differences in brain activation during noxious stimulation.

PubMed

Asghar, Mohammad Sohail; Pereira, Manuel Pedro; Werner, Mads Utke; Mårtensson, Johan; Larsson, Henrik B W; Dahl, Jørgen Berg

2015-01-01

Noxious stimulation of the skin with either chemical, electrical or heat stimuli leads to the development of primary hyperalgesia at the site of injury, and to secondary hyperalgesia in normal skin surrounding the injury. Secondary hyperalgesia is inducible in most individuals and is attributed to central neuronal sensitization. Some individuals develop large areas of secondary hyperalgesia (high-sensitization responders), while others develop small areas (low-sensitization responders). The magnitude of each area is reproducible within individuals, and can be regarded as a phenotypic characteristic. To study differences in the propensity to develop central sensitization we examined differences in brain activity and anatomy according to individual phenotypical expression of secondary hyperalgesia by magnetic resonance imaging. Forty healthy volunteers received a first-degree burn-injury (47 °C, 7 min, 9 cm(2)) on the non-dominant lower-leg. Areas of secondary hyperalgesia were assessed 100 min after the injury. We measured neuronal activation by recording blood-oxygen-level-dependent-signals (BOLD-signals) during mechanical noxious stimulation before burn injury and in both primary and secondary hyperalgesia areas after burn-injury. In addition, T1-weighted images were used to measure differences in gray-matter density in cortical and subcortical regions of the brain. We found significant differences in neuronal activity between high- and low-sensitization responders at baseline (before application of the burn-injury) (p < 0.05). After the burn-injury, we found significant differences between responders during noxious stimulation of both primary (p < 0.01) and secondary hyperalgesia (p ≤ 0.04) skin areas. A decreased volume of the right (p = 0.001) and left caudate nucleus (p = 0.01) was detected in high-sensitization responders in comparison to low-sensitization responders. These findings suggest that brain-structure and neuronal activation to noxious

Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

PubMed Central

Jetten, Nadine; Roumans, Nadia; Gijbels, Marion J.; Romano, Andrea; Post, Mark J.; de Winther, Menno P. J.; van der Hulst, Rene R. W. J.; Xanthoulea, Sofia

2014-01-01

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds. PMID:25068282

Phenotypic variability in patients with ADA2 deficiency due to identical homozygous R169Q mutations.

PubMed

Van Montfrans, Joris M; Hartman, Esther A R; Braun, Kees P J; Hennekam, Eric A M; Hak, Elisabeth A; Nederkoorn, Paul J; Westendorp, Willeke F; Bredius, Robbert G M; Kollen, Wouter J W; Schölvinck, Elisabeth H; Legger, G Elizabeth; Meyts, Isabelle; Liston, Adrian; Lichtenbelt, Klaske D; Giltay, Jacques C; Van Haaften, Gijs; De Vries Simons, Gaby M; Leavis, Helen; Sanders, Cornelis J G; Bierings, Marc B; Nierkens, Stefan; Van Gijn, Marielle E

2016-05-01

To determine the genotype-phenotype association in patients with adenosine deaminase-2 (ADA2) deficiency due to identical homozygous R169Q mutations inCECR1 METHODS: We present a case series of nine ADA2-deficient patients with an identical homozygous R169Q mutation. Clinical and diagnostic data were collected and available MRI studies were reviewed. We performed genealogy and haplotype analyses and measured serum ADA2 activity. ADA2 activity values were correlated to clinical symptoms. Age of presentation differed widely between the nine presented patients (range: 0 months to 8 years). The main clinical manifestations were (hepato)splenomegaly (8/9), skin involvement (8/9) and neurological involvement (8/9, of whom 6 encountered stroke). Considerable variation was seen in type, frequency and intensity of other symptoms, which included aplastic anaemia, acute myeloid leukaemia and cutaneous ulcers. Common laboratory abnormalities included cytopenias and hypogammaglobulinaemia. ADA2 enzyme activity in patients was significantly decreased compared with healthy controls. ADA2 activity levels tended to be lower in patients with stroke compared with patients without stroke. Genealogical studies did not identify a common ancestor; however, based on allele frequency, a North-West European founder effect can be noted. Three patients underwent haematopoietic cell transplantation, after which ADA2 activity was restored and clinical symptoms resolved. This case series revealed large phenotypic variability in patients with ADA2 deficiency though they were homozygous for the same R169Q mutation inCECR1 Disease modifiers, including epigenetic and environmental factors, thus seem important in determining the phenotype. Furthermore, haematopoietic cell transplantation appears promising for those patients with a severe clinical phenotype. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions

Galectin-3 in M2 macrophages plays a protective role in resolution of neuropathology in brain parasitic infection by regulating neutrophil turnover.

PubMed

Quenum Zangbede, Fredice O; Chauhan, Arun; Sharma, Jyotika; Mishra, Bibhuti B

2018-06-26

Macrophages/microglia with M2- activation phenotype are thought to play an important anti-inflammatory and tissue reparative functions in the brain, yet the molecular basis of their functions in the central nervous system (CNS) remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti , we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3 -/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3 deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in-vitro. Furthermore, adoptive transfer of M2 macrophages from Galectin-3 sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3 -/- mice. Together, these results demonstrate for the first time a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuro-inflammatory diseases. SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell protective functions in the CNS microenvironment are undefined. Quenum Zangbede et. al., report that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models

A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment

PubMed Central

Shiran, M R; Chowdry, J; Rostami-Hodjegan, A; Ellis, S W; Lennard, M S; Iqbal, M Z; Lagundoye, O; Seivewright, N; Tucker, G T

2003-01-01

Aims To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT). Methods Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma. Results Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity. Conclusions There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients. PMID:12895196

Overexpression of IL-10 in C2D macrophages promotes a macrophage phenotypic switch in adipose tissue environments.

PubMed

Xie, Linglin; Fu, Qiang; Ortega, Teresa M; Zhou, Lun; Rasmussen, Dane; O'Keefe, Jacy; Zhang, Ke K; Chapes, Stephen K

2014-01-01

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206(+), CD301(+), CD11c(-)CD206(+) (M2) and CD11c(+)CD206(+) (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.

Nanostructured TiO2 surfaces promote polarized activation of microglia, but not astrocytes, toward a proinflammatory profile

NASA Astrophysics Data System (ADS)

de Astis, Silvia; Corradini, Irene; Morini, Raffaella; Rodighiero, Simona; Tomasoni, Romana; Lenardi, Cristina; Verderio, Claudia; Milani, Paolo; Matteoli, Michela

2013-10-01

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The classic activation state (M1) is characterized by high capacity to present antigens, high production of nitric oxide (NO) and reactive oxygen species (ROS) and proinflammatory cytokines. Classically activated cells act as potent effectors that drive the inflammatory response and may mediate detrimental effects on neural cells. The second phenotype (M2) is an alternative, apparently beneficial, activation state, more related to a fine tuning of inflammation, scavenging of debris, promotion of angiogenesis, tissue remodeling and repair. Specific environmental chemical signals are able to induce these different polarization states. We provide here evidence that nanostructured substrates are able, exclusively in virtue of their physical properties, to push microglia toward the proinflammatory activation phenotype, with an efficacy which reflects the graded nanoscale rugosity. The acquisition of a proinflammatory phenotype appears specific for microglia and not astrocytes, indicating that these two cell types, although sharing common innate immune responses, respond differently to external physical stimuli.

VEGF may contribute to macrophage recruitment and M2 polarization in the decidua.

PubMed

Wheeler, Karen C; Jena, Manoj K; Pradhan, Bhola S; Nayak, Neha; Das, Subhendu; Hsu, Chaur-Dong; Wheeler, David S; Chen, Kang; Nayak, Nihar R

2018-01-01

It is increasingly evident that cytokines and growth factors produced in the decidua play a pivotal role in the regulation of the local immune microenvironment and the establishment of pregnancy. One of the major growth factors produced in the decidua is vascular endothelial growth factor (VEGF), which acts not only on endothelial cells, but also on multiple other cell types, including macrophages. We sought to determine whether decidua-derived VEGF affects macrophage recruitment and polarization using human endometrial/decidual tissue samples, primary human endometrial stromal cells (ESCs), and the human monocyte cell line THP1. In situ hybridization was used for assessment of local VEGF expression and immunohistochemistry was used for identification and localization of CD68-positive endometrial macrophages. Macrophage migration in culture was assessed using a transwell migration assay, and the various M1/M2 phenotypic markers and VEGF expression were assessed using quantitative real-time PCR (qRT-PCR). We found dramatic increases in both VEGF levels and macrophage numbers in the decidua during early pregnancy compared to the secretory phase endometrium (non-pregnant), with a significant increase in M2 macrophage markers, suggesting that M2 is the predominant macrophage phenotype in the decidua. However, decidual samples from preeclamptic pregnancies showed a significant shift in macrophage phenotype markers, with upregulation of M1 and downregulation of M2 markers. In THP1 cultures, VEGF treatment significantly enhanced macrophage migration and induced M1 macrophages to shift to an M2 phenotype. Moreover, treatment with conditioned media from decidualized ESCs induced changes in macrophage migration and polarization similar to that of VEGF treatment. These effects were abrogated by the addition of a potent VEGF inhibitor. Together these results suggest that decidual VEGF plays a significant role in macrophage recruitment and M2 polarization, and that inhibition

VEGF may contribute to macrophage recruitment and M2 polarization in the decidua

PubMed Central

Nayak, Neha; Das, Subhendu; Hsu, Chaur-Dong; Wheeler, David S.; Chen, Kang; Nayak, Nihar R.

2018-01-01

It is increasingly evident that cytokines and growth factors produced in the decidua play a pivotal role in the regulation of the local immune microenvironment and the establishment of pregnancy. One of the major growth factors produced in the decidua is vascular endothelial growth factor (VEGF), which acts not only on endothelial cells, but also on multiple other cell types, including macrophages. We sought to determine whether decidua-derived VEGF affects macrophage recruitment and polarization using human endometrial/decidual tissue samples, primary human endometrial stromal cells (ESCs), and the human monocyte cell line THP1. In situ hybridization was used for assessment of local VEGF expression and immunohistochemistry was used for identification and localization of CD68-positive endometrial macrophages. Macrophage migration in culture was assessed using a transwell migration assay, and the various M1/M2 phenotypic markers and VEGF expression were assessed using quantitative real-time PCR (qRT-PCR). We found dramatic increases in both VEGF levels and macrophage numbers in the decidua during early pregnancy compared to the secretory phase endometrium (non-pregnant), with a significant increase in M2 macrophage markers, suggesting that M2 is the predominant macrophage phenotype in the decidua. However, decidual samples from preeclamptic pregnancies showed a significant shift in macrophage phenotype markers, with upregulation of M1 and downregulation of M2 markers. In THP1 cultures, VEGF treatment significantly enhanced macrophage migration and induced M1 macrophages to shift to an M2 phenotype. Moreover, treatment with conditioned media from decidualized ESCs induced changes in macrophage migration and polarization similar to that of VEGF treatment. These effects were abrogated by the addition of a potent VEGF inhibitor. Together these results suggest that decidual VEGF plays a significant role in macrophage recruitment and M2 polarization, and that inhibition

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method.

PubMed

Kotrotsiou, Tzimoula; Tzimoula, Kotrotsiou; Exindari, Maria; Maria, Exindari; Diza, Eudoxia; Eudoxia, Diza; Gioula, Georgia; Georgia, Gioula; Melidou, Angeliki; Angeliki, Melidou; Malisiovas, Nikolaos; Nikolaos, Malisiovas

2015-02-01

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene. Copyright © 2015 Elsevier Inc. All rights reserved.

Macrophage Phenotypes Regulate Scar Formation and Chronic Wound Healing.

PubMed

Hesketh, Mark; Sahin, Katherine B; West, Zoe E; Murray, Rachael Z

2017-07-17

Macrophages and inflammation play a beneficial role during wound repair with macrophages regulating a wide range of processes, such as removal of dead cells, debris and pathogens, through to extracellular matrix deposition re-vascularisation and wound re-epithelialisation. To perform this range of functions, these cells develop distinct phenotypes over the course of wound healing. They can present with a pro-inflammatory M1 phenotype, more often found in the early stages of repair, through to anti-inflammatory M2 phenotypes that are pro-repair in the latter stages of wound healing. There is a continuum of phenotypes between these ranges with some cells sharing phenotypes of both M1 and M2 macrophages. One of the less pleasant consequences of quick closure, namely the replacement with scar tissue, is also regulated by macrophages, through their promotion of fibroblast proliferation, myofibroblast differentiation and collagen deposition. Alterations in macrophage number and phenotype disrupt this process and can dictate the level of scar formation. It is also clear that dysregulated inflammation and altered macrophage phenotypes are responsible for hindering closure of chronic wounds. The review will discuss our current knowledge of macrophage phenotype on the repair process and how alterations in the phenotypes might alter wound closure and the final repair quality.

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

PubMed Central

Puustinen, Pietri; Rytter, Anna; Mortensen, Monika; Kohonen, Pekka; Moreira, José M.

2014-01-01

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation. PMID:24590173

Activation of mTORC1/mTORC2 signaling in pediatric low-grade glioma and pilocytic astrocytoma reveals mTOR as a therapeutic target

PubMed Central

Hütt-Cabezas, Marianne; Karajannis, Matthias A.; Zagzag, David; Shah, Smit; Horkayne-Szakaly, Iren; Rushing, Elisabeth J.; Cameron, J. Douglas; Jain, Deepali; Eberhart, Charles G.; Raabe, Eric H.; Rodriguez, Fausto J.

2013-01-01

Background Previous studies support a role for mitogen-activated protein kinase pathway signaling, and more recently Akt/mammalian target of rapamycin (mTOR), in pediatric low-grade glioma (PLGG), including pilocytic astrocytoma (PA). Here we further evaluate the role of the mTORC1/mTORC2 pathway in order to better direct pharmacologic blockade in these common childhood tumors. Methods We studied 177 PLGGs and PAs using immunohistochemistry and tested the effect of mTOR blockade on 2 PLGG cell lines (Res186 and Res259) in vitro. Results Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 107/177 (59%) PAs and other PLGGs, while p4EBP1 was observed in 35/115 (30%), pElF4G in 66/112 (59%), mTOR (total) in 53/113 (47%), RAPTOR (mTORC1 component) in 64/102 (63%), RICTOR (mTORC2 component) in 48/101 (48%), and pAkt (S473) in 63/103 (61%). Complete phosphatase and tensin homolog protein loss was identified in only 7/101 (7%) of cases. In PA of the optic pathways, compared with other anatomic sites, there was increased immunoreactivity for pS6, pElF4G, mTOR (total), RICTOR, and pAkt (P < .05). We also observed increased pS6 (P = .01), p4EBP1 (P = .029), and RICTOR (P = .05) in neurofibromatosis type 1 compared with sporadic tumors. Treatment of the PLGG cell lines Res186 (PA derived) and Res259 (diffuse astrocytoma derived) with the rapalog MK8669 (ridaforolimus) led to decreased mTOR pathway activation and growth. Conclusions These findings suggest that the mTOR pathway is active in PLGG but varies by clinicopathologic subtype. Additionally, our data suggest that mTORC2 is differentially active in optic pathway and neurofibromatosis type 1–associated gliomas. MTOR represents a potential therapeutic target in PLGG that merits further investigation. PMID:24203892

uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

PubMed

Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario

2017-09-15

In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma. © 2017 UICC.

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

PubMed Central

Luong, Preston M.; Shogan, Benjamin D.; Zaborin, Alexander; Belogortseva, Natalia; Shrout, Joshua D.

2014-01-01

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF. PMID:24244000

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

PubMed

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

2016-10-04

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

(Epi)genotype–phenotype correlations in Beckwith–Wiedemann syndrome

PubMed Central

Mussa, Alessandro; Russo, Silvia; De Crescenzo, Agostina; Freschi, Andrea; Calzari, Luciano; Maitz, Silvia; Macchiaiolo, Marina; Molinatto, Cristina; Baldassarre, Giuseppina; Mariani, Milena; Tarani, Luigi; Bedeschi, Maria Francesca; Milani, Donatella; Melis, Daniela; Bartuli, Andrea; Cubellis, Maria Vittoria; Selicorni, Angelo; Cirillo Silengo, Margherita; Larizza, Lidia; Riccio, Andrea; Ferrero, Giovanni Battista

2016-01-01

Beckwith–Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype–phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms' tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype–phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening. PMID:25898929

The effect of mineral trioxide aggregate on phagocytic activity and production of reactive oxygen, nitrogen species and arginase activity by M1 and M2 macrophages.

PubMed

Rezende, T M B; Vieira, L Q; Cardoso, F P; Oliveira, R R; de Oliveira Mendes, S T; Jorge, M L R; Ribeiro Sobrinho, A P

2007-08-01

To assess the influence of co-culture with mineral trioxide aggregate (MTA) on phagocytosis and the production of reactive oxygen intermediates (ROI) and nitrogen (NO) species and the arginase activity by M1 and M2 peritoneal macrophages. Cellular viability, adherence and phagocytosis of Saccharomyces boulardii were assayed in the presence of MTA. Macrophages were stimulated with zymosan for ROI assays and with Fusobacterium nucleatum and Peptostreptococcus anaerobius and IFN-gamma for NO production and arginase activity, when in contact with capillaries containing MTA. Data were analysed by T, anova, Kruskall-Wallis and Mann-Whitney tests. M2 macrophages displayed greater cellular viability in polypropylene tubes, greater ability to ingest yeast and smaller production of ROI and higher arginase activity when compared with M1 macrophages. Both macrophages, M1 and M2, presented similar cell adherence and NO production. The addition of bacterial preparations to macrophages interfered with NO and arginase productions. MTA did not interfere with any of the parameters measured. Phagocytosis and the ability of the two macrophage subtypes to eliminate microbes were not affected by MTA.

Atherosclerosis following renal injury is ameliorated by pioglitazone and losartan via macrophage phenotype

PubMed Central

Yamamoto, Suguru; Zhong, Jiayong; Yancey, Patricia G.; Zuo, Yiqin; Linton, MacRae F.; Fazio, Sergio; Yang, Haichun; Narita, Ichiei; Kon, Valentina

2016-01-01

Objective Chronic kidney disease (CKD) amplifies atherosclerosis, which involves renin-angiotensin system (RAS) regulation of macrophages. RAS influences peroxisome proliferator-activated receptor-γ (PPARγ), a modulator of atherogenic functions of macrophages, however, little is known about its effects in CKD. We examined the impact of combined therapy with a PPARγ agonist and angiotensin receptor blocker on atherogenesis in a murine uninephrectomy model. Methods Apolipoprotein E knockout mice underwent uninephrectomy (UNx) and treatment with pioglitazone (UNx + Pio), losartan (UNx + Los), or both (UNx + Pio/Los) for 10 weeks. Extent and characteristics of atherosclerotic lesions and macrophage phenotypes were assessed; RAW264.7 and primary peritoneal mouse cells were used to examine pioglitazone and losartan effects on macrophage phenotype and inflammatory response. Results UNx significantly increased atherosclerosis. Pioglitazone and losartan each significantly reduced the atherosclerotic burden by 29.6% and 33.5%, respectively; although the benefit was dramatically augmented by combination treatment which lessened atherosclerosis by 55.7%. Assessment of plaques revealed significantly greater macrophage area in UNx + Pio/Los (80.7 ± 11.4% vs. 50.3 ± 4.2% in UNx + Pio and 57.2 ± 6.5% in UNx + Los) with more apoptotic cells. The expanded macrophage-rich lesions of UNx + Pio/Los had more alternatively activated, Ym-1 and arginine 1-positive M2 phenotypes (Ym-1: 33.6 ± 8.2%, p < 0.05 vs. 12.0 ± 1.1% in UNx; arginase 1: 27.8 ± 0.9%, p < 0.05 vs. 11.8 ± 1.3% in UNx). In vitro, pioglitazone alone and together with losartan was more effective than losartan alone in dampening lipopolysaccharide-induced cytokine production, suppressing M1 phenotypic change while enhancing M2 phenotypic change. Conclusion Combination of pioglitazone and losartan is more effective in reducing renal injury-induced atherosclerosis than either treatment alone. This benefit reflects

Atherosclerosis following renal injury is ameliorated by pioglitazone and losartan via macrophage phenotype.

PubMed

Yamamoto, Suguru; Zhong, Jiayong; Yancey, Patricia G; Zuo, Yiqin; Linton, MacRae F; Fazio, Sergio; Yang, Haichun; Narita, Ichiei; Kon, Valentina

2015-09-01

Chronic kidney disease (CKD) amplifies atherosclerosis, which involves renin-angiotensin system (RAS) regulation of macrophages. RAS influences peroxisome proliferator-activated receptor-γ (PPARγ), a modulator of atherogenic functions of macrophages, however, little is known about its effects in CKD. We examined the impact of combined therapy with a PPARγ agonist and angiotensin receptor blocker on atherogenesis in a murine uninephrectomy model. Apolipoprotein E knockout mice underwent uninephrectomy (UNx) and treatment with pioglitazone (UNx + Pio), losartan (UNx + Los), or both (UNx + Pio/Los) for 10 weeks. Extent and characteristics of atherosclerotic lesions and macrophage phenotypes were assessed; RAW264.7 and primary peritoneal mouse cells were used to examine pioglitazone and losartan effects on macrophage phenotype and inflammatory response. UNx significantly increased atherosclerosis. Pioglitazone and losartan each significantly reduced the atherosclerotic burden by 29.6% and 33.5%, respectively; although the benefit was dramatically augmented by combination treatment which lessened atherosclerosis by 55.7%. Assessment of plaques revealed significantly greater macrophage area in UNx + Pio/Los (80.7 ± 11.4% vs. 50.3 ± 4.2% in UNx + Pio and 57.2 ± 6.5% in UNx + Los) with more apoptotic cells. The expanded macrophage-rich lesions of UNx + Pio/Los had more alternatively activated, Ym-1 and arginine 1-positive M2 phenotypes (Ym-1: 33.6 ± 8.2%, p < 0.05 vs. 12.0 ± 1.1% in UNx; arginase 1: 27.8 ± 0.9%, p < 0.05 vs. 11.8 ± 1.3% in UNx). In vitro, pioglitazone alone and together with losartan was more effective than losartan alone in dampening lipopolysaccharide-induced cytokine production, suppressing M1 phenotypic change while enhancing M2 phenotypic change. Combination of pioglitazone and losartan is more effective in reducing renal injury-induced atherosclerosis than either treatment alone. This benefit reflects mitigation in macrophage cytokine

Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

PubMed Central

Gupta, Mamta; Hendrickson, Andrea E. Wahner; Yun, Seong Seok; Han, Jing Jing; Schneider, Paula A.; Koh, Brian D.; Stenson, Mary J.; Wellik, Linda E.; Shing, Jennifer C.; Peterson, Kevin L.; Flatten, Karen S.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Barr, Sharon

2012-01-01

The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo. PMID:22080480

Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

PubMed Central

Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D.

2016-01-01

The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt- transcription in tested Spt- mutants. PMID:27261007

Lifestyle Advice Combined with Personalized Estimates of Genetic or Phenotypic Risk of Type 2 Diabetes, and Objectively Measured Physical Activity: A Randomized Controlled Trial

PubMed Central

van Sluijs, Esther M. F.; Marteau, Theresa M.; Sutton, Stephen

2016-01-01

Background Information about genetic and phenotypic risk of type 2 diabetes is now widely available and is being incorporated into disease prevention programs. Whether such information motivates behavior change or has adverse effects is uncertain. We examined the effect of communicating an estimate of genetic or phenotypic risk of type 2 diabetes in a parallel group, open, randomized controlled trial. Methods and Findings We recruited 569 healthy middle-aged adults from the Fenland Study, an ongoing population-based, observational study in the east of England (Cambridgeshire, UK). We used a computer-generated random list to assign participants in blocks of six to receive either standard lifestyle advice alone (control group, n = 190) or in combination with a genetic (n = 189) or a phenotypic (n = 190) risk estimate for type 2 diabetes (intervention groups). After 8 wk, we measured the primary outcome, objectively measured physical activity (kJ/kg/day), and also measured several secondary outcomes (including self-reported diet, self-reported weight, worry, anxiety, and perceived risk). The study was powered to detect a between-group difference of 4.1 kJ/kg/d at follow-up. 557 (98%) participants completed the trial. There were no significant intervention effects on physical activity (difference in adjusted mean change from baseline: genetic risk group versus control group 0.85 kJ/kg/d (95% CI −2.07 to 3.77, p = 0.57); phenotypic risk group versus control group 1.32 (95% CI −1.61 to 4.25, p = 0.38); and genetic risk group versus phenotypic risk group −0.47 (95% CI −3.40 to 2.46, p = 0.75). No significant differences in self-reported diet, self-reported weight, worry, and anxiety were observed between trial groups. Estimates of perceived risk were significantly more accurate among those who received risk information than among those who did not. Key limitations include the recruitment of a sample that may not be representative of the UK population, use of self

Lifestyle Advice Combined with Personalized Estimates of Genetic or Phenotypic Risk of Type 2 Diabetes, and Objectively Measured Physical Activity: A Randomized Controlled Trial.

PubMed

Godino, Job G; van Sluijs, Esther M F; Marteau, Theresa M; Sutton, Stephen; Sharp, Stephen J; Griffin, Simon J

2016-11-01

Information about genetic and phenotypic risk of type 2 diabetes is now widely available and is being incorporated into disease prevention programs. Whether such information motivates behavior change or has adverse effects is uncertain. We examined the effect of communicating an estimate of genetic or phenotypic risk of type 2 diabetes in a parallel group, open, randomized controlled trial. We recruited 569 healthy middle-aged adults from the Fenland Study, an ongoing population-based, observational study in the east of England (Cambridgeshire, UK). We used a computer-generated random list to assign participants in blocks of six to receive either standard lifestyle advice alone (control group, n = 190) or in combination with a genetic (n = 189) or a phenotypic (n = 190) risk estimate for type 2 diabetes (intervention groups). After 8 wk, we measured the primary outcome, objectively measured physical activity (kJ/kg/day), and also measured several secondary outcomes (including self-reported diet, self-reported weight, worry, anxiety, and perceived risk). The study was powered to detect a between-group difference of 4.1 kJ/kg/d at follow-up. 557 (98%) participants completed the trial. There were no significant intervention effects on physical activity (difference in adjusted mean change from baseline: genetic risk group versus control group 0.85 kJ/kg/d (95% CI -2.07 to 3.77, p = 0.57); phenotypic risk group versus control group 1.32 (95% CI -1.61 to 4.25, p = 0.38); and genetic risk group versus phenotypic risk group -0.47 (95% CI -3.40 to 2.46, p = 0.75). No significant differences in self-reported diet, self-reported weight, worry, and anxiety were observed between trial groups. Estimates of perceived risk were significantly more accurate among those who received risk information than among those who did not. Key limitations include the recruitment of a sample that may not be representative of the UK population, use of self-reported secondary outcome measures, and

Heterologous Expression of Ralp3 in Streptococcus pyogenes M2 and M6 Strains Affects the Virulence Characteristics

PubMed Central

Siemens, Nikolai; Kreikemeyer, Bernd

2013-01-01

Background Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS). In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i) which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii) is Ralp3 actively lost during evolution in these serotypes. Methodology/Principal Findings We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. Conclusions/Significance Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis. PMID:23424622

Identifying Novel Phenotypes of Vulnerability and Resistance to Activity-Based Anorexia in Adolescent Female Rats

PubMed Central

Barbarich-Marsteller, Nicole C.; Underwood, Mark D.; Foltin, Richard W.; Myers, Michael M.; Walsh, B. Timothy; Barrett, Jeffrey S.; Marsteller, Douglas A.

2018-01-01

Objective Activity-based anorexia is a translational rodent model that results in severe weight loss, hyperactivity, and voluntary self-starvation. The goal of our investigation was to identify vulnerable and resistant phenotypes of activity-based anorexia in adolescent female rats. Method Sprague-Dawley rats were maintained under conditions of restricted access to food (N = 64; or unlimited access, N = 16) until experimental exit, predefined as a target weight loss of 30–35% or meeting predefined criteria for animal health. Nonlinear mixed effects statistical modeling was used to describe wheel running behavior, time to event analysis was used to assess experimental exit, and a regressive partitioning algorithm was used to classify phenotypes. Results Objective criteria were identified for distinguishing novel phenotypes of activity-based anorexia, including a vulnerable phenotype that conferred maximal hyperactivity, minimal food intake, and the shortest time to experimental exit, and a resistant phenotype that conferred minimal activity and the longest time to experimental exit. Discussion The identification of objective criteria for defining vulnerable and resistant phenotypes of activity-based anorexia in adolescent female rats provides an important framework for studying the neural mechanisms that promote vulnerability to or protection against the development of self-starvation and hyperactivity during adolescence. Ultimately, future studies using these novel phenotypes may provide important translational insights into the mechanisms that promote these maladaptive behaviors characteristic of anorexia nervosa. PMID:23853140

Identifying novel phenotypes of vulnerability and resistance to activity-based anorexia in adolescent female rats.

PubMed

Barbarich-Marsteller, Nicole C; Underwood, Mark D; Foltin, Richard W; Myers, Michael M; Walsh, B Timothy; Barrett, Jeffrey S; Marsteller, Douglas A

2013-11-01

Activity-based anorexia is a translational rodent model that results in severe weight loss, hyperactivity, and voluntary self-starvation. The goal of our investigation was to identify vulnerable and resistant phenotypes of activity-based anorexia in adolescent female rats. Sprague-Dawley rats were maintained under conditions of restricted access to food (N = 64; or unlimited access, N = 16) until experimental exit, predefined as a target weight loss of 30-35% or meeting predefined criteria for animal health. Nonlinear mixed effects statistical modeling was used to describe wheel running behavior, time to event analysis was used to assess experimental exit, and a regressive partitioning algorithm was used to classify phenotypes. Objective criteria were identified for distinguishing novel phenotypes of activity-based anorexia, including a vulnerable phenotype that conferred maximal hyperactivity, minimal food intake, and the shortest time to experimental exit, and a resistant phenotype that conferred minimal activity and the longest time to experimental exit. The identification of objective criteria for defining vulnerable and resistant phenotypes of activity-based anorexia in adolescent female rats provides an important framework for studying the neural mechanisms that promote vulnerability to or protection against the development of self-starvation and hyperactivity during adolescence. Ultimately, future studies using these novel phenotypes may provide important translational insights into the mechanisms that promote these maladaptive behaviors characteristic of anorexia nervosa. Copyright © 2013 Wiley Periodicals, Inc.

Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail

PubMed Central

Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

2016-01-01

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro. PMID:27917125

Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail.

PubMed

Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

2016-01-01

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro . HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro .

Whole-Blood Thiopurine S-Methyltransferase Genotype and Phenotype Concordance in Iranian Kurdish Ulcerative Colitis (UC) Patients.

PubMed

Bahrehmand, Fariborz; Vaisi-Raygani, Asad; Kiani, Amir; Bashiri, Homayoun; Zobeiri, Mahdi; Tanhapour, Maryam; Pourmotabbed, Tayebeh

2017-05-01

Thiopurine methyl transferase (TPMT), a drug-metabolizing enzyme, catalyzes methylation and consequently, the metabolism of thiopurine compounds used for treatment of inflammatory bowel disease (IBD). Individuals who are homozygous recessive or have extremely low TPMT activity need to avoid thiopurines because of concern for significant leukopenia. The aim of this research was to determine TPMT phenotypes and genotypes in IBD patients to predict the risk of thiopurine toxicity before treatment. The present case-control study consisted of 210 ulcerative colitis patients and 212 unrelated healthy controls from the population of western Iran. TPMT phenotype and genotype were determined by HPLC and allele specific PCR and PCR-RFLP, respectively. TPMT phenotyping and genotyping were compatible and demonstrated no frequency for deficient, 2.2% for low, and 97.8% for normal-activity which is different compared with the results of other studies. There was a significant negative correlation between TPMT activities as calculated based on nmol6MTG/gHb/h and the Hb levels in both UC (r = -0.54, p < 0.001) and control groups (r = -0.27, p < 0.001). Interestingly, a significant positive correlation between Hb levels and TPMT activities was seen when the enzyme activity was calculated in mU/L in both UC patients (r = 0.14, p = 0.05) and in control subjects (r = 0.43, p < 0.001). The overall concordance rate between TPMT phenotypes and genotypes of mutants to alleles (9 out of 422), based on receiver-operating characteristic (ROC) curve, yielded a sensitivity of 94.7% and specificity of 90% for mU/L and a sensitivity of 85.6% and specificity of 90% for nmol6MTG/gHb/h. The use of mU/L is more appropriate than nmol6MTG/gHb/h for expressing TPMT activity, and there is better correlation between genotypes and phenotypes of TPMT based on mU/L. The frequency of known mutant TPMT alleles in western Iran (Kurd population) is low suggesting low risk of thiopurine drug toxicity in IBD

Phenotype variability and allelic heterogeneity in KMT2B-Associated disease.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Nakagawa, Eiji; Koichihara, Reiko; Sakamoto, Takashi; Mure, Hideo; Morigaki, Ryoma; Koizumi, Hidetaka; Oki, Ryosuke; Montecchiani, Celeste; Caltagirone, Carlo; Orlacchio, Antonio; Hattori, Ayako; Mashimo, Hideaki; Izumi, Yuishin; Mezaki, Takahiro; Kumada, Satoko; Taniguchi, Makoto; Yokochi, Fusako; Saitoh, Shinji; Goto, Satoshi; Kaji, Ryuji

2018-04-05

Mutations in Lysine-Specific Histone Methyltransferase 2B gene (KMT2B) have been reported to be associated with complex early-onset dystonia. Almost all reported KMT2B mutations occurred de novo in the paternal germline or in the early development of the patient. We describe clinico-genetic features on four Japanese patients with novel de novo mutations and demonstrate the phenotypic spectrum of KMT2B mutations. We performed genetic studies, including trio-based whole exome sequencing (WES), in a cohort of Japanese patients with a seemingly sporadic early-onset generalized combined dystonia. Potential effects by the identified nucleotide variations were evaluated biologically. Genotype-phenotype correlations were also investigated. Four patients had de novo heterozygous mutations in KMT2B, c.309delG, c.1656dupC, c.3325_3326insC, and c.5636delG. Biological analysis of KMT2B mRNA levels showed a reduced expression of mutant transcript frame. All patients presented with motor milestone delay, microcephaly, mild psychomotor impairment, childhood-onset generalized dystonia and superimposed choreoathetosis or myoclonus. One patient cannot stand due to axial hypotonia associated with cerebellar dysfunction. Three patients had bilateral globus pallidal deep brain stimulation (DBS) with excellent or partial response. We further demonstrate the allelic heterogeneity and phenotypic variations of KMT2B-associated disease. Haploinsufficiency is one of molecular pathomechanisms underlying the disease. Cardinal clinical features include combined dystonia accompanying mild psychomotor disability. Cerebellum would be affected in KMT2B-associated disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Unbalancing p53/Mdm2/IGF-1R axis by Mdm2 activation restrains the IGF-1-dependent invasive phenotype of skin melanoma

PubMed Central

Worrall, C; Suleymanova, N; Crudden, C; Trocoli Drakensjö, I; Candrea, E; Nedelcu, D; Takahashi, S-I; Girnita, L; Girnita, A

2017-01-01

Melanoma tumors usually retain wild-type p53; however, its tumor-suppressor activity is functionally disabled, most commonly through an inactivating interaction with mouse double-minute 2 homolog (Mdm2), indicating p53 release from this complex as a potential therapeutic approach. P53 and the tumor-promoter insulin-like growth factor type 1 receptor (IGF-1R) compete as substrates for the E3 ubiquitin ligase Mdm2, making their relative abundance intricately linked. Hence we investigated the effects of pharmacological Mdm2 release from the Mdm2/p53 complex on the expression and function of the IGF-1R. Nutlin-3 treatment increased IGF-1R/Mdm2 association with enhanced IGF-1R ubiquitination and a dual functional outcome: receptor downregulation and selective downstream signaling activation confined to the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. This Nutlin-3 functional selectivity translated into IGF-1-mediated bioactivities with biphasic effects on the proliferative and metastatic phenotype: an early increase and late decrease in the number of proliferative and migratory cells, while the invasiveness was completely inhibited following Nutlin-3 treatment through an impaired IGF-1-mediated matrix metalloproteinases type 2 activation mechanism. Taken together, these experiments reveal the biased agonistic properties of Nutlin-3 for the mitogen-activated protein kinase pathway, mediated by Mdm2 through IGF-1R ubiquitination and provide fundamental insights into destabilizing p53/Mdm2/IGF-1R circuitry that could be developed for therapeutic gain. PMID:28092675

Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

PubMed

Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

2010-10-15

Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

Allele-Specific Silencing of Mutant mRNA Rescues Ultrastructural and Arrhythmic Phenotype in Mice Carriers of the R4496C Mutation in the Ryanodine Receptor Gene (RYR2).

PubMed

Bongianino, Rossana; Denegri, Marco; Mazzanti, Andrea; Lodola, Francesco; Vollero, Alessandra; Boncompagni, Simona; Fasciano, Silvia; Rizzo, Giulia; Mangione, Damiano; Barbaro, Serena; Di Fonso, Alessia; Napolitano, Carlo; Auricchio, Alberto; Protasi, Feliciano; Priori, Silvia G

2017-08-18

Mutations in the cardiac Ryanodine Receptor gene ( RYR2 ) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise. We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2 . We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2 -R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2 R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2 -R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities. The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT. © 2017 American Heart Association, Inc.

HOMEOSTATIC REGULATION OF KCC2 ACTIVITY BY THE ZINC RECEPTOR mZnR/GPR39 DURING SEIZURES

PubMed Central

Gilad, David; Shorer, Sharon; Ketzef, Maya; Friedman, Alon; Sekler, Israel; Aizenman, Elias; Hershfinkel, Michal

2015-01-01

The aim of this study was to investigate the role of the synaptic metabotropic zinc receptor mZnR/GPR39 in physiological adaptation to epileptic seizures. We previously demonstrated that synaptic activation of mZnR/GPR39 enhances inhibitory drive in the hippocampus by upregulating neuronal K+/Cl− co-transporter 2 (KCC2) activity. Here, we first show that mZnR/GPR39 knockout (KO) adult mice have dramatically enhanced susceptibility to seizures triggered by a single intraperitoneal injection of kainic acid, when compared to wild type (WT) littermates. Kainate also substantially enhances seizure-associated gamma oscillatory activity in juvenile mZnR/GPR39 KO hippocampal slices, a phenomenon that can be reproduced in WT tissue by extracellular Zn2+ chelation. Importantly, kainate-induced synaptic Zn2+ release enhances surface expression and transport activity of KCC2 in WT, but not mZnR/GPR39 KO hippocampal neurons. Kainate-dependent upregulation of KCC2 requires mZnR/GPR39 activation of the Gαq/phospholipase C/extracellular regulated kinase (ERK1/2) signaling cascade. We suggest that mZnR/GPR39-dependent upregulation of KCC2 activity provides homeostatic adaptation to an excitotoxic stimulus by increasing inhibition. As such, mZnR/GPR39 may provide a novel pharmacological target for dampening epileptic seizure activity. PMID:25562657

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

PubMed

Cyr, A R; Kulak, M V; Park, J M; Bogachek, M V; Spanheimer, P M; Woodfield, G W; White-Baer, L S; O'Malley, Y Q; Sugg, S L; Olivier, A K; Zhang, W; Domann, F E; Weigel, R J

2015-01-22

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors

PubMed Central

Seip, Kotryna; Nygaard, Vigdis; Haugen, Mads H.; Engesæter, Birgit Ø.; Mælandsmo, Gunhild M.; Prasmickaite, Lina

2016-01-01

The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases. PMID:26918352

Active PI3K Pathway Causes an Invasive Phenotype Which Can Be Reversed or Promoted by Blocking the Pathway at Divergent Nodes

PubMed Central

Wallin, Jeffrey J.; Guan, Jane; Edgar, Kyle A.; Zhou, Wei; Francis, Ross; Torres, Anthony C.; Haverty, Peter M.; Eastham-Anderson, Jeffrey; Arena, Sabrina; Bardelli, Alberto; Griffin, Sue; Goodall, John E.; Grimshaw, Kyla M.; Hoeflich, Klaus P.; Torrance, Christopher; Belvin, Marcia; Friedman, Lori S.

2012-01-01

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP3 production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis. PMID:22570710

Role of M2 and M3 muscarinic acetylcholine receptor subtypes in activation of bladder afferent pathways in spinal cord injured rats.

PubMed

Matsumoto, Yoshihiro; Miyazato, Minoru; Yokoyama, Hitoshi; Kita, Masafumi; Hirao, Yoshihiko; Chancellor, Michael B; Yoshimura, Naoki

2012-05-01

To evaluate the role of M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes in the activation of bladder afferent pathways in rats with chronic spinal cord injury (SCI). Adult female Sprague-Dawley rats were spinalized at the T9 level. Continuous cystometry was performed under awake conditions 2 or 4 weeks after SCI. The effects of intravesical administration of an mAChR agonist (oxotremorine-methiodide), a nonselective antagonist (atropine), an M2-selective antagonist (methoctramine), and an M3-selective antagonist (darifenacin) were examined. After cystometry, the bladder was removed and separated into the mucosa and detrusor, and the M2 and M3 mAChR mRNA expression in the mucosa was determined using real-time quantitative polymerase chain reaction. At 2 and 4 weeks after SCI, intravesical administration of a nonselective mAChR agonist (25 μM oxotremorine-methiodide) increased the area under the curve of nonvoiding contractions, although the intercontraction interval of voiding contractions and maximal voiding pressure did not change. This effect was blocked by atropine and methoctramine (10 μM) but not by darifenacin (50 μM). However, mAChR antagonists alone (10-50 μM) had no effect on cystometric parameters. M2 mAChR mRNA expression was increased in the mucosa of SCI rats compared with that in normal rats. Our results suggest that the M2 mAChR subtype plays an important role in bladder afferent activation that enhances detrusor overactivity in SCI rats. However, because mAChR antagonists alone did not affect any cystometric parameters, the muscarinic mechanism controlling bladder afferent activity might not be involved in the emergence of detrusor overactivity in SCI. Copyright © 2012 Elsevier Inc. All rights reserved.

M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis.

PubMed

Shrivastava, Richa; Asif, Mohammad; Singh, Varsha; Dubey, Parul; Ahmad Malik, Showkat; Lone, Mehraj-U-Din; Tewari, Brij Nath; Baghel, Khemraj Singh; Pal, Subhashis; Nagar, Geet Kumar; Chattopadhyay, Naibedya; Bhadauria, Smrati

2018-04-03

Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer, Ti gamma/delta or TNK phenotype.

PubMed

Testa, U; Care, A; Montesoro, E; Fossati, C; Giannella, G; Masciulli, R; Fagioli, M; Bulgarini, D; Habetswallner, D; Isacchi, G

1990-01-01

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10(3) U/ml), alone or in combination with interleukin-1 alpha or beta (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1+, Ti alpha/beta-, Ti gamma/delta-, and CD3- lymphocytes; (b) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, and CD3+ lymphocytes and (c) NKH-1+, Ti alpha/beta+, Ti gamma/delta- and CD3+ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced center.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

PubMed

Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M; Wells, Alan

2016-07-07

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due

mTORC2 activation is regulated by the urokinase receptor (uPAR) in bladder cancer.

PubMed

Hau, Andrew M; Leivo, Mariah Z; Gilder, Andrew S; Hu, Jing-Jing; Gonias, Steven L; Hansel, Donna E

2017-01-01

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer. Copyright © 2016. Published by Elsevier Inc.

Positive and negative modulation of circadian activity rhythms by mGluR5 and mGluR2/3 metabotropic glutamate receptors.

PubMed

Gannon, Robert L; Millan, Mark J

2011-01-01

Glutamate released from retinal ganglion cells conveys information about the daily light:dark cycle to master circadian pacemaker neurons within the suprachiasmatic nucleus that then synchronize internal circadian rhythms with the external day-length. Glutamate activation of ionotropic glutamate receptors in the suprachiasmatic nucleus is well established, but the function of the metabotropic glutamate receptors that are also located in this nucleus is not known. Therefore, in this study we evaluated agonists and antagonists acting at orthosteric or allosteric sites for mGluR5 and mGluR2/3 metabotropic glutamate receptors for their ability to modulate light-induced phase advances and delays of hamster circadian activity rhythms. mGluR5 allosteric antagonists fenobam, MPEP and MTEP, each 10 mg/kg, potentiated light-induced phase advances of hamster circadian activity rhythms, while the mGluR5 agonists CHPG, (S)-3,5-DHPG or positive allosteric modulator CDPPB had no effect. Neither mGluR5 agonists nor antagonists had any effect on light-induced phase delays of activity rhythms. The competitive mGluR2/3 antagonist LY341495, 10 mg/kg, also potentiated light-induced phase advances, but inhibited light-induced phase delays. The mGluR2/3 agonists LY354740 and LY404039 were without effect on phase advances while a third agonist LY379268, 10 mg/kg, inhibited both light-induced advances and delays. Finally, mGluR2/3 agonists LY379268 and LY404039 also inhibited light-induced phase delays of activity rhythms. These results suggest that during light-induced phase advances, mGluR2/3 and mGluR5 receptors act to negatively modulate the effects of light on the circadian pacemaker or its output(s). mGluR5 receptors do not appear to be involved during light-induced phase delays. In contrast, the role for mGluR2/3 receptors during phase delays is more complicated as both agonists and antagonists inhibit light-induced phase delays. Dysfunctions in human circadian rhythms have been

The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity.

PubMed

Paquet, Maryse; Asay, Matthew J; Fam, Sami R; Inuzuka, Hiroyuki; Castleberry, Amanda M; Oller, Heide; Smith, Yoland; Yun, C Chris; Traynelis, Stephen F; Hall, Randy A

2006-10-06

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.

Cytoplasmic p21(CIP1/WAF1), ERK1/2 activation, and cytoskeletal remodeling are associated with the senescence-like phenotype after airborne particulate matter (PM(10)) exposure in lung cells.

PubMed

Sánchez-Pérez, Yesennia; Chirino, Yolanda I; Osornio-Vargas, Álvaro Román; Herrera, Luis A; Morales-Bárcenas, Rocío; López-Saavedra, Alejandro; González-Ramírez, Imelda; Miranda, Javier; García-Cuellar, Claudia María

2014-02-10

The exposure to particulate matter with a mean aerodynamic diameter ≤10 μm (PM10) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10 exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21(CIP1/WAF1), an increase in β-galactosidase activity and changes in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10 concentration (10 μg/cm(2)) for 24 h and 48 h. The results showed that PM10 exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21(CIP1/WAF1) via phosphorylation at Thr(145) and Ser(146) and the phosphorylation of ERK1/2 on Thr(202). Changes in the cell cycle or apoptosis were not observed, but an increase in β-galactosidase activity was detected. The PM10 from CZ caused more dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21(CIP1/WAF1) retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM10. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Streptococcal inhibitor of complement promotes innate immune resistance phenotypes of invasive M1T1 group A Streptococcus.

PubMed

Pence, Morgan A; Rooijakkers, Suzan H M; Cogen, Anna L; Cole, Jason N; Hollands, Andrew; Gallo, Richard L; Nizet, Victor

2010-01-01

Streptococcal inhibitor of complement (SIC) is a highly polymorphic extracellular protein and putative virulence factor secreted by M1 and M57 strains of group A Streptococcus (GAS). The sic gene is highly upregulated in invasive M1T1 GAS isolates following selection of mutations in the covR/S regulatory locus in vivo. Previous work has shown that SIC (allelic form 1.01) binds to and inactivates complement C5b67 and human cathelicidin LL-37. We examined the contribution of SIC to innate immune resistance phenotypes of GAS in the intact organism, using (1) targeted deletion of sic in wild-type and animal-passaged (covS mutant) M1T1 GAS harboring the sic 1.84 allele and (2) heterologous expression of sic in M49 GAS, which does not possess the sic genein its genome. We find that M1T1 SIC production is strongly upregulated upon covS mutation but that the sic gene is not required for generation and selection of covS mutants in vivo. SIC 1.84 bound both human and murine cathelicidins and was necessary and sufficient to promote covS mutant M1T1 GAS resistance to LL-37, growth in human whole blood and virulence in a murine model of systemic infection. Finally, the sic knockout mutant M1T1 GAS strain was deficient in growth in human serum and intracellular macrophage survival. We conclude that SIC contributes to M1T1 GAS immune resistance and virulence phenotypes. Copyright © 2010 S. Karger AG, Basel.

Expression of the Homeobox Gene HOXA9 in Ovarian Cancer Induces Peritoneal Macrophages to Acquire an M2 Tumor-Promoting Phenotype

PubMed Central

Ko, Song Yi; Ladanyi, Andras; Lengyel, Ernst; Naora, Honami

2015-01-01

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8+ tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages. PMID:24332016

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

PubMed

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.

Silencing Nrf2 impairs glioma cell proliferation via AMPK-activated mTOR inhibition

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

Jia, Yue; Wang, Handong, E-mail: njhdwang@hotmail.com; Wang, Qiang

Gliomas are the leading cause of death among adults with primary brain malignancies. Treatment for malignant gliomas remains limited, and targeted therapies have been incompletely explored. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, the role and mechanism of Nrf2 in cancer cell proliferation was investigated in multiple glioma cell lines. We first evaluated the expression patterns of Nrf2 in four glioma cell lines and found all four cell lines expressed Nrf2, but the highest level was observed in U251 cells. We further evaluatedmore » the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA (shRNA). We found that Nrf2 depletion inhibited glioma cell proliferation. Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA. Moreover, suppression of Nrf2 expression could lead to ATP depletion (with concomitant rise in AMP/ATP ratio) and consequently to AMPK-activated mTOR inhibition. Finally, activation of adenosine monophosphate–activated protein kinase (AMPK) by treated with phenformin, an AMPK agonist, can mimic the inhibitory effect of Nrf2 knockdown in U251 cells. In conclusion, our findings will shed light to the role and mechanism of Nrf2 in regulating glioma proliferation via ATP-depletion-induced AMPK activation and consequent mTOR inhibition, a novel insight into our understanding the role and mechanism of Nrf2 in glioma pathoetiology. To our knowledge, this is also the first report to provide a rationale for the implication of cross-linking between Nrf2 and mTOR signaling.« less

Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli.

PubMed

Cheng, Jun; Ye, Ying; Wang, Ying-ying; Li, Hui; Li, Xu; Li, Jia-bin

2008-02-01

The aim of the present study was to study the phenotypic and molecular characterization of 5 novel CTX-M-beta-1actamases carried by 5 Klebsiella pneumoniae isolates and 3 Escherichia coli isolates collected from 4 hospitals in Hefei, China. The purified PCR products were ligated with pGEM-Teasy vectors, expressed, and sequenced. The complete genes of the CTX-M-beta-lactamases were ligated with the pHSG398 vector to express prokaryotic recombinant proteins. Plasmids were extracted by rapid alkaline lysis protocol, and the PCR method was performed to determine whether the prokaryotic expression was successful or not. Antimicrobial susceptibility was tested and the phenotypes of transformants were determined according to criteria recommended by the Clinical and Laboratory Standards Institute. The kinetic parameters of enzymes were confirmed. The isoelectric points (pI) were determined by isoelectric focusing assay. Pulsed-field gel electrophoresis and plasmid profiling were performed. The PCR products had 1101 nucleotides and were determined as CTX-M-46, CTX-M-47, CTX-M-48, CTX-M-49, and CTX-M-50. All strains were resistant to cefotaxime, but most of them were susceptible or intermediate to ceftazidime. The phenotypes of novel enzymes were determined as extended-spectrum-beta-lactamases (ESBL). Penicillin G, cephalothin, cefuroxime, and cefotaxime were determined to good substrates, whereas ceftazidime hydrolysis was not detected. The pI of the 5 novel CTX-M-beta-lactamases were 8.0. CTX-M-derivatives could be the multiplex genesis in our area. This is the first report of these 5 novel plasmid-mediated CTX-M ESBL produced from China in the world. Molecular typing reveals notably different origin in genes encoding different CTX-M variants of 8 strains.

Parkin Regulates the Activity of Pyruvate Kinase M2*

PubMed Central

Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

2016-01-01

Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

Cytochrome P450 2D6 variants in a Caucasian population: Allele frequencies and phenotypic consequences

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

Sachse, C.; Brockmoeller, J.; Bauer, S.

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015,more » respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of.005 (*1 x 2), .013 (* 2 x 2), and .001 (*4 x 2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T{sub 1957}C), *2B (additional C{sub 2558}T), and *4E (additional C{sub 2938}T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EN/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment. 35 refs., 4 figs., 5 tabs.« less

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a "neglected" IL-2low Bimhigh phenotype, poor CTL function and cell death.

PubMed

Holz, Lauren E; Benseler, Volker; Vo, Michelle; McGuffog, Claire; Van Rooijen, Nico; McCaughan, Geoffrey W; Bowen, David G; Bertolino, Patrick

2012-10-01

The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Accuracy of various human NAT2 SNP genotyping panels to infer rapid, intermediate and slow acetylator phenotypes

PubMed Central

Hein, David W; Doll, Mark A

2012-01-01

Aim Humans exhibit genetic polymorphism in NAT2 resulting in rapid, intermediate and slow acetylator phenotypes. Over 65 NAT2 variants possessing one or more SNPs in the 870-bp NAT2 coding region have been reported. The seven most frequent SNPs are rs1801279 (191G>A), rs1041983 (282C>T), rs1801280 (341T>C), rs1799929 (481C>T), rs1799930 (590G>A), rs1208 (803A>G) and rs1799931 (857G>A). The majority of studies investigate the NAT2 genotype assay for three SNPs: 481C>T, 590G>A and 857G>A. A tag-SNP (rs1495741) recently identified in a genome-wide association study has also been proposed as a biomarker for the NAT2 phenotype. Materials & methods Sulfamethazine N-acetyltransferase catalytic activities were measured in cryopreserved human hepatocytes from a convenience sample of individuals in the USA with an ethnic frequency similar to the 2010 US population census. These activities were segregated by the tag-SNP rs1495741 and each of the seven SNPs described above. We assessed the accuracy of the tag-SNP and various two-, three-, four- and seven-SNP genotyping panels for their ability to accurately infer NAT2 phenotype. Results The accuracy of the various NAT2 SNP genotype panels to infer NAT2 phenotype were as follows: seven-SNP: 98.4%; tag-SNP: 77.7%; two-SNP: 96.1%; three-SNP: 92.2%; and four-SNP: 98.4%. Conclusion A NAT2 four-SNP genotype panel of rs1801279 (191G>A), rs1801280 (341T>C), rs1799930 (590G>A) and rs1799931 (857G>A) infers NAT2 acetylator phenotype with high accuracy, and is recommended over the tag-, two-, three- and (for economy of scale) the seven-SNP genotyping panels, particularly in populations of non-European ancestry. PMID:22092036

ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype.

PubMed

Miglionico, Rocchina; Ostuni, Angela; Armentano, Maria Francesca; Milella, Luigi; Crescenzi, Elvira; Carmosino, Monica; Bisaccia, Faustino

2017-01-01

Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype. Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype. To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21 Cip p53 independent; and 4) downregulation of lamin A/C. These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

2014-01-01

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

Expression of M2-polarized macrophages is associated with poor prognosis for advanced epithelial ovarian cancer.

PubMed

Lan, Chunyan; Huang, Xin; Lin, Suxia; Huang, Huiqiang; Cai, Qichun; Wan, Ting; Lu, Jiabin; Liu, Jihong

2013-06-01

Macrophages are polarized into two functionally distinct forms, M1 and M2, in response to different microenvironment. Tumor-associated macrophages (TAMs) generally have M2 phenotype and promote tumor progression. Few studies to date have described the infiltration of M2-polarized macrophages in ovarian cancer. We used two macrophages markers, CD68 and CD163, to analyze the expression of TAMs and to clarify the relationship between the M2 form and survival in advanced ovarian cancer. Clinical data of 110 patients with stages III-IV epithelial ovarian cancer at Sun Yat-sen University Cancer Center between 1999 and 2007 were retrospectively reviewed. Immunohistochemical staining of CD68 and CD163 was performed. Correlations between macrophage density and patient survival were analyzed. Our data showed that no significant difference was observed in survival between patients in the high- and the low-CD68 expression groups. In contrast, the progression-free survival (PFS) rates (p = 0.003) and overall survival (OS) rates (p = 0.004) were significantly higher in the low-CD163 expression group than in the high-CD163 expression group, respectively. Similarly, we also observed significantly improved 3-year PFS (49.8% vs. 11.0%, p < 0.001) and OS (77.4% vs. 45.0%, p < 0.001) rates in patients in the low-CD163/CD68 ratio group when compared with the high-CD163/CD68 ratio group. Multivariate analysis identified the density of CD163-positive cells as well as the ratio of CD163/CD68 as negative predictors for PFS and OS, respectively. Our results show that the infiltration of CD163-positive M2 macrophages as well as activation of macrophages towards the M2 phenotype may contribute to poor survival in advanced ovarian cancer.

Parkin Regulates the Activity of Pyruvate Kinase M2.

PubMed

Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

2016-05-06

Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of PARP activity in lung cancer-induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.

PubMed

Chacon-Cabrera, Alba; Mateu-Jimenez, Mercè; Langohr, Klaus; Fermoselle, Clara; García-Arumí, Elena; Andreu, Antoni L; Yelamos, Jose; Barreiro, Esther

2017-12-01

Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1 -/- ) and Parp-2 -/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1 -/- , and Parp-2 -/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1 -/- and Parp-2 -/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention. © 2017 Wiley Periodicals, Inc.

Glucose pathways adaptation supports acquisition of activated microglia phenotype.

PubMed

Gimeno-Bayón, J; López-López, A; Rodríguez, M J; Mahy, N

2014-06-01

With its capacity to survey the environment and phagocyte debris, microglia assume a diversity of phenotypes to respond specifically through neurotrophic and toxic effects. Although these roles are well accepted, the underlying energetic mechanisms associated with microglial activation remain largely unclear. This study investigates microglia metabolic adaptation to ATP, NADPH, H(+) , and reactive oxygen species production. To this end, in vitro studies were performed with BV-2 cells before and after activation with lipopolysaccharide + interferon-γ. Nitric oxide (NO) was measured as a marker of cell activation. Our results show that microglial activation triggers a metabolic reprogramming based on an increased glucose uptake and a strengthening of anaerobic glycolysis, as well as of the pentose pathway oxidative branch, while retaining the mitochondrial activity. Based on this energy commitment, microglial defense capacity increases rapidly as well as ribose-5-phosphate and nucleic acid formation for gene transcription, essential to ensure the newly acquired functions demanded by central nervous system signaling. We also review the role of NO in this microglial energy commitment that positions cytotoxic microglia within the energetics of the astrocyte-neuron lactate shuttle. Copyright © 2014 Wiley Periodicals, Inc.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes

PubMed Central

Mircheff, Austin K.; Wang, Yanru; Schechter, Joel E.; Li, Meng; Tong, Warren; Attar, Mayssa; Chengalvala, Murty; Harmuth, Joe; Prusakiewicz, Jeffery J.

2016-01-01

Purpose To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. Methods Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1° ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). Results Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1° ATD-eyes and exacerbated 1° ATD-LG atrophy. IMIP were evident in 2° ATD-LG as well as 1° ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. Conclusions The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms. PMID:27423911

Recognition of TLR2 N-Glycans: Critical Role in ArtinM Immunomodulatory Activity

PubMed Central

da Silva, Thiago Aparecido; Ruas, Luciana Pereira; Nohara, Lilian L.; de Almeida, Igor Correia; Roque-Barreira, Maria Cristina

2014-01-01

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties. PMID:24892697

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

PubMed

De Angelis, Federica; Marinelli, Sara; Fioretti, Bernard; Catacuzzeno, Luigi; Franciolini, Fabio; Pavone, Flaminia; Tata, Ada Maria

2014-06-01

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments. © 2013 Wiley Periodicals, Inc.

Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells.

PubMed

Hujber, Zoltán; Petővári, Gábor; Szoboszlai, Norbert; Dankó, Titanilla; Nagy, Noémi; Kriston, Csilla; Krencz, Ildikó; Paku, Sándor; Ozohanics, Olivér; Drahos, László; Jeney, András; Sebestyén, Anna

2017-06-02

Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming. HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13 C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well. Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13 C atoms from 13 C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo. Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

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

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependentmore » phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. �

Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

PubMed Central

Yang, Shenglan; Gong, Wei; Wang, Yan; Cianflone, Katherine; Tang, Jiarong; Wang, Dao Wen

2012-01-01

Background MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes. PMID:22761738

Mdm2 mediates FMRP- and Gp1 mGluR-dependent protein translation and neural network activity.

PubMed

Liu, Dai-Chi; Seimetz, Joseph; Lee, Kwan Young; Kalsotra, Auinash; Chung, Hee Jung; Lu, Hua; Tsai, Nien-Pei

2017-10-15

Activating Group 1 (Gp1) metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, elicits translation-dependent neural plasticity mechanisms that are crucial to animal behavior and circuit development. Dysregulated Gp1 mGluR signaling has been observed in numerous neurological and psychiatric disorders. However, the molecular pathways underlying Gp1 mGluR-dependent plasticity mechanisms are complex and have been elusive. In this study, we identified a novel mechanism through which Gp1 mGluR mediates protein translation and neural plasticity. Using a multi-electrode array (MEA) recording system, we showed that activating Gp1 mGluR elevates neural network activity, as demonstrated by increased spontaneous spike frequency and burst activity. Importantly, we validated that elevating neural network activity requires protein translation and is dependent on fragile X mental retardation protein (FMRP), the protein that is deficient in the most common inherited form of mental retardation and autism, fragile X syndrome (FXS). In an effort to determine the mechanism by which FMRP mediates protein translation and neural network activity, we demonstrated that a ubiquitin E3 ligase, murine double minute-2 (Mdm2), is required for Gp1 mGluR-induced translation and neural network activity. Our data showed that Mdm2 acts as a translation suppressor, and FMRP is required for its ubiquitination and down-regulation upon Gp1 mGluR activation. These data revealed a novel mechanism by which Gp1 mGluR and FMRP mediate protein translation and neural network activity, potentially through de-repressing Mdm2. Our results also introduce an alternative way for understanding altered protein translation and brain circuit excitability associated with Gp1 mGluR in neurological diseases such as FXS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

SNARE-dependent upregulation of KCC2 activity following metabotropic zinc receptor (mZnR/GPR39) activation in rat cortical neurons in vitro

PubMed Central

Saadi, Robert A.; He, Kai; Hartnett, Karen A.; Kandler, Karl; Hershfinkel, Michal; Aizenman, Elias

2012-01-01

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 via an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor mZnR/GPR39. Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture and then tested the hypothesis that the increase in KCC2 activity is mediated through a SNARE-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist MCPG. Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx via the co-transporter using an intracellular pH sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts via mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and SNAP-25. Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons

Seizure-dependent mTOR activation in 5-HT neurons promotes autism-like behaviors in mice.

PubMed

McMahon, John J; Yu, Wilson; Yang, Jun; Feng, Haihua; Helm, Meghan; McMahon, Elizabeth; Zhu, Xinjun; Shin, Damian; Huang, Yunfei

2015-01-01

Epilepsy and autism spectrum disorder (ASD) are common comorbidities of one another. Despite the prevalent correlation between the two disorders, few studies have been able to elucidate a mechanistic link. We demonstrate that forebrain specific Tsc1 deletion in mice causes epilepsy and autism-like behaviors, concomitant with disruption of 5-HT neurotransmission. We find that epileptiform activity propagates to the raphe nuclei, resulting in seizure-dependent hyperactivation of mTOR in 5-HT neurons. To dissect whether mTOR hyperactivity in 5-HT neurons alone was sufficient to recapitulate an autism-like phenotype we utilized Tsc1flox/flox;Slc6a4-cre mice, in which mTOR is restrictively hyperactivated in 5-HT neurons. Tsc1flox/flox;Slc6a4-cre mice displayed alterations of the 5-HT system and autism-like behaviors, without causing epilepsy. Rapamycin treatment in these mice was sufficient to rescue the phenotype. We conclude that the spread of seizure activity to the brainstem is capable of promoting hyperactivation of mTOR in the raphe nuclei, which in turn promotes autism-like behaviors. Thus our study provides a novel mechanism describing how epilepsy can contribute to the development of autism-like behaviors, suggesting new therapeutic strategies for autism. Copyright © 2015 Elsevier Inc. All rights reserved.

Seizure-dependent mTOR activation in 5-HT neurons promotes autism-like behaviors in mice

PubMed Central

McMahon, John J.; Yu, Wilson; Yang, Jun; Feng, Haihua; Helm, Meghan; McMahon, Elizabeth; Zhu, Xinjun; Shin, Damian; Huang, Yunfei

2014-01-01

Epilepsy and autism spectrum disorder (ASD) are common comorbidities of one another. Despite the prevalent correlation between the two disorders, few studies have been able to elucidate a mechanistic link. We demonstrate that forebrain specific Tsc1 deletion in mice causes epilepsy and autism-like behaviors, concomitant with disruption of 5-HT neurotransmission. We find that epileptiform activity propagates to the raphe nuclei, resulting in seizure-dependent hyperactivation of mTOR in 5-HT neurons. To dissect whether mTOR hyperactivity in 5-HT neurons alone was sufficient to recapitulate an autism-like phenotype we utilized Tsc1flox/flox;Slc6a4-cre mice, in which mTOR is restrictively hyperactivated in 5-HT neurons. Tsc1flox/flox;Slc6a4-cre mice displayed alterations of the 5-HT system and autism-like behaviors, without causing epilepsy. Rapamycin treatment in these mice was sufficient to rescue the phenotype. We conclude that the spread of seizure activity to the brainstem is capable of promoting hyperactivation of mTOR in the raphe nuclei, which in turn promotes autism-like behaviors. Thus our study provides a novel mechanism describing how epilepsy can contribute to the development of autism-like behaviors, suggesting new therapeutic strategies for autism. PMID:25315683

Aberrant let7a/HMGA2 signaling activity with unique clinical phenotype in JAK2-mutated myeloproliferative neoplasms

PubMed Central

Chen, Chih-Cheng; You, Jie-Yu; Lung, Jrhau; Huang, Cih-En; Chen, Yi-Yang; Leu, Yu-Wei; Ho, Hsing-Ying; Li, Chian-Pei; Lu, Chang-Hsien; Lee, Kuan-Der; Hsu, Chia-Chen; Gau, Jyh-Pyng

2017-01-01

High mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that is negatively regulated by let-7 microRNA through binding to it’s 3′-untranslated region. Transgenic mice expressing Hmga2 with a truncation of its 3′-untranslated region has been shown to exhibit a myeloproliferative phenotype. To decipher the let-7-HMGA2 axis in myeloproliferative neoplasms, we employed an in vitro model supplemented with clinical correlation. Ba/F3 cells with inducible JAK2V617F expression (Ton.JAK2.V617F cells) showed upregulation of HMGA2 with concurrent let-7a repression. Ton.JAK2.V617F cells treated with a let-7a inhibitor exhibited further escalation of Hmga2 expression, while a let-7a mimic diminished the Hmga2 transcript level. Hmga2 overexpression conferred JAK2-mutated cells with a survival advantage through inhibited apoptosis. A pan-JAK inhibitor, INC424, increased the expression of let-7a, downregulated the level of Hmga2, and led to increased apoptosis in Ton.JAK2.V617F cells in a dose-dependent manner. In samples from 151 patients with myeloproliferative neoplasms, there was a modest inverse correlation between the expression levels of let-7a and HMGA2. Overexpression of HMGA2 was detected in 29 (19.2%) of the cases, and it was more commonly seen in patients with essential thrombocythemia than in those with polycythemia vera (26.9% vs. 12.7%, P=0.044). Patients with upregulated HMGA2 showed an increased propensity for developing major thrombotic events, and they were more likely to harbor one of the 3 driver myeloproliferative neoplasm mutations in JAK2, MPL and CALR. Our findings suggest that, in a subset of myeloproliferative neoplasm patients, the let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. PMID:28057739

Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model

PubMed Central

Kuen, Janina; Darowski, Diana; Kluge, Tobias

2017-01-01

Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs and monocytes. Using this model, we analyzed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analyzing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated. 3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activated spheroid polarized MDMs or block immune suppressive factors such as Arginase-I. PMID:28750018

Topical corticosteroids do not revert the activated phenotype of eosinophils in eosinophilic esophagitis but decrease surface levels of CD18 resulting in diminished adherence to ICAM-1, ICAM-2, and endothelial cells.

PubMed

Lingblom, Christine; Bergquist, Henrik; Johnsson, Marianne; Sundström, Patrik; Quiding-Järbrink, Marianne; Bove, Mogens; Wennerås, Christine

2014-12-01

Swallowed topical corticosteroids are the standard therapy for eosinophilic esophagitis (EoE) in adults. Eosinophils in the blood of untreated EoE patients have an activated phenotype. Our aim was to determine if corticosteroids restore the phenotype of eosinophils to a healthy phenotype and if certain cell-surface molecules on blood eosinophils correlate with eosinophilic infiltration of the esophagus. Levels of eight surface markers on eosinophils from treated and untreated EoE patients were determined by flow cytometry and analyzed using multivariate methods of pattern recognition. Corticosteroid-treated EoE patients' eosinophils had decreased levels of CD18 compared to both untreated patients and healthy controls, but maintained their activated phenotype. CD18 expression correlated positively with eosinophil numbers in the esophagus and promoted the adherence of eosinophils to ICAM-1, ICAM-2, and to endothelial cells. The diminished expression of CD18 may be one mechanism behind the reduced entry of eosinophils into the esophagus in corticosteroid-treated EoE patients.

Investigation on Quantitative Structure Activity Relationships and Pharmacophore Modeling of a Series of mGluR2 Antagonists

PubMed Central

Zhang, Meng-Qi; Zhang, Xiao-Le; Li, Yan; Fan, Wen-Jia; Wang, Yong-Hua; Hao, Ming; Zhang, Shu-Wei; Ai, Chun-Zhi

2011-01-01

MGluR2 is G protein-coupled receptor that is targeted for diseases like anxiety, depression, Parkinson’s disease and schizophrenia. Herein, we report the three-dimensional quantitative structure–activity relationship (3D-QSAR) studies of a series of 1,3-dihydrobenzo[ b][1,4]diazepin-2-one derivatives as mGluR2 antagonists. Two series of models using two different activities of the antagonists against rat mGluR2, which has been shown to be very similar to the human mGluR2, (activity I: inhibition of [3H]-LY354740; activity II: mGluR2 (1S,3R)-ACPD inhibition of forskolin stimulated cAMP.) were derived from datasets composed of 137 and 69 molecules respectively. For activity I study, the best predictive model obtained from CoMFA analysis yielded a Q2 of 0.513, R2 ncv of 0.868, R2 pred = 0.876, while the CoMSIA model yielded a Q2 of 0.450, R2 ncv = 0.899, R2 pred = 0.735. For activity II study, CoMFA model yielded statistics of Q2 = 0.5, R2 ncv = 0.715, R2 pred = 0.723. These results prove the high predictability of the models. Furthermore, a combined analysis between the CoMFA, CoMSIA contour maps shows that: (1) Bulky substituents in R7, R3 and position A benefit activity I of the antagonists, but decrease it when projected in R8 and position B; (2) Hydrophilic groups at position A and B increase both antagonistic activity I and II; (3) Electrostatic field plays an essential rule in the variance of activity II. In search for more potent mGluR2 antagonists, two pharmacophore models were developed separately for the two activities. The first model reveals six pharmacophoric features, namely an aromatic center, two hydrophobic centers, an H-donor atom, an H-acceptor atom and an H-donor site. The second model shares all features of the first one and has an additional acceptor site, a positive N and an aromatic center. These models can be used as guidance for the development of new mGluR2 antagonists of high activity and selectivity. This work is the first report on 3

Investigation on quantitative structure activity relationships and pharmacophore modeling of a series of mGluR2 antagonists.

PubMed

Zhang, Meng-Qi; Zhang, Xiao-Le; Li, Yan; Fan, Wen-Jia; Wang, Yong-Hua; Hao, Ming; Zhang, Shu-Wei; Ai, Chun-Zhi

2011-01-01

MGluR2 is G protein-coupled receptor that is targeted for diseases like anxiety, depression, Parkinson's disease and schizophrenia. Herein, we report the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a series of 1,3-dihydrobenzo[ b][1,4]diazepin-2-one derivatives as mGluR2 antagonists. Two series of models using two different activities of the antagonists against rat mGluR2, which has been shown to be very similar to the human mGluR2, (activity I: inhibition of [(3)H]-LY354740; activity II: mGluR2 (1S,3R)-ACPD inhibition of forskolin stimulated cAMP.) were derived from datasets composed of 137 and 69 molecules respectively. For activity I study, the best predictive model obtained from CoMFA analysis yielded a Q(2) of 0.513, R(2) (ncv) of 0.868, R(2) (pred) = 0.876, while the CoMSIA model yielded a Q(2) of 0.450, R(2) (ncv) = 0.899, R(2) (pred) = 0.735. For activity II study, CoMFA model yielded statistics of Q(2) = 0.5, R(2) (ncv) = 0.715, R(2) (pred) = 0.723. These results prove the high predictability of the models. Furthermore, a combined analysis between the CoMFA, CoMSIA contour maps shows that: (1) Bulky substituents in R(7), R(3) and position A benefit activity I of the antagonists, but decrease it when projected in R(8) and position B; (2) Hydrophilic groups at position A and B increase both antagonistic activity I and II; (3) Electrostatic field plays an essential rule in the variance of activity II. In search for more potent mGluR2 antagonists, two pharmacophore models were developed separately for the two activities. The first model reveals six pharmacophoric features, namely an aromatic center, two hydrophobic centers, an H-donor atom, an H-acceptor atom and an H-donor site. The second model shares all features of the first one and has an additional acceptor site, a positive N and an aromatic center. These models can be used as guidance for the development of new mGluR2 antagonists of high activity and selectivity

Genotype-phenotype associations in obesity dependent on definition of the obesity phenotype.

PubMed

Kring, Sofia Inez Iqbal; Larsen, Lesli Hingstrup; Holst, Claus; Toubro, Søren; Hansen, Torben; Astrup, Arne; Pedersen, Oluf; Sørensen, Thorkild I A

2008-01-01

In previous studies of associations of variants in the genes UCP2, UCP3, PPARG2, CART, GRL, MC4R, MKKS, SHP, GHRL, and MCHR1 with obesity, we have used a case-control approach with cases defined by a threshold for BMI. In the present study, we assess the association of seven abdominal, peripheral, and overall obesity phenotypes, which were analyzed quantitatively, and thirteen candidate gene polymorphisms in these ten genes in the same cohort. Obese Caucasian men (n = 234, BMI >or= 31.0 kg/m(2)) and a randomly sampled non-obese group (n = 323), originally identified at the draft board examinations, were re-examined at median ages of 47.0 or 49.0 years by anthropometry and DEXA scanning. Obesity phenotypes included BMI, fat body mass index, waist circumference, waist for given BMI, intra-abdominal adipose tissue, hip circumference and lower body fat mass (%). Using logistic regression models, we estimated the odds for defined genotypes (dominant or recessive genetic transmission) in relation to z-scores of the phenotypes. The minor (rare) allele for SHP 512G>C (rs6659176) was associated with increased hip circumference. The minor allele for UCP2 Ins45bp was associated with increased BMI, increased abdominal obesity, and increased hip circumference. The minor allele for UCP2 -866G>A (rs6593669) was associated with borderline increased fat body mass index. The minor allele for MCHR1 100213G>A (rs133072) was associated with reduced abdominal obesity. None of the other genotype-phenotype combinations showed appreciable associations. If replicated in independent studies with focus on the specific phenotypes, our explorative studies suggest significant associations between some candidate gene polymorphisms and distinct obesity phenotypes, predicting beneficial and detrimental effects, depending on compartments for body fat accumulation. Copyright 2008 S. Karger AG, Basel.

SATB2-associated syndrome presenting with Rett-like phenotypes.

PubMed

Lee, J S; Yoo, Y; Lim, B C; Kim, K J; Choi, M; Chae, J-H

2016-06-01

The SATB2-associated syndrome (SAS) was proposed recently, after the SATB2 gene was initially discovered to be associated with isolated cleft palate. This syndrome is characterized by intellectual disability with delayed speech development, facial dysmorphism, cleft or high-arched palate, and dentition problems. Here, we describe two novel SATB2 sequence variants in two unrelated patients presenting with Rett-like phenotypes. We performed trio-based whole-exome sequencing in a 17-month-old girl presenting with severe retardation and Rett-like phenotypes, which revealed a de novo missense variant in SATB2 (p.Glu396Gln). Moreover, targeted sequencing of the SATB2 gene was performed in a 2-year-old girl with severe psychomotor retardation, facial hypotonia, and cleft palate who also exhibited some features of Rett syndrome. A nonsense variant in SATB2 was identified in this patient (p.Arg459*). This study expanded the clinical and genetic spectrum of SAS. SATB2 variants should be considered in cases with psychomotor retardation alone or in any cases with Rett-like phenotypes, regardless of the typical features of SAS such as cleft palate. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Punicalagin, a PTP1B inhibitor, induces M2c phenotype polarization via up-regulation of HO-1 in murine macrophages.

PubMed

Xu, Xiaolong; Guo, Yuhong; Zhao, Jingxia; He, Shasha; Wang, Yan; Lin, Yan; Wang, Ning; Liu, Qingquan

2017-09-01

Current data have shown that punicalagin (PUN), an ellagitannin isolated from pomegranate, possesses anti-inflammatory and anti-oxidant properties; however, its direct targets have not yet been reported. This is the first report that PTP1B serves as a direct target of PUN, with IC 50 value of 1.04μM. Results from NPOI further showed that the K on and K off of PUN-PTP1B complex were 3.38e2M -1 s -1 and 4.13e-3s -1 , respectively. The active site Arg24 of PTP1B was identified as a key binding site of PUN by computation simulation and point mutation. Moreover, inhibition of PTP1B by PUN promoted an M2c-like macrophage polarization and enhanced anti-inflammatory cytokines expression, including IL-10 and M-CSF. Based on gene expression profile, we elucidated that PUN treatment significantly up-regulated 275 genes and down-regulated 1059 genes. M1-like macrophage marker genes, such as Tlr4, Irf1/2, Hmgb1, and Stat1 were down-regulated, while M2 marker genes, including Tmem171, Gpr35, Csf1, Il1rn, Cebpb, Fos, Vegfα, Slc11a1, and Bhlhe40 were up-regulated in PUN-treated macrophages. Hmox-1, a gene encoding HO-1 protein, was preferentially expressed with 16-fold change. Inhibition of HO-1 obviously restored PUN-induced M2 polarization and IL-10 secretion. In addition, phosphorylation of both Akt and STAT3 contributed to PUN-induced HO-1 expression. This study provided new insights into the mechanisms of PUN-mediated anti-inflammatory and anti-oxidant activities and provided new therapeutic strategies for inflammatory diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

The UK MRC Mitochondrial Disease Patient Cohort Study: clinical phenotypes associated with the m.3243A>G mutation--implications for diagnosis and management.

PubMed

Nesbitt, Victoria; Pitceathly, Robert D S; Turnbull, Doug M; Taylor, Robert W; Sweeney, Mary G; Mudanohwo, Ese E; Rahman, Shamima; Hanna, Michael G; McFarland, Robert

2013-08-01

Population-based studies suggest the m.3243A>G mutation in MTTL1 is the most common disease-causing mtDNA mutation, with a carrier rate of 1 in 400 people. The m.3243A>G mutation is associated with several clinical syndromes including mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS), maternally inherited deafness and diabetes (MIDD) and progressive external ophthalmoplegia (PEO). Many patients affected by this mutation exhibit a clinical phenotype that does not fall within accepted criteria for the currently recognised classical mitochondrial syndromes. We have defined the phenotypic spectrum associated with the m.3243A>G mtDNA mutation in 129 patients, from 83 unrelated families, recruited to the Mitochondrial Disease Patient Cohort Study UK. 10% of patients exhibited a classical MELAS phenotype, 30% had MIDD, 6% MELAS/MIDD, 2% MELAS/chronic PEO (CPEO) and 5% MIDD/CPEO overlap syndromes. 6% had PEO and other features of mitochondrial disease not consistent with another recognised syndrome. Isolated sensorineural hearing loss occurred in 3%. 28% of patients demonstrated a panoply of clinical features, which were not consistent with any of the classical syndromes associated with the m.3243A>G mutation. 9% of individuals harbouring the mutation were clinically asymptomatic. Following this study we propose guidelines for screening and for the management of confirmed cases.

Effects of clopidogrel and clarithromycin on the disposition of sibutramine and its active metabolites M1 and M2 in relation to CYP2B6*6 polymorphism.

PubMed

Pan, Wei; Bae, Soo-Kyung; Shim, Eon-Jeong; Park, Sung-Eun; Lee, Sang-Seop; Park, Soo-Jin; Yeo, Chang-Woo; Zhou, Hong-Hao; Shon, Ji-Hong; Shin, Jae-Gook

2013-02-01

Plasma concentrations of sibutramine and its two active metabolites after single oral dose of sibutramine were determined in Korean healthy male subjects with different CYP2B6 genotypes (CYP2B6*1/*1, *1/*6 and *6/*6), either alone or after four-day pretreatment with clopidogrel or clarithromycin. The pretreatment with clopidogrel and clarithromycin raised the mean area under the concentration-time curve (AUC) of sibutramine by 163% and 255%, respectively. Co-administration of clarithromycin, combined with CYP2B6*6/*6 genotype, led to highest concentration of sibutramine. The molar sum AUC (M1 + M2) was raised by 35% in the clopidogrel phase but not significantly affected by clarithromycin or CYP2B6 genotype. The CYP2B6*6/*6 subjects in the clopidogrel phase showed the highest molar AUC (M1 + M2) among three genotype groups throughout the three phases. The exposure of sibutramine and its metabolites seemed to be associated with the CYP2B6 genotype. The treatment of clopidogrel significantly altered the disposition of active metabolites as well as sibutramine, but clarithromycin only affects the disposition of sibutramine. These results suggest that the perturbation of CYP2B6 activity may contribute to the inter-individual variation of sibutramine drug responses although the clinical relevance is remained to be established.

Increased intermediate M1-M2 macrophage polarization and improved cognition in mild cognitive impairment patients on ω-3 supplementation.

PubMed

Famenini, Sam; Rigali, Elizabeth A; Olivera-Perez, Henry M; Dang, Johnny; Chang, Michael To; Halder, Ramesh; Rao, Rammohan V; Pellegrini, Matteo; Porter, Verna; Bredesen, Dale; Fiala, Milan

2017-01-01

Monocyte/macrophages of patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) are defective in phagocytosis and degradation amyloid β 1-42 (Aβ 1-42 ), but are improved by ω-3 fatty acids (ω-3s). The hypothesis of this study was that active Aβ 1-42 phagocytosis by macrophages prevents brain amyloidosis and thus maintains cognition. We studied the effects of self-supplementation with a drink with ω-3s, antioxidants, and resveratrol on Mini-Mental State Examination (MMSE) scores, macrophage M1M2 phenotype [the ratio of inflammatory cluster of differentiation (CD)54+CD80 and proresolution markers CD163+CD206], and Aβ 1-42 phagocytosis in patients initially diagnosed as having MCI or subjective cognitive impairment (SCI). At baseline, the median MMSE score in patients in both the apolipoprotein E (ApoE) ε3/ε3 and ApoE ε3/ε4 groups was 26.0 and macrophage Aβ 1-42 phagocytosis was defective. The MMSE rate of change increased in the ApoE ε3/ε3 group a median 2.2 points per year (P = 0.015 compared to 0) but did not change in the ApoE ε3/ε4 group (P = 0.014 between groups). In the ApoE ε3/ε3 group, all patients remained cognitively stable or improved; in the ApoE ε3/ε4 group, 1 recovered from dementia, but 3 lapsed into dementia. The macrophage phenotype polarized in patients bearing ApoE ε3/ε3 to an intermediate (green zone) M1-M2 type at the rate of 0.226 U/yr, whereas in patients bearing ApoE ε3/ε4, polarization was negative (P = 0.08 between groups). The baseline M1M2 type in the extreme M1 (red zone) or M2 (white zone) was unfavorable for cognitive outcome. Aβ 1-42 phagocytosis increased in both ApoE groups (P = 0.03 in each groups). In vitro, the lipidic mediator resolvin D1 (RvD1) down regulated the M1 type in patients with ApoE ε3/ε3 but in some patients with ε3/ε4, paradoxically up-regulated the M1 type. Antioxidant/ω-3/resveratrol supplementation was associated with favorable immune and cognitive responses in Apo

Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever

PubMed Central

Durbin, Anna P.; Vargas, Maria José; Wanionek, Kimberli; Hammond, Samantha N.; Gordon, Aubree; Rocha, Crisanta; Balmaseda, Angel; Harris, Eva

2008-01-01

In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14+, CD32+, CD86+, CD11c+). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis. PMID:18452966

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

PubMed Central

Takeda, Makoto; Pekosz, Andrew; Shuck, Kevin; Pinto, Lawrence H.; Lamb, Robert A.

2002-01-01

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31 virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31 virus. We found that the M2-del29–31 virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31 virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31 virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2–Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells

PubMed Central

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state. PMID:29410668

The underlying mechanism of proinflammatory NF-κB activation by the mTORC2/Akt/IKKα pathway during skin aging

PubMed Central

Choi, Yeon Ja; Moon, Kyoung Mi; Chung, Ki Wung; Jeong, Ji Won; Park, Daeui; Kim, Dae Hyun; Yu, Byung Pal; Chung, Hae Young

2016-01-01

Mammalian target of rapamycin complex 2 (mTORC2), one of two different enzymatic complexes of mTOR, regulates a diverse set of substrates including Akt. mTOR pathway is one of well-known mediators of aging process, however, its role in skin aging has not been determined. Skin aging can be induced by physical age and ultraviolet (UV) irradiation which are intrinsic and extrinsic factors, respectively. Here, we report increased mTORC2 pathway in intrinsic and photo-induced skin aging, which is implicated in the activation of nuclear factor-κB (NF-κB). UVB-irradiated or aged mice skin revealed that mTORC2 activity and its component, rictor were significantly upregulated which in turn increased Akt activation and Akt-dependent IκB kinase α (IKKα) phosphorylation at Thr23 in vivo. We also confirmed that UVB induced the mTORC2/Akt/IKKα signaling pathway with HaCaT human normal keratinocytes. The increased mTORC2 signaling pathway during skin aging were associated to NF-κB activation. Suppression of mTORC2 activity by the treatment of a mTOR small inhibitor or knockdown of RICTOR partially rescued UVB-induced NF-κB activation through the downregulation of Akt/IKKα activity. Our data demonstrated the upregulation of mTORC2 pathway in intrinsic and photo-induced skin aging and its role in IKKα/NF-κB activation. These data not only expanded the functions of mTOR to skin aging but also revealed the therapeutic potential of inhibiting mTORC2 in ameliorating both intrinsic skin aging and photoaging. PMID:27486771

PPARδ induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTOR activation.

PubMed

Yuan, Hongyan; Lu, Jin; Xiao, Junfeng; Upadhyay, Geeta; Umans, Rachel; Kallakury, Bhaskar; Yin, Yuhzi; Fant, Michael E; Kopelovich, Levy; Glazer, Robert I

2013-07-15

The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In this study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathologic changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to 5 months. Histopathologic changes occurred concurrently with an increase in an inflammatory, invasive, metabolic, and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning 1 week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTOR signaling that were attenuated by treatment with the mTOR inhibitor everolimus. Our findings are the first to show a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. ©2013 AACR.

Study of patients with Hyper-IgM type IV phenotype who recovered spontaneously during late childhood and review of the literature.

PubMed

Karaca, Neslihan Edeer; Durandy, Anne; Gulez, Nesrin; Aksu, Guzide; Kutukculer, Necil

2011-08-01

Hyper-IgM syndromes are characterized by normal or elevated serum IgM levels with the absence or reduced levels of other immunoglobulins. There are some patients with defective class-switch recombination (CSR) who do not have CD40L, CD40, AID, and UNG defects. The aim of this study is to determine the B-cell functions of patients with Hyper-IgM type 4 phenotype. Ten patients (seven males and three females) 84.2 ± 16.5 months of age with initial low serum IgG and IgA and high or normal IgM levels were included. Clinically, 50% had recurrent upper respiratory tract, 10% urinary tract, 10% lower respiratory tract infections, and 30% had mixed type infections. Lymphoid hyperplasia, overt autoimmune manifestations, or malignancy was not noted. Seven of 10 patients were studied twice; at the age of 34.2 ± 13.7 and at 86.6 ± 12.3 months. Absolute lymphocyte counts and lymphocyte subsets were normal in all cases. All of them had normal expression of CD40 on B cells and CD40L on activated T cells for males. At first examination, all patients had normal in vitro sCD40L+rIL-4-induced B cell proliferation response and somatic hypermutation but CSR towards IgE was absent. AID and UNG genes did not show any abnormalities. All showed improvement in both clinical findings and Ig levels during the follow-up period of 55.8 ± 14.8 months. Ages for normalization of IgG and IgA were 68.2 ± 8.7 and 70.2 ± 21.6 months, respectively. During the second evaluation: In vitro sCD40L+rIL-4-induced B-cell proliferation was normal in all cases, whereas CSR was still abnormal in five of eight patients. Two of the patients had an increase in in vitro CSR response but still low IgG2 subclass levels. Three patients with initially absent in vitro CSR response also normalized. Clinical manifestations and immunoglobulin levels of the patients with Hyper-IgM type 4 phenotype recovered in late childhood at about 6 years of age. There was a transient CSR defect which was

Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type, which mediates inhibition of tumour immune-evasion via the Toll-like receptor 2 pathway.

PubMed

Liu, Yi; Zhang, Lingyun; Zhu, Xiangxiang; Wang, Yuehua; Liu, WenWei; Gong, Wei

2015-11-01

Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti-cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin-6 (IL-6), IL-12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr-1(+) CD11b(+) monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll-like receptor 2 (TLR2) signal of Gr-1(+) CD11b(+) MDSCs and increased their M1-type macrophage characteristics, such as producing IL-12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr-1(+) CD11b(+) MDSCs by pAbM treatment had less ability to convert the CD4(+) CD25(-) cells into CD4(+) CD25(+) phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti-tumour effect of pAbM was dependent on Gr-1(+ ) CD11b(+) monocytes, nether CD8(+) T cells nor CD4(+) T cells. In addition to, pAbM did not inhibit tumour growth in TLR2(-/-) mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti-tumour effects through the opposite of the suppressive function of Gr-1(+) CD11b(+) MDSCs. © 2015 John Wiley & Sons Ltd.

Interleukin-4 Ameliorates the Functional Recovery of Intracerebral Hemorrhage Through the Alternative Activation of Microglia/Macrophage.

PubMed

Yang, Jianjing; Ding, Saidan; Huang, Weilong; Hu, Jiangnan; Huang, Shengwei; Zhang, Yu; Zhuge, Qichuan

2016-01-01

Neuro-inflammation plays an important role in the recovery of brain injury after stroke. Microglia/macrophage is the major executor in the neuro-inflammation, which can be polarized into two distinct phenotypes: injurious/toxic classical activation (M1 phenotype) and protective alternative activation (M2 phenotype). Here, we investigated whether intracerebral administration of interleukin-4 (IL-4) at an early stage could affect the activation of microglia/macrophage and the corresponding outcome after intracerebral hemorrhage (ICH). The neuro-behavior was recorded between different groups in the rat ICH model. The M1 and M2 markers were then determined by qRT-PCR, western blotting, ELISA, and immunofluorescence, respectively. We observed aberrant activation of microglia/macrophage after ICH. After intracerebral injection of IL-4, M1 activation was greatly inhibited while M2 activation was enhanced, along with improving neurobehavioral recovery from deficits after ICH. Our study showed that early intracerebral injection of IL-4 potentially promotes neuro-functional recovery, probably through enhancing the alternative activation of microglia/macrophage.

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells*

PubMed Central

Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S.

2016-01-01

The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca2+] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca2+-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca2+ entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca2+-dependent up-regulation of AQP5. These important findings reveal that the Ca2+-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. PMID:26903518

COX-2/mPGES-1/PGE2 cascade activation mediates uric acid-induced mesangial cell proliferation.

PubMed

Li, Shuzhen; Sun, Zhenzhen; Zhang, Yue; Ruan, Yuan; Chen, Qiuxia; Gong, Wei; Yu, Jing; Xia, Weiwei; He, John Ci-Jiang; Huang, Songming; Zhang, Aihua; Ding, Guixia; Jia, Zhanjun

2017-02-07

Hyperuricemia is not only the main feature of gout but also a cause of gout-related organ injuries including glomerular hypertrophy and sclerosis. Uric acid (UA) has been proven to directly cause mesangial cell (MC) proliferation with elusive mechanisms. The present study was undertaken to examined the role of inflammatory cascade of COX-2/mPGES-1/PGE2 in UA-induced MC proliferation. In the dose- and time-dependent experiments, UA increased cell proliferation shown by the increased total cell number, DNA synthesis rate, and the number of cells in S and G2 phases in parallel with the upregulation of cyclin A2 and cyclin D1. Interestingly, UA-induced cell proliferation was accompanied with the upregulation of COX-2 and mPGES-1 at both mRNA and protein levels. Strikingly, inhibition of COX-2 via a specific COX-2 inhibitor NS-398 markedly blocked UA-induced MC proliferation. Meanwhile, UA-induced PGE2 production was almost entirely abolished. Furthermore, inhibiting mPGES-1 by a siRNA approach in MCs also ameliorated UA-induced MC proliferation in line with a significant blockade of PGE2 secretion. More importantly, in gout patients, we observed a significant elevation of urinary PGE2 excretion compared with healthy controls, indicating a translational potential of this study to the clinic. In conclusion, our findings indicated that COX-2/mPGES-1/PGE2 cascade activation mediated UA-induced MC proliferation. This study offered new insights into the understanding and the intervention of UA-related glomerular injury.

DEL phenotype.

PubMed

Kwon, Dong H; Sandler, S G; Flegel, Willy A

2017-09-01

DEL red blood cells (RBCs) type as D- by routine serologic methods and are transfused routinely, without being identified as expressing a very weak D antigen, to D- recipients. DEL RBCs are detected only by adsorption and elution of anti-D or by molecular methods. Most DEL phenotypes have been reported in population studies conducted in East Asia, although DEL phenotypes have been detected also among Caucasian individuals. Approximately 98 percent of DEL phenotypes in East Asians are associated with the RHD*DEL1 or RHD*01EL.01 allele. The prevalence of DEL phenotypes has been reported among D- Han Chinese (30%), Japanese (28%), and Korean (17%) populations. The prevalence of DEL phenotypes is significantly lower among D- Caucasian populations (0.1%). Among the 3-5 percent of African individuals who are D-, there are no reports of the DEL phenotype. Case reports from East Asia indicate that transfusion of DEL RBCs to D- recipients has been associated with D alloimmunization. East Asian immigrants constitute 2.1 percent of the 318.9 million persons residing in the United States, and an estimated 2.8 percent are blood donors. Using these statistics, we estimate that 68-683 units of DEL RBCs from donors of East Asian ancestry are transfused as D- annually in the United States. Given the reports from East Asia of D alloimmunization attributed to transfusion of DEL RBCs, one would expect an occasional report of D alloimmunization in the United States following transfusion of DEL RBCs to a D- recipient. If such cases do occur, the most likely reason that they are not detected is the absence of active post-transfusion monitoring for formation of anti-D.

Regulation of immunophenotype modulation of monocytes-macrophages from M1 into M2 by prostate cancer cell-culture supernatant via transcription factor STAT3.

PubMed

Solís-Martínez, R; Cancino-Marentes, M; Hernández-Flores, G; Ortiz-Lazareno, P; Mandujano-Álvarez, G; Cruz-Gálvez, C; Sierra-Díaz, E; Rodríguez-Padilla, C; Jave-Suárez, L F; Aguilar-Lemarroy, A; Bravo-Cuellar, A

2018-04-01

Transcription factor STAT3 has a prominent innate immunity effect on cancer progression. We determined the regulation of STAT3 in the immunophenotype modulation of macrophages from M1 into M2 induced by the cell-culture supernatant of the Prostate-Cancer line PC3. Monocytes-macrophages from healthy donors were cultured in the supernatant of PC3 cells, membrane proteins, and intracytoplasmic and phosphorylated STAT3 were measured using flow cytometry, while cytokines and growth factors were studied using luminescence. Cytotoxicity and nitric oxide were evaluated via colorimetric assays. The supernatant of PC3 prostate-tumor cells effectively induced macrophages toward an M2 profile, and the expression of phosphorylated STAT3 in the monocytes-macrophages notably increased, and mainly related to IL-10. In the group of monocytes-macrophages treated with a STAT3 inhibitor, the macrophages were induced toward an M1 phenotype. In this study, we showed that the secretion profile of PC3 prostate-cancer cells induces a change in macrophage phenotype from M1 into M2, and that the phenomenon is related to phosphorylation of transcription factor STAT3 and IL-10. Copyright © 2018. Published by Elsevier B.V.

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down.

PubMed

Forrester, Alison R; Elias, Martina S; Woodward, Emma L; Graham, Mark; Williams, Faith M; Reynolds, Nick J

2014-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

PubMed Central

Forrester, Alison R.; Elias, Martina S.; Woodward, Emma L.; Graham, Mark; Williams, Faith M.; Reynolds, Nick J.

2014-01-01

Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. Objective To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Methods Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. Results In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD > β-NF > ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Conclusion Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. PMID:24161567

Effects of type 1 and type 2 diabetes on the pharmacokinetics of tramadol enantiomers in patients with neuropathic pain phenotyped as cytochrome P450 2D6 extensive metabolizers.

PubMed

de Moraes, Natália Valadares; Lauretti, Gabriela Rocha; Lanchote, Vera Lucia

2014-09-01

The aim of this study was to evaluate the influence of poorly controlled type 1 (T1DM) and type 2 diabetes mellitus (T2DM) on the pharmacokinetics and metabolism of tramadol enantiomers in patients with neuropathic pain. Nondiabetic patients (control group, n = 12), patients with T1DM (n = 9) or T2DM (n = 9), all with neuropathic pain and phenotyped as cytochrome P450 2D6 extensive metabolizers, received a single oral dose of 100 mg racemic tramadol. Serial blood samples were collected over a 24-h period. Patients with T1DM showed reduced Cmax of both tramadol enantiomers. The plasma concentrations of the active (+)-M1 were significantly reduced in T1DM (area under the curve plasma concentration versus time (AUC∞ ): 313.1 ng·h/ml) when compared with nondiabetic patients (AUC∞ : 1246.6 ng·h/ml). The fraction unbound of (+)-M1 was increased in patients with T1DM. Patients with T1DM and T2DM showed reduced AUC and increased fraction unbound of (-)-M1. The reduced total plasma concentrations of the active (+)-M1 in patients with T1DM may not be of clinical relevance because they are counterbalanced by the increased fraction unbound. © 2014 Royal Pharmaceutical Society.

Same MSH2 Gene Mutation But Variable Phenotypes in 2 Families With Lynch Syndrome: Two Case Reports and Review of Genotype-Phenotype Correlation.

PubMed

Liccardo, Raffaella; De Rosa, Marina; Duraturo, Francesca

2018-01-01

Lynch syndrome is an autosomal dominant syndrome that can be subdivided into Lynch syndrome I, or site-specific colonic cancer, and Lynch syndrome II, or extracolonic cancers, particularly carcinomas of the stomach, endometrium, biliary and pancreatic systems, and urinary tract. Lynch syndrome is associated with point mutations and large rearrangements in DNA MisMatch Repair ( MMR ) genes. This syndrome shows a variable phenotypic expression in people who carry pathogenetic mutations. So far, a correlation in genotype-phenotype has not been definitely established. In this study, we describe 2 Lynch syndrome cases presenting with the same genotype but different phenotypes and discuss possible reasons for this.

Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity

PubMed Central

Carroll, Bernadette; Maetzel, Dorothea; Maddocks, Oliver DK; Otten, Gisela; Ratcliff, Matthew; Smith, Graham R; Dunlop, Elaine A; Passos, João F; Davies, Owen R; Jaenisch, Rudolf; Tee, Andrew R; Sarkar, Sovan; Korolchuk, Viktor I

2016-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling. DOI: http://dx.doi.org/10.7554/eLife.11058.001 PMID:26742086

Genetic and phenotypic characterization of resistance to macrolides in Streptococcus pyogenes from Argentina.

PubMed

Martínez, Silvia; Amoroso, Ana M; Famiglietti, Angela; de Mier, Carmen; Vay, Carlos; Gutkind, Gabriel O

2004-01-01

Five hundred and seventy-eight strains of group A streptococci (GAS) isolated mostly from paediatric pharyngeal swabs were tested to evaluate their susceptibility to erythromycin. Resistant strains were then tested for their MICs to erythromycin and clindamycin, their phenotype of resistance to macrolides-lincosamides-streptogramin (MLS(B)) and for the presence of macrolide resistance genes. The rate of resistance to erythromycin was 8.2%. Constitutive, inducible and M phenotypes of resistance were detected in 2.1, 2.1 and 95.8% of resistant strains, respectively. All M phenotypes harboured the mefA gene, whereas constitutive and inducible phenotypes had ermB and ermTR genes, respectively.

Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages.

PubMed

Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, B P; Di Stefano, V; Minutoli, L

2009-08-01

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. LPS-stimulated (1 microg.mL(-1)) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32-128 microg.mL(-1)) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kappaB-alpha (IkappaB-alpha) levels were evaluated by Western blot analysis. Nuclear factor kappaB (NF-kappaB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-alpha (TNF-alpha) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 microg.mL(-1)) significantly inhibited COX-2 (LPS = 18 +/- 2.1; flavocoxid = 3.8 +/- 0.9 integrated intensity), 5-LOX (LPS = 20 +/- 3.8; flavocoxid = 3.1 +/- 0.8 integrated intensity) and iNOS expression (LPS = 15 +/- 1.1; flavocoxid = 4.1 +/- 0.4 integrated intensity), but did not modify COX-1 expression. PGE(2) and LTB(4) levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IkappaB-alpha protein (LPS = 1.9 +/- 0.2; flavocoxid = 7.2 +/- 1.6 integrated intensity), blunted increased NF-kappaB binding activity (LPS = 9.2 +/- 2; flavocoxid = 2.4 +/- 0.7 integrated intensity) and the enhanced

Magel2 knockout mice manifest altered social phenotypes and a deficit in preference for social novelty.

PubMed

Fountain, M D; Tao, H; Chen, C-A; Yin, J; Schaaf, C P

2017-07-01

MAGEL2 is one of five protein-coding, maternally imprinted, paternally expressed genes in the Prader-Willi syndrome (PWS)-critical domain on chromosome 15q11-q13. Truncating pathogenic variants of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG) (OMIM #615547), a neurodevelopmental disorder related to PWS. Affected individuals manifest a spectrum of neurocognitive and behavioral phenotypes, including intellectual disability and autism spectrum disorder (ASD). Magel2 knockout mice carrying a maternally inherited, imprinted wild-type (WT) allele and a paternally inherited Magel2-lacZ knock-in allele, which abolishes endogenous Magel2 gene function, exhibit several features reminiscent of the human Prader-Willi phenotypes, including neonatal growth retardation, excessive weight gain after weaning and increased adiposity in adulthood. They were shown to have altered circadian rhythm, reduced motor activity and reduced fertility. An extensive assessment for autism-like behaviors in this mouse model was warranted, because of the high prevalence of ASD in human patients. The behavior of Magel2 knockout mice and their WT littermates were assayed via open field, elevated plus maze, tube, three-chamber and partition tests. Our studies confirm decreased horizontal activity of male and female mice and increased vertical activity of females, in the open field. Both sexes spent more time in the open arm of the elevated plus maze, suggestive of reductions in anxiety. Both sexes displayed a lack of preference for social novelty, via a lack of discrimination between known and novel partners in the partition test. The in-depth investigation of behavioral profiles caused by Magel2 loss-of-function helps to elucidate the etiology of behavioral phenotypes both for SHFYNG and PWS in general. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Peroxisome proliferator-activated receptor δ (PPARδ) induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTor activation

PubMed Central

Yuan, Hongyan; Lu, Jin; Xiao, Junfeng; Upadhyay, Geeta; Umans, Rachel; Kallakury, Bhaskar; Yin, Yuhzi; Fant, Michael E.; Kopelovich, Levy; Glazer, Robert I.

2013-01-01

The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In the present study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathological changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to five months. Histopathological changes occurred concurrently with an increase in an inflammatory, invasive, metabolic and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning one week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTor signaling that were attenuated by treatment with the mTor inhibitor everolimus. Our findings are the first to demonstrate a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease. PMID:23811944

Long non-coding RNA cox-2 prevents immune evasion and metastasis of hepatocellular carcinoma by altering M1/M2 macrophage polarization.

PubMed

Ye, Yibiao; Xu, Yunxiuxiu; Lai, Yu; He, Wenguang; Li, Yanshan; Wang, Ruomei; Luo, Xinxi; Chen, Rufu; Chen, Tao

2018-03-01

Macrophages have been shown to demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. We investigate long non-coding RNA (lncRNA) cox-2 in macrophage polarization and the regulatory mechanism functions in hepatocellular carcinoma (HCC). Lipopolysaccharide (LPS) was used to induce RAW264.7 macrophages into M1 type, and IL-4 was to induce RAW264.7 macrophages into M2 type. We selected mouse hepatic cell line Hepal-6 and hepatoma cell line HepG2 for co-incubation with M1 or M2 macrophages. Quantitative real-time PCR was used to detect the expressions of lncRNA cox-2 and mRNAs. ELISA was conducted for testing IL-12 and IL-10 expressions; Western blotting for epithelial mesenchymal transition related factors (E-cadherin and Vimentin). An MTT, colony formation assay, flow cytometry, transwell assay, and stretch test were conducted to test cell abilities. The M1 macrophages had higher lncRNA cox-2 expression than that in the non-polarized macrophages and M2 macrophages. The lncRNA cox-2 siRNA decreased the expression levels of IL-12, iNOS, and TNF-α in M1 macrophages, increased the expression levels of IL-10, Arg-1, and Fizz-1 in M2 macrophages (all P < 0.05). The lncRNA cox-2 siRNA reduces the ability of M1 macrophages to inhibit HCC cell proliferation, invasion, migration, EMT, angiogenesis and facilitate apoptosis while strengthening the ability of M2 macrophages to promote proliferation HCC cell growth and inhibit apoptosis. These findings indicate that lncRNA cox-2 inhibits HCC immune evasion and tumor growth by inhibiting the polarization of M2 macrophages. © 2017 Wiley Periodicals, Inc.

Genetic and pharmacological evidence that G2019S LRRK2 confers a hyperkinetic phenotype, resistant to motor decline associated with aging

PubMed Central

Longo, Francesco; Russo, Isabella; Shimshek, Derya R.; Greggio, Elisa; Morari, Michele

2014-01-01

The leucine-rich repeat kinase 2 mutation G2019S in the kinase-domain is the most common genetic cause of Parkinson's disease. To investigate the impact of the G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed in knock-in (KI) mice bearing this kinase-enhancing mutation. Two cohorts of G2019S KI mice and wild-type littermates (WT) were subjected to behavioral tests, specific for akinesia, bradykinesia and overall gait ability, at different ages (3, 6, 10, 15 and 19 months). The motor performance of G2019S KI mice remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. Several lines of evidence suggest that enhanced LRRK2 kinase activity is the main contributor to the observed hyperkinetic phenotype of G2019S KI mice: i) KI mice carrying a LRRK2 kinase-dead mutation (D1994S KD) showed a similar progressive motor decline as WT; ii) two LRRK2 kinase inhibitors, H-1152 and Nov-LRRK2-11, acutely reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or D1994S KD animals. LRRK2 target engagement in vivo was further substantiated by reduction of LRRK2 phosphorylation at Ser935 in the striatum and cortex at efficacious doses of Nov-LRRK2-11, and in the striatum at efficacious doses of H-1152. In summary, expression of the G2019S mutation in the mouse LRRK2 gene confers a hyperkinetic phenotype that is resistant to age-related motor decline, likely via enhancement of LRRK2 kinase activity. This study provides an in vivo model to investigate the effects of LRRK2 inhibitors on motor function. PMID:25107341

mActive: A Randomized Clinical Trial of an Automated mHealth Intervention for Physical Activity Promotion.

PubMed