Cell Death Of CD4 Research Articles

HIV associated cell death: Peptide-induced apoptosis restricts viral transmission.

The human immunodeficiency virus (HIV) is still a global pandemic and despite the successful use of anti-retroviral therapy, a well-established cure remains to be identified. Viral modulation of cell death has a significant role in HIV pathogenesis. Here we sought to understand the major mechanisms of HIV-induced death of lymphocytes and the effects on viral transmission. Flow cytometry analysis of lymphocytes from five latent HIV-infected patients, and HIV IIIB-infected MT2 cells demonstrated both necrosis and apoptosis to be the major mechanisms of cell death in CD4+ and CD4-/CD8- lymphocytes. Significantly, pro-apoptotic tumor necrosis factor (TNF) peptide (P13) was found to inhibit HIV-related cell death and reduced viral transmission. Whereas pro-necrotic TNF peptide (P16) had little effect on HIV-related cell death and viral transmission. Understanding mechanisms by which cell death can be manipulated may provide additional drug targets to reduce the loss of CD4+ cells and the formation of a viral reservoir in HIV infection.

STING modulates necrotic cell death in CD4 T cells via activation of PARP-1/PAR following acute systemic inflammation

Regulated cell death profoundly affects on the progress of inflammatory and immune responses in various acute inflammatory diseases, as seen in sepsis and trauma. However, the mechanisms underlying CD4 T cells death have not yet been fully addressed. We demonstrated that interferon genes (STING) promoted excessive Poly (ADP-ribose) polymerase 1 (PARP-1) activity stimulated by endotoxin, which in turn induced apoptosis-inducing factor (AIF)-independent but PARP-1 dependent programmed cell death. Elevated PARP-1 activity triggered a cascade of molecular events, including PAR polymer release from the nucleus and the nicotinamide adenine dinucleotide (NAD+) and ATP depletion. Interestingly, translocation of AIF, a biochemical signature for PARP-1-dependent parthanatos, was not observed in the present study, suggesting a non-canonical mechanism of CD4 T cells parthanatos. In this study, we also identify a STING-mediated mechanism of necrotic cell death in CD4 T cells in septic animals. Furthermore, we revealed wider effects of STING on the mortality in mice when PARP-1 gene inhibited. These findings reveal that STING signaling and targeting PARP-1/PAR pathway in CD4 T cells may present a new therapeutic strategy for the treatment of acute systemic inflammatory diseases.

The Interplay of HIV and Autophagy in Early Infection.

HIV/AIDS is still a global threat despite the notable efforts made by the scientific and health communities to understand viral infection, to design new drugs or to improve existing ones, as well as to develop advanced therapies and vaccine designs for functional cure and viral eradication. The identification and analysis of HIV-1 positive individuals that naturally control viral replication in the absence of antiretroviral treatment has provided clues about cellular processes that could interact with viral proteins and RNA and define subsequent viral replication and clinical progression. This is the case of autophagy, a degradative process that not only maintains cell homeostasis by recycling misfolded/old cellular elements to obtain nutrients, but is also relevant in the innate and adaptive immunity against viruses, such as HIV-1. Several studies suggest that early steps of HIV-1 infection, such as virus binding to CD4 or membrane fusion, allow the virus to modulate autophagy pathways preparing cells to be permissive for viral infection. Confirming this interplay, strategies based on autophagy modulation are able to inhibit early steps of HIV-1 infection. Moreover, autophagy dysregulation in late steps of the HIV-1 replication cycle may promote autophagic cell-death of CD4+ T cells or control of HIV-1 latency, likely contributing to disease progression and HIV persistence in infected individuals. In this scenario, understanding the molecular mechanisms underlying HIV/autophagy interplay may contribute to the development of new strategies to control HIV-1 replication. Therefore, the aim of this review is to summarize the knowledge of the interplay between autophagy and the early events of HIV-1 infection, and how autophagy modulation could impair or benefit HIV-1 infection and persistence, impacting viral pathogenesis, immune control of viral replication, and clinical progression of HIV-1 infected patients.

Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall Skeletons (Nr-CWS)

Nocardia rubra cell wall skeleton (Nr-CWS) has been reported to have innate immunostimulating and anti-tumor activities. However, the immunomodulatory effects of Nr-CWS on CD8+ T cells and their related mechanisms are still unknown. In this work, our team purified CD8+T cells from spleen cells and explored the phenotype and function of NR-CWS in vitro on CD8+T cells. We observed that Nr-CWS can significantly up-regulate the expression of CD69 and CD25 on CD8+T cells, with no significant effect on apoptosis or cell death of CD8+T cells that occurs in vitro during culture. In addition, the effect of perforin and granzyme B was increased after Nr-CWS treatment, but did not substantially alter the expression of TRAIL and FasL. A variety of cytokine analyses have shown that of the cytokines examined (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6 and IL-10), only IFN-γ and TNF The increase in -α was more pronounced, and the effect of Nr-CWS in CD8+T cell culture medium on CD8+ T cells was independent of Th cells. Our results demonstrated that Nr-CWS could up-regulate CD69 and CD25 expression on CD8+T cells, promoting IFN-γ and TNF-α secretion, and enhancing perforin and granzyme B production. Thus Nr-CWS may have Immunoaugmenting therapeutic activity via an increase in CD8+T cells response.

Abstract A210: Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor immunity – A resistance mechanism to PD-1 checkpoint blockade in melanoma

Abstract Background: Tumor surface PD-L1 (B7-H1) signals through T-cell surface receptor PD-1 to suppress antitumor immunity. PD-1 inhibitors like pembrolizumab block this pathway to prevent tumor escape from immunosurveillance. These PD-1 checkpoint inhibitors achieve objective response in 20-30% of patients with melanoma (1-3). As the majority of these patients are resistant to this treatment, there is a critical need to combat PD-1 checkpoint inhibitor resistance in melanoma. What is the mechanism of PD-1 checkpoint inhibitor resistance in melanoma? Our group has reported that soluble PD-L1 (sPD-L1, sB7-H1) is elevated in the serum of patients with PD-1 inhibitor-resistant melanoma and non-small cell lung cancer (NSCLC) (4). We have also shown that soluble PD-L1 is produced by cleavage from other tumor types and leads to apoptosis in CD8+ T-cells (5). The aim of this study is to confirm the source of sPD-L1 in human melanoma and its relation to outcomes. Methods: B7H1-expressing transgenic melanoma cells (Mel-B7H1) were treated with an array of protease inhibitors over 24 hours. Cells were then pelleted and supernatants were analyzed by ELISA for sPD-L1 (sB7-H1) production. Tumor serum samples were also analyzed by ELISA for sPD-L1 production.Tumor pathology slides from patients with melanoma (serum donors from the above study) were stained by immunohistochemistry with antibodies against proteases ADAM10 or ADAM17. Slides were visualized by microscopy for the presence of these antigens. Activated human CD8+ T-cells were cultured with recombinant human B7-H1 (PD-L1) protein, sPD-L1-rich, or sPD-L1-depleted supernatants from a tumor cell line in the presence of placebo or PD-1 checkpoint inhibitor antibodies. Cell survival was measured at 48 hours by flow cytometry.Results: ADAM10 inhibitor GI254023X, ADAM17 inhibitor TAPI-0, and broad metalloprotease inhibitor TAPI-2 inhibited Mel-B7H1 production of sB7H1 (p<0.001, 0.095, and 0.001 respectively). Specific inhibitors of other metalloproteases did not significantly alter sB7H1 production. PD-L1 transcription levels were not significantly altered. Tumor pathology slides from patients with high serum sPD-L1 exhibited high ADAM10 and/or ADAM17 staining. Conversely, tumors from patients with low-sPD-L1levels tended to exhibit low ADAM10 and/or ADAM17 staining. Recombinant PD-L1 and PD-L1-rich supernatants induced cell death of CD8+ T-cells, whereas PBS control solution and PD-L1-depleted supernatants did not. Supraphysiologic doses of pembrolizumab decreased cell death in PD-L1-rich supernatant-treated CD8+ T-cells. Conclusions: Matrix metalloprotease inhibitors ADAM10 and ADAM17 mediate sPD-L1 production from melanoma cells. In samples from patients with melanoma, immunohistochemical staining of these proteases in tumor tissue correlates with serum sPD-L1 levels. As we have shown previously, sPD-L1 induces apoptosis of activated CD8+ T-cells, which may explain PD-1 inhibitor resistance in most patients with melanoma.We posit that sPD-L1 outcompetes PD-1 inhibitors to suppress tumor immunosurveillance. Our results suggest that the combination of ADAM10/17 inhibitor and PD-1 checkpoint inhibitor may overcome this mechanism of PD-1 resistance in melanoma and other cancers. Future studies will determine (A) how a combined ADAM10/17 inhibitor and PD-1 inhibitor may function in clinical use, (B) how tumor-derived sPD-L1 signals downstream to cause apoptosis in T-cells, and (C) whether surface ADAM10/17 predict surface PD-L1 staining in melanoma.

IRF-5 Promotes Cell Death in CD4 T Cells during Chronic Infection

The transcription factor interferon regulatory factor 5 (IRF-5) plays an important function in innate immunity and in initiating pro-inflammatory responses against pathogens. IRF-5 is constitutively expressed in several cell types, including plasmacytoid dendritic cells, monocytes, and B cells. We have previously reported that IRF-5 is also expressed in Tcells during infection. The role of IRF-5 in Tcells is yet unknown. Here, we demonstrate that IRF-5 is increasingly expressed in interferon (IFN)-γ+ CD4 Tcells over the course of L.donovani infection. This transcription factor is induced by apoptotic material via Toll-like receptor 7 (TLR7) and promotes the expression of death receptor 5 (DR5). IRF-5 activation sensitizes CD4 Tcells to cell death. Because tissue disruption and chronic inflammation are common characteristics of persistent infections, activation of IRF-5 in CD4 Tcells may represent a common pathway that leads to suppression of protective CD4 Tcell responses, favoring the establishment of chronic infection.

TGF-β and NFAT control T cell receptor restimulation-induced cell death of CD4+CD25+Foxp3+ regulatory cells

Abstract One of the key questions in the area of immunomodulation is how CD4+CD25+Foxp3+ regulatory cells (Tregs) are controlled. Here we studied how their survival is regulated in the context of T cell Receptor re-stimulation apoptosis. We show that both mouse and human Tregs and iTregs exhibit increased resistance to T cell Receptor restimulation-induced cell death (RICD) compared to conventional CD4+ cells. The enhanced survival was dependent on TGF-β signaling that results in reduced nuclear translocation of nuclear factor of activated T-cells (NFAT), as well as up-regulation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) in Tregs. Tregs also showed significantly decreased susceptibility to α-CD3 antibody mediated apoptosis compared to the effector cells in vivo. While ionomycin increased Treg apoptosis, NFAT activation inhibitors reduced the apoptosis levels of effector cells almost to Treg levels, demonstrating that reduced NFAT activity contributes to decreased RICD in Tregs. Finally, RICD resistance in Tregs manifests itself as increased protection of these cells from cell death during mouse oropharyngeal candidiasis (OPC) infection and inflammatory bowel disease (IBD). Protection from cell death was abrogated in the absence of TGF-β signaling in Tregs. Taken together, our data unravel the previously unrecognized role of TGF-β in promoting Treg survival during TCR restimulation, through regulation of multiple pathways involving NFAT and cFLIP in Tregs. This novel mechanism that offers a selective survival advantage to Tregs may have broader ramifications in immune homeostasis during inflammation.

Differential adjuvant activities of TLR7 and TLR9 agonists inversely correlate with nitric oxide and PGE2 production.

Activation of different pattern recognition receptors causes distinct profiles of innate immune responses, which in turn dictate the adaptive immune response. We found that mice had higher CD4+ T cell expansion to an immunogen, ovalbumin, when coadministered with CpG than with CL097 in vivo. To account for this differential adjuvanticity, we assessed the activities of CpG and CL097 on antigen-specific CD4+ T cell expansion in vitro using an OT-II CD4+ T cell/bone marrow-derived dendritic cell (DC) co-culture system. Unexpectedly, ovalbumin-stimulated expansion of OT-II CD4+ T cells in vitro was potently suppressed by both TLR agonists, with CL097 being stronger than CpG. The suppression was synergistically reversed by co-inhibition of cyclooxygenases 1 and 2, and inducible nitric oxide (NO) synthase. In addition, stimulation of OT-II CD4+ T cell/DC cultures with CL097 induced higher levels of CD4+ T cell death than stimulation with CpG, and this CD4+ T cell turnover was reversed by NO and PGE2 inhibition. Consistently, the co-cultures stimulated with CL097 produced higher levels of prostaglandin E2 (PGE2) and NO than stimulation with CpG. CL097 induced higher PGE2 production in DC cultures and higher IFN-γ in the OT-II CD4+ T cell/DC cultures, accounting for the high levels of PGE2 and NO. This study demonstrates that the adjuvant activities of immunostimulatory molecules may be determined by differential induction of negative regulators, including NO and PGE2 suppressing clonal expansion and promoting cell death of CD4+ T cells.

Longitudinal changes in plasma Caspase-1 and Caspase-3 during the first 2 years of HIV-1 infection in CD4Low and CD4High patient groups.

Over 95% of CD4 cell death occurs by Caspase-1-mediated pyroptosis during HIV infection. Caspase-3-mediated apoptosis accounts for the death in a small proportion of infected CD4 cells. To date, there have been no reports on the dynamics of Caspase-1 and Caspase-3 and their relationship with disease progression in acute HIV-1 infection. In this study, two distinct HIV-1 patient groups were enrolled. The CD4High group maintained a CD4 level above450 cells/μl while CD4 levels in the CD4Low group dropped below 250 cells/μl within 2 years after infection. Blood samples were collected at 1, 2, 3, 4, 6, 12 and 24 months after HIV infection. Plasma Caspase-1 and Caspase-3 levels in the two patients groups were determined by a single-step ELISA using commercially available monoclonal antibodies. The results showed that Caspase-1 and Caspase-3 levels in the CD4High group increased rapidly and then decreased within a short time during early HIV-1 infection. In contrast, Caspase-1 and Caspase-3 levels in the CD4Low group were obviously increased after 1 year of HIV-1 infection.

Microrna-17-92 Cluster: Novel Target for Controlling Gvhd While Preserving GVL Effect

Graft-versus-host disease (GVHD) remains a life threatening complication after allogeneic hematopoietic stem cell transplantation (HCT). Donor T cells are the key pathogenic effectors in the induction of GVHD. MicroRNAs (miRs) have been shown to play an important role in orchestrating immune response, among which miR-17-92 cluster is one of the best characterized miR clusters that encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1 and 92-1. Although regulatory functions of miR-17-92 cluster have been elaborated in a variety of immune responses including anti-infection, anti-tumor, and autoimmunity, the role of this miR cluster in the modulation of T-cell response to alloantigens and the development of GVHD has not been explored previously. Based on the previous report that miR-17-92 promotes Th1 responses and inhibits induced regulatory T-cell (iTreg) differentiation in vitro, we hypothesized that blockade of miR-17-92 would constrain T-cell alloresponse and attenuate GVHD.To evaluate the function of miR-17-92 on T-cell alloresponse, we utilized the mice with miR-17-92 conditional knock-out (KO) on T cells as donors, and compared the alloresponse of WT and KO T cells after allogeneic bone marrow transplantation (allo-BMT). We observed that KO T cells had substantially reduced ability to proliferate and produce IFNγ as compared to WT counterparts 4 days after cell transfer. Interestingly, CD4 but not CD8 KO T cells had increased cell death in the population of fast-dividing T cells. Thus, miR-17-92 cluster promotes activation and expansion of both CD4 and CD8 T cells, and inhibits activation-induced cell death of CD4 but not CD8 T cells at the early stage of alloresponse in vivo. We further evaluated the role of miR-17-92 on T cells in the development of acute GVHD in a fully MHC-mismatched BMT model. In sharp contrast to WT T cells that caused severe GVHD and resulted in 100% mortality of the recipients, KO T cells were impaired in causing severe GVHD reflected by mild clinical manifestations and no mortality. These observations were extended to MHC-matched but minor antigen-mismatched as well as haploidentical BMT models that are more clinically relevant. We next addressed the critical question whether T cells deficient for miR-17-92 are still capable of mediating graft-versus-leukemia (GVL) effect. Using A20 lymphoma and P815 mastocytoma cell lines, we demonstrated that the KO T cells essentially retained the GVL activity in MHC-mismatched and haploidentical BMT model, respectively.Mechanistic studies revealed that miR-17-92 promoted CD4 T-cell proliferation, survival, migration to target organs, and Th1-differentiation, but reduced Th2-differentiation and iTreg generation. However, miR-17-92 had less impact on CD8 T-cell proliferation, survival, IFNγ production, and cytolytic activity reflected by granzyme B and CD107a expression. Moreover, miR-17-92 negatively regulated TNFα production by both CD4 and CD8 T cells. We therefore conclude that miR-17-92 cluster is required for T cells to induce severe GVHD, but it is dispensable for T cells to mediate the GVL effect.To increase translational potential of our findings, we designed the locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, which have been reported to be the key members in this cluster. We observed that the treatment with anti-miR-17 significantly inhibited T-cell expansion and IFNγ production in response to alloantigen in vivo, and anti-miR-19 was more effective. Furthermore, our ongoing experiment showed the treatment with anti-miR-17 or anti-miR-19 was able to considerably attenuate the severity of GVHD as compared to scrambled antagomir in a MHC-mismatched BMT model.Taken together, the current work reveals that miR-17-92 cluster is essential for T-cell alloresponse and GVHD development, and validates miR-17-92 cluster as promising therapeutic target for the control of GVHD while preserving GVL activity in allogeneic HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Palmitoylation is required for Fas receptor clustering and cell death in CD4+ T cells (LYM7P.722)

Abstract Fas-Fas ligand (FasL) interactions are critical for apoptosis and T cell homeostasis, with deficiencies in either Fas or FasL leading to autoimmunity in both mice and humans. Fas can trigger apoptotic cell death directly through recruitment of a death-inducing signaling complex (DISC) consisting of FADD and Caspase-8. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis in CD4+ T cells, with the Fas-sensitive memory population having increased lipid raft receptor localization. Palmitoylation of human Fas at cysteine 199 (Cys-194 in mouse) is required for localization to lipid rafts. To investigate the role of palmitoylation in Fas signaling, we developed a transgenic mouse defective in palmitoylation of Fas receptor (Fas C194→V) backcrossed to the Fas-defective lpr/lpr mouse (FasC194Vlpr/lpr). These mice developed autoimmunity characteristic of lpr mice, but CD4-CD8- “Double-Negative” (DN) T cells did not accumulate. T cells expressing C194V mutant Fas were resistant to Fas- and TCR-induced cell death, indicating lipid raft localization is required for the apoptotic function of Fas, but not accumulation of DN T cells. High-resolution PALM imaging of cells expressing wild-type or C194V mutant Fas revealed receptor clustering into higher-order oligomers at the plasma membrane required palmitoylation. These findings identify palmitoylation as a key post-translational modification of Fas that controls the apoptotic signal.

Enhanced proliferation and defective activation-induced cell death of CD4+ T cells in childhood asthma

Hyperactivation of CD4+ T cells in peripheral blood and airway tissues has been suggested to play a key role in the development and maintenance of chronic inflammation in childhood asthma. However, the underlying mechanisms are not yet clear. To investigate alterations in serum levels of T helper cell-related cytokines, mitogen-stimulated CD4+ T cell proliferation and activation-induced cell death (AICD) in childhood asthma. 21 children with untreated asthma and 21 healthy volunteers (age and gender matched) participated in this study. Th1/Th2/Th17 cytokines in serum were analyzed by flow cytometry. CD4+ T cells were isolated from participants by using immuno-magnetic beads and were stimulated by phytohemagglutinin (PHA). Cell proliferation was evaluated with a Cell Counting Kit-8 (CCK-8). Activation induced cell death (AICD) of CD4+ T cells was also induced by PHA and apoptosis was assayed by annexin V/PI staining. Quantitative RT-PCR was carried out to analyze Fas and FasL mRNA expression. FLIPL, caspase-8 and Bcl-2 were detected by western blot analysis. In children with asthma, the proliferative capacity of CD4+ T cells was enhanced and AICD was inhibited significantly, while serum IL-4, IL-10 and TNF were markedly higher compared with the control group. Fas mRNA expression in the asthma group was obviously lower than that in the control group, while no change was detected in FasL mRNA expression. Western blot analysis showed that the levels of the anti-apoptotic proteins, FLIPL and Bcl-2 in CD4+ T cells of the asthma group were significantly higher than in the control group. Spearman's correlation tests showed that only IL-4 correlated positively with FLIPL and Bcl-2 expression, while IL-10 and TNF were unrelated to FLIPL and Bcl-2 expression. These results indicate that enhanced proliferation and defective AICD of CD4+ T cells influence the T cell-mediated inflammatory reaction in childhood asthma and that increased IL-4, FLIPL and Bcl-2 expression and decreased Fas expression jointly participate in these changes in cell proliferation and apoptosis.ression and decreased Fas expression jointly participate in these changes in cell proliferation and apoptosis.

Assessing main death pathways in T lymphocytes from HIV infected individuals

Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV-infected individuals. The combination of three factors: (i) antibody staining to identify CD3(+) CD4(+) and CD3(+) CD8(+) cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC-1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase-independent and -dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z-VAD-fmk or Q-VD-oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q-VD-oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC-1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T-cells, JC-1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T-cell death mechanisms in HIV-infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T-cells require further evaluation.

HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration

Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4(+) T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4(+) T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4(+) T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

ASPECTS OF CYCLON AND BDNF GENE EXPRESSION IN SCHIZOPHRENIA PATIENTS

The pathogenesis of the schizophrenic illness is still not fully elucidated. Many studies have been conducted revealing different aspects but may be the studies of greatest significance are studying the genetic aspects of expression of trophic factors and enzymes associated with nervous system development and plasticity. In this relation we aimed at measuring the Cyclon and BDNF genes expression in blood of patients suffering from schizophrenia and to test for correlation between them. Our result did not reveal correlation in spite of their connection with the disease. The pathogenesis of the schizophrenic illness is still not fully elucidated. In this relation many studies have been performed examining the function of different systems in the organism. One of these directions is associated with the activity of the immune system and its effects on neural plasticity, glial activation and eventually the associated functional disorders of CNS. In this regard several studies for genetic associations with schizophrenia a linkage is found with components of the IL-3 signaling pathway – the gene coding for IL-3 (3) and the genes coding for the two subunits of the IL-3 receptor (4,11) In 2008 Akemi Hoshino e Hodaka Fujii(10) identified the gene CCDC86 to be directly induced by IL-3 in a IL-3 dependent pro-B cell line (14 ) and named it Nyclon (cytokine induced protein with coiled-coil domain)(10). In a consequent study the gene was identified to be important for the phenomenon of activation induced cell death (AICD) in T lymphocytes (14). The transgenic overexpression of Cyclon enhances the activation-induced cell death of CD4+ and CD8+ T lymphocytes and targeted deletion of Cyclon allele leads to resistance to AICD and lower expression of Fas (CD95) (14 ). These experiments demonstrate that Cyclon modulates the activation induced cell death and that this effect is mediated through induction of the expression of the cell membrane pro-apoptic death receptor Fas. This receptor is a member of the Tumor Necrosis Factor Receptor Superfamily, member 6, and plays pivotal role in regulation of programmed cell death and the proper physiological functioning of the immune system (19). Cyclon is also expressed in the central nervous system – in neurons and glia, and probably in these cells it also regulates the Fas expression and hence the process of programmed cell death. Neuronal and glial apoptosis are processes definitely associated with the pathogenesis of the disease and therefore it is possible that Cyclon also plays important role in the pathogenesis of schizophrenia. Another factor associated with the cell differentiation and apoptosis of cells in the nervous system is BDNF. Concerning the genetic link of BDNF with the pathogenesis of schizophrenia of interest is a study of Agartz et al. (1). The authors found an association between a polymorphism of BDNF and brain morphology in patient with schizophrenia. In a MRI study of the volume of cerebellar tonsils, hemispheres and subregions of vermis, striatal structures, hippocampus and corpus callosum differences between patients and healthy controls were found. A significant difference in the volume of frontal gray matter is associated with BDNF 11757 G/C polymorphism, and 270 C/T

Anti-latency agents to purge HIV reservoirs

The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with highly active anti-retroviral therapy (HAART), since latently infected cells remain a permanent source of viral reactivation. HIV establishes a persistent infection in CD4+ T lymphocytes (and to a lesser extent in macrophages as well), creating a persistent reservoir consisting mainly of latently infected resting memory CD4+ T cell. Although pre- and post-integration latencies have been described in HIV-1, the reservoir that appears to be the major barrier to eradication is composed of latently infected cells carrying an integrated provirus that is transcriptionally silent. It has been suggested that reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus. Immunoactivation therapy to reduce the latent pool of HIV by treatment with the anti-CD3 antibody OKT-3 alone or in combination with interleukin-2, substantially failed to significantly decrease the viral reservoir. Non-specific T-cell activation may induce high-level viral replication above a level that can be fully contained by ART, while increasing the susceptibility of uninfected cells. Selective targeting of HIV provirus via agents that induce the expression of quiescent HIV, but have limited effects on the uninfected host cell is an alternate approach to attack latent HIV. Activation from latency to completion of the replication cycle should result in lytic cell death of CD4+ T cells. Multiple mechanisms that contribute to the maintenance of proviral latency could be targeted to activate the latent virus. As examples of potentially useful agents, IL–7 can reactivate HIV–1 in latently infected cells in vitro through the induction of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) signalling pathway. The use of different chemical compounds targeting the PKC signalling pathway (prostratin, bryostatin) has also been proposed as a means of reactivating viral reservoirs. Finally, HDAC blocking is an attractive potential means of inducing broad reactivation of HIV–1 reservoirs, and promising results have been achieved using the HDAC inhibitor vorinostat.

An Artificial Antigen-presenting Cell with Paracrine Delivery of IL-2 Impacts the Magnitude and Direction of the T Cell Response

Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells. Paracrine delivery of IL-2 upon T cell contact with paAPCs induces significant IL-2 accumulation in the synaptic contact region. This accumulation increases CD25 (the inducible IL-2 Rα chain) on responding T cells and increases proliferation of CD8+ T cells in vitro to levels 10 times that observed with equivalent amounts of bulk IL-2. These CD8+ T cell responses critically depend upon close contact of T cells and the paAPCs and require sustained release of low levels of IL-2. The same conditions promote activation-induced cell death in CD4+ T cells. These findings provide insight into the response of T cell subsets to paracrine IL-2.

Inflammatory Cytokines Determine the Susceptibility of Human CD8 T Cells to Fas-mediated Activation-induced Cell Death through Modulation of FasL and c-FLIPS Expression

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affects their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokine IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death. Human CD8 T cells activated in the presence of IL-1/6/23 underwent significantly less cell death after activation as compared with those activated in IL-12. This was due to reduced susceptibility to Fas-mediated activation-induced cell death (AICD). Mechanistically, the reduced level of cell death in CD8 T cells activated in IL-1/6/23 was a result of a low level of FasL expression and high level of c-FLIP(S) expression. When the effect of IL-1, IL-6, and IL-23 individually was examined, IL-1 or IL-6 alone was sufficient to inhibit CD8 T cell death that occurs after activation in IL-12. IL-1, but not IL-6, inhibited expression of FasL, whereas IL-6, but not IL-1, increased c-FLIP(S) expression. Our findings show that the presence of IL-1 and/or IL-6 during activation of human CD8 T cells attenuates Fas-mediated AICD, whereas IL-12 increases the susceptibility of activated CD8 T cells to this form of cell death.

Macromolecular synthesis inhibitors perturb glucocorticoid receptor trafficking

The ability of inhibitors of transcription and translation to prevent glucocorticoid-induced apoptosis has been interpreted to indicate that the cell death machinery requires de novo protein synthesis. The transcriptional inhibitors actinomycin D (Act D) and DRB as well as the translational inhibitors CHX and puromycin inhibited early loss of mitochondrial membrane integrity in a dose-dependent manner. This effect was not observed with the transcriptional inhibitor α-amanitin suggesting they may have additional effects. Their role in the glucocorticoid receptor (GR) intracellular trafficking was therefore investigated. Here, we show that Act D and CHX reduced glucocorticoid binding, GR turnover and impaired GR nuclear translocation. We performed the same experiments in different thymocyte subpopulations of Balb/c mice. At the highest dose tested, actinomycin D and cycloheximide abolished glucocorticoid-induced cell death of CD4+CD8+ and CD4+CD8−. In all subsets, Act D, DRB, as well as CHX and puromycin prevented receptor nuclear translocation, indicating a general alteration of GR trafficking. Overall, our data support a direct effect of macromolecular inhibitors on GR activation and trafficking. Finally, direct alterations of the functional properties of the glucocorticoid receptor might be responsible for cell death prevention by actinomycin D, DRB, cycloheximide and puromycin.