Peritoneal B1 and B2 cells respond differently to LPS and IL-21 stimulation.

Peritoneal B cells can be divided into B1 cells (CD11b+CD19+) and B2 cells (CD11b-CD19+) based on CD11b expression. B1 cells play a crucial role in the innate immune response by producing natural antibodies and cytokines. B2 cells share similar traits with B1 cells, influenced by the peritoneal environment. However, the response of both B1 and B2 cells to the same stimuli in the peritoneum remains uncertain. We isolated peritoneal B1 and B2 cells from mice and assessed differences in Interleukin-10(IL-10) secretion, apoptosis, and surface molecule expression following exposure to LPS and Interleukin-21(IL-21). Our findings indicate that B1 cells are potent IL-10 producers, possessing surface molecules with an IgMhiCD43+CD21low profile, and exhibit a propensity for apoptosis in vitro. Conversely, B2 cells exhibit lower IL-10 production and surface markers characterized as IgMlowCD43-CD21hi, indicative of some resistance to apoptosis. LPS stimulates MAPK phosphorylation in B1 and B2 cells, causing IL-10 production. Furthermore, LPS inhibits peritoneal B2 cell apoptosis by enhancing Bcl-xL expression. Conversely, IL-21 has no impact on IL-10 production in these cells. Nevertheless, impeding STAT3 phosphorylation permits IL-21 to increase IL-10 production in peritoneal B cells. Moreover, IL-21 significantly raises apoptosis levels in these cells, a process independent of STAT3 phosphorylation and possibly linked to reduced Bcl-xL expression. This study elucidates the distinct functional and response profiles of B1 and B2 cells in the peritoneum to stimuli like LPS and IL-21, highlighting their differential roles in immunological responses and B cell diversity.

Virus-mediated inactivation of anti-apoptotic Bcl-2 family members promotes Gasdermin-E-dependent pyroptosis in barrier epithelial cells.

Two sets of innate immune proteins detect pathogens. Pattern recognition receptors (PRRs) bind microbial products, whereas guard proteins detect virulence factor activities by the surveillance of homeostatic processes within cells. While PRRs are well known for their roles in many types of infections, the role of guard proteins in most infectious contexts remains less understood. Here, we demonstrated that inhibition of protein synthesis during viral infection is sensed as a virulence strategy and initiates pyroptosis in human keratinocytes. We identified the BCL-2 family members MCL-1 and BCL-xL as sensors of translation shutdown. Virus- or chemical-induced translation inhibition resulted in MCL-1 depletion and inactivation of BCL-xL, leading to mitochondrial damage, caspase-3-dependent cleavage of gasdermin E, and release of interleukin-1α (IL-1α). Blocking this pathway enhanced virus replication in an organoid model of human skin. Thus, MCL-1 and BCL-xL can act as guard proteins within barrier epithelia and contribute to antiviral defense.

Development of Conformational Antibodies to Detect Bcl-xL's Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

Varicella-Zoster Virus infected human neurons are resistant to apoptosis.

Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.

Bcl-XL: A multifunctional anti-apoptotic protein.

B-cell lymphoma-extra large (Bcl-XL) is one of the anti-apoptotic proteins of the Bcl-2 family that is localized in the mitochondria. Bcl-XL is one of the key regulators of apoptosis that can also regulate other important cellular functions. Bcl-XL is overexpressed in many cancers, and its inhibitors have shown good therapeutic effects. Bcl-XL interacts with Beclin 1, a key factor regulating autophagy. Bcl-XL is essential for the survival of neurons and plays protective roles in neuronal injuries. It can promote the growth of neurons and the correct formation of neural networks, enhance synaptic plasticity, and control neurotoxicity. Bcl-XL can also promote the transport of Ca2+ to mitochondria, increase the production of ATP, and improve metabolic efficiency. In addition, targeting Bcl-XL has shown potential value in autoimmune diseases and aging. In this review, we summarize the functions of Bcl-XL in cancer, autophagy, Ca2+ signaling, neuroprotection, neuronal growth and synaptic plasticity, energy metabolism, immunity, and senescence as revealed by investigations conducted in the past 10 years. Moreover, we list some inhibitors that have been developed based on the functions of Bcl-XL.

MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase.

Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.

1,25-Dihydroxy Vitamin D3 Modulates Endometriosis-Related Features of Human Endometriotic Stromal Cells.

PROBLEM: We aimed to evaluate modulatory effects of vitamin D3 on endometriosis-related features of endometriotic stromal cells. METHOD OF STUDY: The effect of vitamin D3 on adhesion, invasion, proliferation, apoptosis, cytokine production, and angiogenesis potential of the eutopic (EuESCs), ectopic (EESCs), and control (CESCs) stromal cells from 25 women with and 20 women without endometriosis was investigated. RESULTS: In all groups, vitamin D3 significantly increased cell adhesion (P = 0.0013-0.042), while decreased invasion (P = 0.026-0.031) and proliferation (P = 0.0013-0.039) of EESCs and EuESCs. Such treatment also resulted in a significant decrease in IL-6 production by EESCs (P = 0.039), but had no significant effect on the IL-8 production. This vitamin also caused significant decrease in Bcl-2 gene expression by EuESCs (P = 0.04) and Bcl-xL by EESCs (P = 0.044-0.009). In addition, vitamin D3 treatment reduced VEGF-A gene expression by EESCs (P = 0.046-0.009). CONCLUSION: Based on substantial favourable in vitro effects of vitamin D3 in endometriosis-related features of human endometriotic stromal cells, further investigations on therapeutic potential of this hormone in endometriosis are warranted.

IL-10/Janus kinase/signal transducer and activator of transcription 3 signaling dysregulates Bim expression in autoimmune lymphoproliferative syndrome.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder of T cell homeostasis caused by mutations that impair FAS-mediated apoptosis. A defining characteristic of ALPS is the expansion of double negative T cells (DNTC). Relatively little is known about how defective FAS-driven cell death and the Bcl-2 apoptotic pathway intersect in ALPS patients. OBJECTIVE: We studied changes in Bcl-2 family member expression in ALPS to determine whether the Bcl-2 pathway might provide a therapeutic target. METHODS: We used flow cytometry to analyze the expression of pro- and anti-apoptotic Bcl-2 family members in T cells from 12 ALPS patients and determined the in vitro sensitivity of ALPS DNTC to the pro-apoptotic BH3 mimetic, ABT-737. RESULTS: The pro-apoptotic molecule, Bim, was significantly elevated in DNTC. Although no general pattern of individual anti-apoptotic Bcl-2 family members emerged, increased expression of Bim was always accompanied by increased expression of at least 1 anti-apoptotic Bcl-2 family member. Strikingly, Bim levels in DNTC correlated significantly with serum IL-10 in ALPS patients, and IL-10 was sufficient to mildly induce Bim in normal and ALPS T cells via a Janus kinase/signal transducer and activator of transcription 3-dependent mechanism. Finally, ABT-737 preferentially killed ALPS DNTC in vitro. CONCLUSION: Combined, these data show that an IL-10/Janus kinase/signal transducer and activator of transcription 3 pathway drives Bim expression in ALPS DNTC, which renders them sensitive to BH3 mimetics, uncovering a potentially novel therapeutic approach to ALPS.

Dendritic cell maturation and survival are differentially regulated by TNFR1 and TNFR2.

The capacity of dendritic cells (DC) to regulate adaptive immunity is controlled by their maturation state and lifespan. Although TNF is a well-known maturation and survival factor for DC, the role of the two TNFR, TNFR1 and TNFR2, in mediating these effects is poorly understood. By using unique TNF variants that selectively signal through TNFR1 and/or TNFR2, we demonstrate differential functions of TNFR in human monocyte-derived and blood CD1c(+) DC. Activation of TNFR1, but not TNFR2, efficiently induced DC maturation, as defined by enhanced expression of cell surface maturation markers (CD83, CD86, and HLA-DR) as well as enhanced T cell stimulatory capacity. In contrast, both TNFR1 and TNFR2 significantly protected DC against cell death, indicating that innate signals can promote DC survival in the absence of DC maturation. We further show differential activation of NF-κB signaling pathways by the TNFR: TNFR1 activated both the p65 and p52 pathways, whereas TNFR2 triggered p52, but not p65, activation. Accordingly, the p65 NF-κB pathway only played a role in the prosurvival effect of TNFR1. However, cell death protection through both TNFR was mediated through the Bcl-2/Bcl-xL pathway. Taken together, our data show that TNFR1, but not TNFR2, signaling induces DC maturation, whereas DC survival can be mediated independently through both TNFR. These data indicate differential but partly overlapping responses through TNFR1 and TNFR2 in both inflammatory and conventional DC, and they demonstrate that DC maturation and DC survival can be regulated through independent signaling pathways.

Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering CD1d trafficking through the endocytic pathway.

NKT cells are a unique subset of T cells that recognize glycolipid Ags presented in the context of CD1d molecules. NKT cells mount strong antitumor responses and are a major focus in developing effective cancer immunotherapy. It is known that CD1d molecules are constantly internalized from the cell surface, recycled through the endocytic compartments, and re-expressed on the cell surface. However, little is known about the regulation of CD1d-mediated Ag processing and presentation in B cell lymphoma. Prosurvival factors of the Bcl-2 family, such as Bcl-xL, are often upregulated in B cell lymphomas and are intimately linked to sphingolipid metabolism, as well as the endocytic compartments. We hypothesized that Bcl-xL can regulate CD1d-mediated Ag presentation to NKT cells. We found that overexpression or induction of Bcl-xL led to increased Ag presentation to NKT cells. Conversely, the inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Furthermore, knockdown of Bcl-xL resulted in the loss of CD1d trafficking to lysosome-associated membrane protein 1(+) compartments. Rab7, a late endosomal protein, was upregulated and CD1d molecules accumulated in the Rab7(+) late endosomal compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated Ag processing and presentation to NKT cells by altering the late endosomal compartment and changing the intracellular localization of CD1d.

IL-33/ST2 axis promotes mast cell survival via BCLXL.

Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues. MC express the IL-33 receptor IL-1 receptor-related protein ST2 at high level, and this IL-1 family cytokine both activates MC directly and primes them to respond to other proinflammatory signals. Whether IL-33 and ST2 play a role in MC survival remains to be defined. In skin-derived human MC, we found that IL-33 attenuated MC apoptosis without altering proliferation, an effect mediated principally through the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). Murine MC demonstrated a similar mechanism, dependent entirely on ST2. In line with these observations, St2(-/-) mice exhibited reduced numbers of tissue MC in inflamed arthritic joints, in helminth-infected intestine, and in normal peritoneum. To confirm an MC-intrinsic role for ST2 in vivo, we performed peritoneal transfer of WT and St2(-/-) MC. In St2(-/-) hosts treated with IL-33 and in WT hosts subjected to thioglycollate peritonitis, WT MC displayed a clear survival advantage over coengrafted St2(-/-) MC. IL-33 blockade specifically attenuated this survival advantage, confirming IL-33 as the relevant ST2 ligand mediating MC survival in vivo. Together, these data reveal a cell-intrinsic role for the IL-33/ST2 axis in the regulation of apoptosis in MC, identifying thereby a previously unappreciated pathway supporting expansion of the MC population with inflammation.

Induction of Bcl-xL-specific cytotoxic T lymphocytes in mice.

The induction of active immunity against tumour-associated antigens to prevent relapse of cancer is a promising approach but has so far shown only low efficacy. This low efficacy may in part be due to clonal escape of tumour cell variants by the downregulation of antigen expression or inflammation-induced dedifferentiation. Identification of novel tumour-associated antigens that at the same time are essential for continued tumour cell survival is thus critical for the development of active cancer vaccinations. At the same time, identification of novel endogenous murine tumour antigens will help improve preclinical development of cancer immunotherapy. The anti-apoptotic protein Bcl-xL has been suggested to be such an essential tumour antigen, but the lack of well-defined murine epitopes have delayed preclinical studies of Bcl-xL-targeting cancer vaccines. Here, we report the identification of two novel murine tumour-associated epitopes TAYQSFEQV and AFFSFGGAL derived from mouse Bcl-xL. Dendritic cell (DC)-based vaccination induced CD8(+) T cells capable of producing IFN-γ upon restimulation with these epitopes. Thus, our data may benefit the design of future immunotherapy strategies by providing a preclinical model for cancer vaccination with an endogenous tumour antigen that can be combined with other cancer treatments.

Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

Helicobacter pylori and mucosa-associated lymphoid tissue: what's new.

Low-grade mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach, gastric MALT lymphoma, is associated with Helicobacter pylori infection. The eradication of H pylori using antibiotics is successful in 60% to 80% of affected patients. In contrast to the previous paradigm, we and other investigators have shown that a certain proportion of patients with H pylori-positive early-stage diffuse large B-cell lymphoma (DLBCL) of the stomach with histological evidence of MALT lymphoma, including high-grade transformed gastric MALT lymphoma and gastric DLBCL(MALT), achieved long-term complete pathological remission (pCR) after first-line H pylori eradication therapy, indicating that the loss of H pylori dependence and high-grade transformation are separate events in the progression of gastric lymphoma. In addition, patients with H pylori-positive gastric DLBCL without histological evidence of MALT (gastric pure DLBCL) may also respond to H pylori eradication therapy. A long-term follow-up study showed that patients who achieved pCR remained lymphoma free. Gastric MALT lymphoma is indirectly influenced by H pylori infection through T-cell stimulation, and recent studies have shown that H pylori-triggering chemokines and their receptors, H pylori-associated epigenetic changes, H pylori-regulated miRNA expression, and tumor infiltration by CD4+CD25+ regulatory T cells contribute to lymphomagenesis of gastric MALT lymphoma. Recent studies have also demonstrated that the translocation of CagA into B lymphocytes inhibits apoptosis through p53 accumulation, BAD phosphorylation, and the up-regulation of Bcl-2 and Bcl-XL expression. In gastric MALT lymphoma, CagA may stimulate lymphomagenesis directly, through the regulation of signal transduction, and intracellular CagA is associated with H pylori dependence. These findings represent a substantial paradigm shift compared with the classical theory of H pylori-reactive T cells contributing indirectly to the development of MALT lymphoma. In conclusion, a wide range of H pylori-related gastric lymphomas have been identified. The use of antibiotics as the sole first-line therapy for early-stage gastric pure DLBCL requires validation in a prospective study. The clinical and biological significance of the CagA oncoprotein in the lymphomagenesis of gastric MALT lymphoma warrants further study.

Role of fatty-acid synthesis in dendritic cell generation and function.

Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4(+) and CD8(+) T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-γ production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.

Critical role of B cell lymphoma 10 in BAFF-regulated NF-κB activation and survival of anergic B cells.

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase ß, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells.

P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic ß cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-x(L) and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-x(L) genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-x(L) and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo.

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.

The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells.

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.