Fas Ligand Expression Research Articles

Gene Expression of Indoleamine 2,3 Dioxygenase 1, Insulin-Growth Factor 1 and Red/IK Cytokine in Alopecia Areata

Alopecia areata (AA) is a chronic, T-cell mediated autoimmune disease directed against the hair follicle, which partially evolves due to a loss of the immune privilege of the anagen hair follicle. The immune privilege is maintained by several factors, including a downregulation of MHC class I and II, local immunosupressants and expression of Fas ligand. The purpose of the study was to evaluate several factors involved in the collapse and restoration of the immune privilege. We investigated IDO1, IGF1 and red/IK gene expression in lesional and perilesionalscalp biopsies from alopecia areata patients. Seven paired punch-biopsies were taken from the active edge of alopecic plaque and from the perilesional scalp. Expression of IDO1, IGF1 and red/IK genes was performed by qRT-PCR. In lesional tissue, IGF1, IDO1 and red/IK genes showed an increase in the mRNA levels as compared with the perilesional scalp. By comparing the pairs of data for the investigated genes, IDO1was statistically upregulated in the lesional area. No significant differences were observed between the gene expression in mild or severe AA, from the lesional or perilesional areas. IDO1 mRNA expression was higher in patients with a relapse duration of less than 6 months as compared to patients with a relapse duration of more than 6 months; levels of IGF1 and red/IK mRNA are increased in lesionals compared to perilesional scalp area.

Gene Expression of Indoleamine 2,3 Dioxygenase 1, Insulin-Growth Factor 1 and Red/IK Cytokine in Alopecia Areata

Alopecia areata (AA) is a chronic, T-cell mediated autoimmune disease directed against the hair follicle, which partially evolves due to a loss of the immune privilege of the anagen hair follicle. The immune privilege is maintained by several factors, including a downregulation of MHC class I and II, local immunosupressants and expression of Fas ligand. The purpose of the study was to evaluate several factors involved in the collapse and restoration of the immune privilege. We investigated IDO1, IGF1 and red/IK gene expression in lesional and perilesionalscalp biopsies from alopecia areata patients. Seven paired punch-biopsies were taken from the active edge of alopecic plaque and from the perilesional scalp. Expression of IDO1, IGF1 and red/IK genes was performed by qRT-PCR. In lesional tissue, IGF1, IDO1 and red/IK genes showed an increase in the mRNA levels as compared with the perilesional scalp. By comparing the pairs of data for the investigated genes, IDO1was statistically upregulated in the lesional area. No significant differences were observed between the gene expression in mild or severe AA, from the lesional or perilesional areas. IDO1 mRNA expression was higher in patients with a relapse duration of less than 6 months as compared to patients with a relapse duration of more than 6 months; levels of IGF1 and red/IK mRNA are increased in lesionals compared to perilesional scalp area.

Pilot investigation of the association between serum stress neuropeptide levels and lymphocyte expression of fas and fas ligand in critical illness.

In critical illness, apoptotic loss of immunocytes is associated with immunosuppression. To explore expression of Fas/Fas ligand (FasL) on B and T cells from critically ill patients without sepsis compared to matched controls and associations with disease severity and neuropeptide Y (NPY), cortisol, adrenocorticotropic hormone (ACTH), and prolactin (PRL) levels. Repeated-measures correlational design with 36 critically ill patients (14-day follow-up) and 36 controls. Disease severity was assessed using the Multiple Organ Dysfunction Score (MODS) and Multi Organ Failure scale. Fas/FasL values were standardized for viable cell counts. An enzyme-linked immunosorbent assay (NPY) and electrochemiluminescence immunoassay (cortisol, ACTH, and PRL) were employed. Fas and FasL expression on T-helper (p < .0001-.03) and T-cytotoxic cells (p < .0001-.002) and Fas expression on B cells (p < .0001-.03) were higher in patients. MODS severity was associated with FasL expression on cytotoxic T cells (r = .752-.902, p = .023-.037). There was an inverse association between Day 1 NPY levels and Fas expression on T-helper cells (r = -.447, p = .019). On the day of maximum severity, we report for the first time an inverse association between NPY levels and FasL expression on helper (r = -.733, p = .016) and cytotoxic (r = -.862, p = .003) T cells. Cortisol levels were positively associated with counts of FasL-positive helper (r = .828) and cytotoxic (r = .544, p < .05) T cells. Results suggest a potential role for stress neuropeptides in lymphocyte survival and activation in critical illness.

Glycyrrhetinic Acid inhibits cell growth and induces apoptosis in ovarian cancer a2780 cells.

Accumulating evidence indicates that glycyrrhizin (GZ) and its hydrolyzed metabolite 18-β glycyrrhetinic acid (GA) exhibit anti-inflammatory and anticancer activities. The objective of this study was to examine the in vitro cytotoxic activity of GA on human ovarian cancer A2780 cells. A2780 cells were cultured in RPMI1640 containing 10% fetal bovine serum. Cells were treated with different doses of GA and cell viability and proliferation were detected by dye exclusion and 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays. Apoptosis induction and expression of Fas and Fas ligand (FasL) were analyzed by flow cytometry. We observed that GA decreases cell viability and suppressed cells proliferation in a dose-dependent manner as detected by dye-exclusion and XTT assayes. In addition, our flow cytometry data show that GA not only induces apoptosis in A2780 cells but also upregulates both Fas and FasL on these cells in a dose-dependent manner. we demonstrate that GA causes cell death in A2780 cells by inducing apoptosis.

Dysfunction of bone marrow vascular niche in acute graft-versus-host disease after MHC-haploidentical bone marrow transplantation.

Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4+, but not CD8+ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4+ donor T cells through the Fas/FasL pathway.

The retinal pigment epithelium (RPE) induces FasL and reduces iNOS and Cox2 in primary monocytes.

Retinal pigment epithelium (RPE) cells may alter the phenotype of monocytes by soluble factors that may be influenced by stimulation of the RPE. Since RPE cells carry the toll-like receptor-3 (TLR3) that detects and reacts to viral infection through binding of dsRNA we investigated the effects of RPE cells with or without TLR3 stimulation on blood-derived monocytes with respect to regulation of pro-/anti-inflammatory cytokines, anti-angiogenic factors and migratory properties. Primary RPE cells were prepared from porcine eyes; monocytes were prepared from porcine blood. TLR3 activation was induced by polyinosinic:polycytidylic acid (Poly I:C). RPE cells were stimulated with Poly I:C in different concentrations for 24 hours and a cell culture supernatant was applied to the monocytes. Expression of CD14 and Fas ligand (FasL) was determined via flow cytometry. The expression of IL-6, IL-1ß, TNFα, Cox2, iNOS and IL-10 was determined via quantitative RT-PCR. Migration was determined using Boyden chamber experiments. The supernatant of RPE cells, irrespective of TLR3 activation, induced FasL expression in the monocytes. Expression of iNOS and Cox2 was reduced by RPE cells and the reduction of Cox2 but not if iNOS was lost under TLR3 activation. No induction of IL-6, IL-1ß, IL-10 or TNFα by the RPE was seen. TLR3-activated RPE cells induced monocyte migration. RPE cells induce an upregulation of FasL and a downregulation of iNOS and Cox2 without upregulating inflammatory cytokines, possibly inducing an anti-angiogenic phenotype in the monocytes. This phenotype is still upheld after challenging RPE cells with dsRNA, mimicking a viral infection.

Lymphopenia associated with highly virulent H5N1 virus infection due to plasmacytoid dendritic cell-mediated apoptosis of T cells.

Although lymphopenia is a hallmark of severe infection with highly pathogenic H5N1 and the newly emerged H7N9 influenza viruses in humans, the mechanism(s) by which lethal H5N1 viruses cause lymphopenia in mammalian hosts remains poorly understood. Because influenza-specific T cell responses are initiated in the lung draining lymph nodes (LNs), and lymphocytes subsequently traffic to the lungs or peripheral circulation, we compared the immune responses in the lung draining LNs postinfection with a lethal A/HK/483/97 or nonlethal A/HK/486/97 (H5N1) virus in a mouse model. We found that lethal H5N1, but not nonlethal H5N1, virus infection in mice enhances Fas ligand (FasL) expression on plasmacytoid dendritic cells (pDCs), resulting in apoptosis of influenza-specific CD8(+) T cells via a Fas-FasL-mediated pathway. We also found that pDCs, but not other DC subsets, preferentially accumulate in the lung draining LNs of lethal H5N1 virus-infected mice, and that the induction of FasL expression on pDCs correlates with high levels of IL-12p40 monomer/homodimer in the lung draining LNs. Our data suggest that one of the mechanisms of lymphopenia associated with lethal H5N1 virus infection involves a deleterious role for pDCs.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a novel mechanism regulating the tumor endothelial barrier and T cell homing to tumors. Selective expression of the death mediator Fas ligand (FasL/CD95L) was detected in the vasculature of many human and mouse solid tumors but not in normal vasculature, and in these tumors it was associated with scarce CD8+ infiltration and predominance of FoxP3+ T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression on endothelial cells, which acquired the ability to kill effector CD8+ T cells, but not Treg cells, due to higher levels of cFLIP expression in Tregs. In the mouse, genetic or pharmacologic suppression of FasL produced a significant increase in the influx of tumor-rejecting CD8+ over FoxP3+ T cells. Pharmacologic inhibition of VEGF and PGE2 attenuated tumor endothelial FasL expression, produced a significant increase in the influx of tumor-rejecting CD8+ over FoxP3+ T cells, which was FasL-dependent, and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which plays a critical role in establishing immune tolerance and determining the fate of tumors.

Tu1677 Upregulation of Fas Ligand Expression Promotes Colon Cancer Cell Migration and Metastasis In Vitro and In Vivo

Introduction: We earlier established that sprouty-2 is overexpressed in human colon cancer and up-regulates c-met and thereby increases the growth phenotype and metastatic potential of colon cancer cells (Oncogene, 29: 5241-53, 2010). To our knowledge, this was the first study to demonstrate the tumor oncogene phenotype of sprouty-2 in colon cancer metastasis. On the contrary, cancer protective role of sprouty-2 was noted in breast, liver and prostate cancers. However, a recent report on non-small cell lung cancer (NSCLC) discovered that silencing of sprouty-2 reduced c-met expression and thus confirmed our findings in colorectal cancer (PNAS, 110: 8573-78, 2013). We further extended our studies to demonstrate that this pathway modulates gastrointestinal specific microRNAs (miRNAs) and regulates various oncogenes and tumor suppressor genes in colon cancer cells. Methods: RNA from control and shRNA sprouty-2 transfected cells were labeled with Hy5 and Hy3 respectively and hybridized to miRCURY LNA arrays. Unsupervised miRNA clustering was carried out and a heat map prepared with Java TreeView. MicroRNA targets were identified by Real-time PCR, western blotting and 3'UTR luciferase assays. Migratory phenotype of colon cancer cells was confirmed by cell migration and invasion assays in trans-well chambers. Cancer cells were transfected by miRNA mimics to assess the expression levels of target genes. Human colon cancer tissue microarrays were utilized to assess the expression of miRNA target genes. Human tumor sections were evaluated by immunofluorescence and immunohistochemical staining. Results: Sprouty-2 shRNA transfected stable cell lines demonstrated upregulation of tumor suppressive miRNAs and down-regulation of oncogenic miRNAs. Transfection of miRNA mimics regulated AKT, p21, N-cadherin and E-cadherin expression in western blotting and RTPCR experiments. Both shRNA sprouty-2 and miRNA mimics blocked cell migration and invasion in cancer cells. AKT was upregulated whereas E-cadherin was downregulated at the invasive fronts in human colon tumors. Conclusion: Sprouty-2/ AKT/E-cadherin expression levels could be used to risk-stratify patients for CRC recurrence and sprouty-2 axis regulation may lead to the development of therapeutics in colon and lung cancer.

Phenethyl isothiocyanate inhibits growth of human chronic myeloid leukemia K562 cells via reactive oxygen species generation and caspases.

Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochromec as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrinIX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

CD4+ T cells from food allergy model are resistant to TCR-dependent apoptotic induction

BackgroundCD4+ T cell polarization plays a critical role in the pathogenesis of allergy. How to modulate the skewed CD4+ T cell polarization is less clear. The specific immunotherapy (SIT) is the only specific remedy for the treatment of allergic diseases; the therapeutic effect is to be improved. ObjectivesThis study aims to investigate the role of interleukin (IL)-18 in enhancing the therapeutic effect of SIT. MethodsA peanut allergy mouse model was developed and treated with SIT or/and IL-18. CD4+ T cell apoptosis was assessed by flow cytometry. The expression of Fas ligand (FasL) was observed by quantitative real time RT-PCR and Western blotting. Interferon-γ in the culture medium was determined by enzyme-linked immunosorbent assay. The fasL gene promoter methylation in CD4+ T cells was assessed by methylation specific PCR. ResultsThe results showed that lower levels of IL-18 were detected in allergic mice; administration of IL-18 significantly enhanced the therapeutic effect of SIT on suppressing the allergic inflammation in the mouse intestine. In the cell culture studies, IL-18 increased the TCR-dependent CD4+ T cell apoptosis, the expression of FasL in CD4+ T cells, the production of Interferon-γ and the demethylation of the FasL promoter in CD4+ T cells. ConclusionsAdministration of IL-18 enhances the effect of SIT on suppressing allergic inflammation in the mouse intestine via enhancing the TCR-dependent CD4+ T cell apoptosis.

Antibody ligation of CM1 on cisplatin-exposed HeLa cells induces apoptosis through reactive oxygen species-dependent Fas ligand expression

Centrocyte/centroblast marker 1 (CM1) has been identified as a pro-apoptosis molecule on B-cell lymphoma cells as well as several types of cancer cells. In this study, we investigated its signaling mechanism in HeLa cells after treatment with cisplatin in order to potentially identify a new therapeutic target. The CM1 molecule was induced on the surface of cisplatin-exposed HeLa cells. In these cells, ligation of CM1 with anti-CM1 monoclonal antibodies inhibited cell proliferation and produced reactive oxygen species. Fas ligand (FasL) expression was upregulated without upregulating Fas in cisplatin-exposed HeLa cells after CM1 stimulation. Pretreatment with N-acetylcysteine, a pan-capase inhibitor, and ZB4, an antagonistic anti-Fas antibody, effectively inhibited the apoptotic effect triggered by CM1. CM1 ligation induced apoptosis through disruption of the mitochondrial membrane potential, decreased Bcl-2 and phosphorylated ERK expression. These findings identify CM1 as a potential new therapeutic target related to cisplatin-exposed cervical cancer.

Expression of Fas (CD95/APO-1) and Fas Ligand (FasL) in Experimentally-Induced Acute Pancreatitis

ABSTRACTIntroduction: Acinar cell death is a crucial event in acute pancreatitis (AP) and may occur either by apoptosis or necrosis. The aim of this study was to investigate the expression of the apoptosis associated proteins Fas and FasL in experimentally induced severe AP. Methods: AP was induced in 30 rats by injecting 0.2 ml of 4.5% sodium taurocholate solution into the biliopancreatic duct. Sham operated animals (n = 30) and 10 normal controls were used for comparisons. Animals were killed at 6, 12, 24, 48, 72 hr and 1 week after operation (five animals at each time point) and both serum and pancreatic tissue were obtained. The severity of AP was graded by morphological evaluation and by measuring serum amylase levels. Acinar cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Tissue expressions of Fas and FasL were evaluated by immunohistochemistry. Results: Sodium taurocholate injection resulted in severe acute necrotizing pancreatitis as early as six hr after taurocholate infusion with gradually increasing severity and a peak at 72 hr, and a significant increase of serum amylase at 6 and 12 hr. Apoptotic acinar cells were observed between 48 and 72 hr. The expression of both Fas and FasL in pancreatic tissue was induced in comparison with normal controls. Fas expression in AP was higher and statistically significant at 24 hr whereas FasL expression was consistently lower with a statistical significance observed at 12 hr when compared to sham-operated animals suggesting Fas upregulation and FasL downregulation in this model of AP. Conclusions: Induction and sequential changes in the expressions of Fas and FasL occur during taurocholate induced severe AP in rats and their temporal modulation might associate with acinar cell death by apoptosis.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Overexpression of Fas and FasL Is Associated with Infectious Complications and Severity of Experimental Severe Acute Pancreatitis by Promoting Apoptosis of Lymphocytes

This study investigated the relationship of Fas and Fas ligand (FasL) expression and apoptosis of lymphocytes in relation to the pathogenic immune response and infectious complications observed in experimental severe acute pancreatitis in mice. Forty male Balb/c mice were randomly divided into control, mild (MAP), and severe acute pancreatitis (SAP) groups. Overexpression of Fas/FasL messenger ribonucleic acid (mRNA) and protein was observed in spleen-derived lymphocytes in SAP (p < 0.01). Apoptosis of these resulted in a depletion of circulating lymphocytes in this group (p < 0.05). A further significant change in the SAP group with infectious complications was observed. A positive relationship was found between the Fas/FasL expression and lymphocyte apoptosis, and negative relationships were observed between Fas/FasL expression and CD4+ and CD19+ lymphocytes and the CD4+/CD8+ ratio in SAP mice (p < 0.01). The results suggest that the overexpression of Fas/FasL is associated with infectious complications and severity of experimental severe acute pancreatitis by promoting apoptosis of lymphocytes.

Intestinal epithelial apoptosis initiates gut mucosal injury during extracorporeal membrane oxygenation in the newborn piglet

Neonates and young infants exposed to extracorporeal circulation during extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass are at risk of developing a systemic inflammatory response syndrome with multi-organ dysfunction. We used a piglet model of ECMO to investigate the hypothesis that epithelial apoptosis is an early event that precedes villous damage during ECMO-related bowel injury. Healthy 3-week-old piglets were subjected to ECMO for up to 8 h. Epithelial apoptosis was measured in histopathological analysis, nuclear imaging, and terminal deoxynucleotidyl transferase dUTP nick end labeling. Plasma intestinal fatty acid-binding protein (I-FABP) levels were measured by enzyme immunoassay. Intestinal mast cells were isolated by fluorescence-assisted cell sorting. Cleaved caspase-8, caspase-9, phospho-p38 MAPK, and fas ligand expression were investigated by immunohistochemistry, western blots, and reverse transcriptase-quantitative PCR. Piglet ECMO was associated with increased gut epithelial apoptosis. Extensive apoptotic changes were noted on villus tips and in scattered crypt cells after 2 h of ECMO. After 8 h, the villi were denuded and apoptotic changes were evident in a majority of crypt cells. Increased circulating I-FABP levels, a marker of gut epithelial injury, showed that epithelial injury occurred during ECMO. We detected increased cleaved caspase-8, but not cleaved caspase-9, in epithelial cells indicating that the extrinsic apoptotic pathway was active. ECMO was associated with increased fas ligand expression in intestinal mast cells, which was induced through activation of the p38 mitogen-activated protein kinase. We conclude that epithelial apoptosis is an early event that initiates gut mucosal injury in a piglet model of ECMO.

CD4+ T cells in HIV infection show increased levels of expression of a receptor for vasoactive intestinal peptide, VPAC2

Immune activation is a strong predictor of disease outcome in HIV infection and promotes the loss of CD4+ T cells. The neuropeptide, vasoactive intestinal peptide (VIP), has immune-modulating properties with specific receptors identified on lymphocytes; VPAC1 and VPAC2. Studies have shown that VIP limits immune activation and apoptosis in T cells by decreasing the expression of the apoptosis signaling molecule Fas ligand (FasL). VIP receptor surface expression has not been investigated by flow cytometry in the context of HIV infection and may represent a novel target for immune-modulating therapy. Eighty-seven untreated HIV-infected individuals with CD4 counts >200 and 57 uninfected controls were recruited from a primary health clinic in Cape Town, South Africa. Flow cytometry was used to determine levels of expression of VPAC1 and VPAC2, as well as FasL on CD4+ T cells, and these results were correlated with the immune activation phenotype %CD38+CD8+ T cells. VPAC2 expression was significantly increased in the HIV group (mean %VPAC2+CD4+ cells 19.25 vs. control 12.56; p≤0.0001), but no difference in VPAC1 expression was observed. VPAC2 correlated positively with FasL (r=0.310; p=0.001), and there was a significant inverse correlation between FasL and the CD4 count (r=-0.211; p=0.013) and a direct correlation with %CD38+CD8+ T cells (r=0.39; p≤0.0001). Thus, higher levels of immune activation correlated with higher levels of the death-signaling FasL and lower CD4 counts. VPAC2 may provide a novel target for the selective limitation of CD4+ T-cell death in HIV infection.

Gene expression of pro- and anti-apoptotic proteins in rock bream (Oplegnathus fasciatus) infected with megalocytivirus (family Iridoviridae)

Viruses belonging to the genus Megalocytivirus cause diseases in marine fishes primarily in East and Southeast Asian countries. Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock beam (Oplegnathus fasciatus) in Korea. In this study, we assessed apoptosis-related gene expression patterns in Megalocytivirus-infected rock bream in high mortality and low mortality conditions to determine important apoptosis-related factors, which may affect fish survival/or death. In the high mortality group (100% mortality at 15 dpi), significantly high levels of perforin, granzyme, Fas ligand and caspase 9 expression (5.6-, 10.2-, 13.4- and 4.2-fold, respectively) were observed in the kidney at 8 dpi. Basal expression levels of Fas and caspase 3 were observed at 8 d (1.5-/0.7-fold) and 10 dpi (1.3-/0.6-fold), accompanied by heavy viral loads (8.12 × 106–2.21 × 107/μl). Inhibitor of apoptosis 1 (IAP1) was highly expressed (3.5- to 4.8-fold) at 1 d and 4 dpi; however, IAP1 was reduced when fish died at 8 d and 10 dpi (1.7- to 2.0-fold), which was not significantly different from that of the control group. A similar expression pattern was observed in the low mortality group (18% expected mortality at 30 dpi), which was characterised by a delayed lower magnitude of expression with lower viral loads than the high mortality group. Perforin, granzyme and Fas ligand expression was significantly higher in the low mortality group than in the control group at several sampling points until 30 dpi. Fas and caspases 8, 9 and 3 expression levels showed no statistical significance until 30 dpi. In the low mortality group, significantly higher IAP1 expression compared with the control was observed at 10 d (2.2-fold), 20 d (3.6-fold) and 22 dpi (2.0-fold). In summary, perforin- and granzyme-related apoptosis initiation signals were activated; however, the Fas-induced apoptosis pathway did not efficiently respond. Upregulated IAP1 in RBIV-infected rock bream, which was reported for the first time in this study, exhibited inhibited apoptotic responses in RBIV-infected fish. Although it remains unclear whether apoptosis inhibition aids or impedes fish survival, our data clearly show that the apoptotic response is inhibited in RBIV-infected rock bream.

Association of genetic variants in apoptosis genes FAS and FASL with radiation-induced late toxicity after prostate cancer radiotherapy

Fas ligand (FASL) triggers apoptotic cell death by cross-linking with its receptor FAS, and after irradiation, expression of FAS and FASL is increased. In the present study, we investigated the association between common polymorphisms in the genes for FAS and FASL and the risk of late side effects after radiotherapy for prostate cancer. The role of FAS (- 1377G > A, rs2234767 and - 670A > G, rs1800682) and FASL (- 844C > T, rs763110) gene polymorphisms in the development of high-grade late rectal and/or urinary toxicity (defined as late toxicity EORTC/RTOG grade ≥ 2) was analyzed in 607 prostate cancer patients treated with radiotherapy. DNA was isolated and the selected polymorphisms were determined by 5'-nuclease (TaqMan) assays. After a median follow-up time of 82months, high-grade late rectal and/or urinary toxicity was observed in 175patients (29.7 %). Univariate analysis revealed a significantly decreased risk of high-grade late toxicity in carriers of the FASL - 844T allele. After adjusting for covariates, patients harboring at least one - 844T allele (CT or TT genotype) remained at decreased risk of high-grade late toxicity compared with patients harboring the CC genotype [hazard ratio (HR) 0.585, 95 %CI 0.39-0.878; p = 0.010]. For patients with the - 844TT genotype, the HR was 0.404 (95 %CI 0.171-0.956; p = 0.039) in multivariate analysis. No significant associations were found for the remaining polymorphisms analyzed. These results provide the first evidence that the presence of the FASL - 844T variant allele may have a protective effect against the development of high-grade late rectal and/or urinary side effects after prostate cancer radiotherapy.

Telomerase governs immunomodulatory properties of mesenchymal stem cells by regulating FAS ligand expression

Bone marrow mesenchymal stem cells (BMMSCs) are capable of differentiating into multiple cell types and regulating immune cell response. However, the mechanisms that govern the immunomodulatory properties of BMMSCs are still not fully elucidated. Here we show that telomerase-deficient BMMSCs lose their capacity to inhibit T cells and ameliorate the disease phenotype in systemic sclerosis mice. Restoration of telomerase activity by telomerase reverse transcriptase (TERT) transfection in TERT−/− BMMSCs rescues their immunomodulatory functions. Mechanistically, we reveal that TERT, combined with β-catenin and BRG1, serves as a transcriptional complex, which binds the FAS ligand (FASL) promoter to upregulate FASL expression, leading to an elevated immunomodulatory function. To test the translational value of these findings in the context of potential clinical therapy, we used aspirin treatment to upregulate telomerase activity in BMMSCs, and found a significant improvement in the immunomodulatory capacity of BMMSCs. Taken together, these findings identify a previously unrecognized role of TERT in improving the immunomodulatory capacity of BMMSCs, suggesting that aspirin treatment is a practical approach to significantly reduce cell dosage in BMMSC-based immunotherapies.Subject Categories Stem Cells; Immunology