anti-Fas Ligand Research Articles

Pharmacological Treatment of Degenerative Cervical Myelopathy: A Critical Review of Current Evidence.

Degenerative cervical myelopathy (DCM) is the leading cause of spinal cord dysfunction in adults, representing substantial morbidity and significant financial and resource burdens. Typically, patients with progressive DCM will eventually receive surgical treatment. Nonetheless, despite advancements in pharmacotherapeutics, evidence for pharmacological therapy remains limited. Health professionals from various fields would find interest in pharmacological agents that could benefit patients with mild DCM or enhance surgical outcomes. This review aims to consolidate all clinical and experimental evidence on the pharmacological treatment of DCM. We conducted a comprehensive narrative review that presents all pharmacological agents that have been investigated for DCM treatment in both humans and animal models. Riluzole exhibits effectiveness solely in rat models, but not in treating mild DCM in humans. Cerebrolysin emerges as a potential neuroprotective agent for myelopathy in animals but had contradictory results in clinical trials. Limaprost alfadex demonstrates motor function improvement in animal models and exhibits promising outcomes in a small clinical trial. Glucocorticoids not only fail to provide clinical benefits but may also lead to adverse events. Cilostazol, anti-Fas ligand antibody, and Jingshu Keli display promise in animal studies, while erythropoietin, granulocyte colony-stimulating factor and limaprost alfadex exhibit potential in both animal and human research. Existing evidence mainly rests on weak clinical data and animal experimentation. Current pharmacological efforts target ion channels, stem cell differentiation, inflammatory, vascular, and apoptotic pathways. The inherent nature and pathogenesis of DCM offer substantial prospects for developing neurodegenerative or neuroprotective therapies capable of altering disease progression, potentially delaying surgical intervention, and optimizing outcomes for those undergoing surgical decompression.

Luminescent Nanocomposites for Biomedical Applications

In this work, we pursue a concept of the multimodal drug preparation based on LuAG:Ce3+ nanoparticles. The functionality studied in this article is focused on the bioimaging of living cells via cathodoluminescence (CL) of scintillating nanoparticles attached to the cell surface. LuAG:Ce3+ scintillating nanoparticles were used as a core; amorphous or mesoporous SiO2 was used as a shell encapsulating the core to increase the surface reactivity; anti-Fas ligand (FasL) antibody was then attached to the outer shell in order to specifically bond the nanocomposite to the cell receptors; finally, polyethylene glycol molecules were attached to increase the biocompatibility of the nanocomposites. CL bioimaging was tested on yeasts Saccharomyces cerevisiae and leukocytes. It was found that the nanocomposites are able to attach to the cells via sorption or bonding to the receptors. The prepared nanocomposite shows stable luminescence under 10-keV electron irradiation, thus making a prospective candidate for this application and future multimodal drug design.

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus.

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1–3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL (mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.

Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor-Dependent Death of Retinal Endothelial Cells.

PurposeCluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox.MethodsHuman RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry.ResultsLigation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner.ConclusionsCluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.

Treatment of surgical brain injury by immune tolerance induced by intrathymic and hepatic portal vein injection of brain antigens.

Surgical brain injury (SBI) defines complications induced by intracranial surgery, such as cerebral edema and other secondary injuries. In our study, intrathymic and hepatic portal vein injection of allogeneic myelin basic protein (MBP) or autogeneic brain cell suspensions were administered to a standard SBI model. Serum pro-inflammatory IL-2, anti-inflammatory IL-4 concentrations and the CD4+T/CD8+T ratio were measured at 1, 3, 7, 14 and 21 d after surgery to verify the establishment of immune tolerance. Furthermore, we confirmed neuroprotective effects by evaluating neurological scores at 1, 3, 7, 14 and 21 d after SBI. Anti-Fas ligand (FasL) immunohistochemistry and TUNEL assays of brain sections were tested at 21 d after surgery. Intrathymic injections of MBP or autogeneic brain cell suspensions functioned by both suppressing secondary inflammatory reactions and improving prognoses, whereas hepatic portal vein injections of autogeneic brain cell suspensions exerted a better effect than MBP. Intrathymic and hepatic portal vein injections of MBP had equal effects on reducing secondary inflammation and improving prognoses. Otherwise, hepatic portal vein injections of autogeneic brain cell suspensions had better outcomes than intrathymic injections of autogeneic brain cell suspensions. Moreover, the benefit of injecting antigens into the thymus was outweighed by hepatic portal vein injections.

Apoptotic CD8 T-lymphocytes disable macrophage-mediated immunity to Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. CD8 T-lymphocytes help to control infection, but apoptosis of CD8 T cells disrupts immunity and efferocytosis can enhance parasite infection within macrophages. Here, we investigate how apoptosis of activated CD8 T cells affects M1 and M2 macrophage phenotypes. First, we found that CD8 T-lymphocytes and inflammatory monocytes/macrophages infiltrate peritoneum during acute T. cruzi infection. We show that treatment with anti-Fas ligand (FasL) prevents lymphocyte apoptosis, upregulates type-1 responses to parasite antigens, and reduces infection in macrophages cocultured with activated CD8 T cells. Anti-FasL skews mixed M1/M2 macrophage profiles into polarized M1 phenotype, both in vitro and following injection in infected mice. Moreover, inhibition of T-cell apoptosis induces a broad reprogramming of cytokine responses and improves macrophage-mediated immunity to T. cruzi. The results indicate that disposal of apoptotic CD8 T cells increases M2-macrophage differentiation and contributes to parasite persistence.

Pentoxifylline and prostaglandin E1 action on ischemia and reperfusion of small intestine tissue in rats. An immunohistochemical study.

To investigate the action of pentoxifylline (PTX) and prostaglandin E1 (PGE1) on ischemia and reperfusion of small intestine tissue in rats, using immunohistochemical analysis. Thirty-five Wistar rats were distributed as follows: group A (n=10): subjected to intestinal ischemia and reperfusion for 60 min, with no drugs; group B (n=10): PTX given during tissue ischemia and reperfusion; group C (n=10): PGE1 given during tissue ischemia and reperfusion; group D (n=5): sham. A segment of the small intestine was excised from each euthanized animal and subjected to immunohistochemical examination. Mean number of cells expressing anti-FAS ligand in the crypts was highest in Group A (78.9 ± 17.3), followed by groups B (16.7 ± 2.8), C (11.3 ± 1.8), and D (2.5 ± 0.9), with very significant differences between groups (p<0.0001). The use of pentoxifylline or prostaglandin E1 proved beneficial during tissue reperfusion. The immunohistochemical results demonstrated a decrease in apoptotic cells, while protecting other intestinal epithelium cells against death after reperfusion, allowing these cells to renew the epithelial tissue.

Solanum lyratumExtracts Induce Extrinsic and Intrinsic Pathways of Apoptosis in WEHI-3 Murine Leukemia Cells and Inhibit Allograft Tumor

We investigated the molecular mechanisms of cell cycle arrest and apoptotic death induced by Solanum lyratum extracts (SLE) or diosgenin in WEHI-3 murine leukemia cells in vitro and antitumor activity in vivo. Diosgenin is one of the components of SLE. Our study showed that SLE and diosgenin decreased the viable WEHI-3 cells and induced G0/G1 phase arrest and apoptosis in concentration- or time-dependent manners. Both reagents increased the levels of ROS production and decreased the mitochondrial membrane potential (ΔΨm). SLE- and diosgenin-triggered apoptosis is mediated through modulating the extrinsic and intrinsic signaling pathways. Intriguingly, the p53 inhibitor (pifithrin-α), anti-Fas ligand (FasL) mAb, and specific inhibitors of caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), and caspase-3 (z-DEVD-fmk) blocked SLE- and diosgenin-reduced cell viability of WEHI-3 cells. The in vivo study demonstrated that SLE has marked antitumor efficacy against tumors in the WEHI-3 cell allograft model. In conclusion, SLE- and diosgenin-induced G0/G1 phase arrest and triggered extrinsic and intrinsic apoptotic pathways via p53 activation in WEHI-3 cells. SLE also exhibited antitumor activity in vivo. Our findings showed that SLE may be potentially efficacious in the treatment of leukemia in the future.

Autoimmune lymphoproliferative syndrome due to FAS mutations outside the signal-transducing death domain: molecular mechanisms and clinical penetrance

PurposeAutoimmune lymphoproliferative syndrome is a disorder of lymphocyte apoptosis. Although FAS molecules bearing mutations in the signal-transducing intracellular death domain exhibit dominant-negative interference with FAS-mediated apoptosis, mechanisms for pathology of non-death domain FAS mutations causing autoimmune lymphoproliferative syndrome are poorly defined. MethodsRNA stability, protein expression, ligand binding, and ability to transmit apoptosis signals by anti-FAS antibody or FAS ligand were determined for a cohort of 39 patients with non-death domain autoimmune lymphoproliferative syndrome. Correlations between mutation type and disease penetrance were established in mutation-positive family members. ResultsFrameshifts or transcriptional stop mutations before exon 7 resulted in messenger RNA haploinsufficiency, whereas an amino-terminal signal sequence mutation and certain intracellular truncations prevented cell surface localization of FAS. All resulted in decreased FAS localization, inability to bind FAS ligand, and reduced FAS ligand-induced apoptosis. Extracellular missense mutations and in-frame deletions expressed defective FAS protein, failed to bind FAS ligand, and exhibited dominant-negative interference with FAS-mediated apoptosis. Mutation-positive relatives with haploinsufficient or extracellular mutations had lower penetrance of autoimmune lymphoproliferative syndrome clinical phenotypes than did relatives with death domain mutations. ConclusionWe have defined molecular mechanisms by which non-death domain FAS mutations result in reduced lymphocyte apoptosis, established a hierarchy of genotype-phenotype correlation among mutation-positive relatives of patients with autoimmune lymphoproliferative syndrome, and demonstrated that FAS haploinsufficiency can lead to autoimmune lymphoproliferative syndrome.Genet Med 2012:14(1):81–89

Abstract B211: Augmentation of Fas (CD95) signaling pathway sensitizes non-small cell lung cancer (NSCLC) cells.

Abstract Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a subset of NSCLC harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR kinase inhibitors, suggesting that other factors may play a role in determining survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) functions as a transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been described in numbers of cancers. Our previous report showed that suppression of Stat3 activity by a pharmacological approach, WP1066, sensitized NSCLC cells. Along with Stat3 inhibition, we also found that WP1066 treatment up-regulated the expression of Fas, a death receptor, regulating numbers of physiological and pathological process of cell death. Although Fas-mediated apoptosis pathway has been studied mostly in the immune system, the identification of Fas mutations in non-lymphoid malignancies, such as NSCLC, suggests its implication in the pathogenesis of non-lymphoid malignancies as well. Results from this study showed that levels of Fas were increased in cells with down-regulated Stat3 by small interfering RNA (siRNA), suggesting that Stat3 may play an inhibitory role to regulate the expression of Fas. To examine whether the Fas pathway is responsible for the WP1066-mediated cytotoxicity, Fas apoptosis signaling was induced by anti-Fas antibody and Fas ligand. Our results revealed that NSCLC cells showed different degrees of sensitivity to anti-Fas antibody but resistance to Fas ligand. However, augmentation of Fas expression by WP1066 remarkably increased the sensitivity of NSCLC cells to both anti-Fas antibody and Fas ligand. Taken together, our findings suggest that regulation of the Fas system may be a potential strategy to treat NSCLC and the combined use of a Fas inducer, such as WP1066, may further strengthen its efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B211.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

The Triterpenoid CDDO-Me Delays Murine Acute Graft-versus-Host Disease with the Preservation of Graft-versus-Tumor Effects after Allogeneic Bone Marrow Transplantation

The occurrence of acute graft-versus-host disease (aGVHD) and tumor relapse represent the two major obstacles impeding the efficacy of allogeneic bone marrow transplantation (BMT) in cancer. We have previously shown that the synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) can inhibit murine early aGVHD, but antitumor effects were not assessed. In the current study, we found that a new derivative of CDDO, CDDO-Me, had an increased ability to inhibit allogeneic T cell responses and induce cell death of alloreactive T cells in vitro. Administration of CDDO-Me to mice following allogeneic BMT resulted in significant and increased protection from lethal aGVHD compared to CDDO. This correlated with reduced TNF-alpha production, reduced donor T cell proliferation, and decreased adhesion molecule (alpha(4)beta(7) integrin) expression on the donor T cells. CDDO-Me was also superior to CDDO in inhibiting leukemia growth in vitro. When CDDO-Me was administered following an allogeneic BMT to leukemia-bearing mice, significant increases in survival were observed. These findings suggest that CDDO-Me is superior to CDDO in delaying aGVHD, while preserving or possibly even augmenting GVT effects.

Increased Killing of Liver NK Cells by Fas/Fas Ligand and NKG2D/NKG2D Ligand Contributes to Hepatocyte Necrosis in Virus-Induced Liver Failure

The role of liver NK cells in virus-induced severe viral hepatitis and, subsequently, hepatic failure is not well defined. In this study, we investigated the role of liver NK cells in the development of hepatocyte necrosis in fulminant hepatic failure (FHF) and acute-on-chronic liver failure (ACLF) because of viral infection. A mouse model of FHF induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of liver NK cells. Samples from patients with hepatitis B virus-related ACLF (HBV-ACLF) were examined. After MHV-3 infection, the number of NK cells in livers of BALB/cJ mice increased markedly, peaked at 48 h postinfection, and remained at a high level until sacrifice. In peripheral blood, spleen, and bone marrow, this number decreased significantly. Expression of CD69, cytotoxic activity, and intracellular IFN-gamma and TNF-alpha production by liver NK cells at 48 h postinfection were all significantly upregulated. Depletion of NK cells 24 h post-MHV-3 infection increased the mice survival from 0 of 18 (0%) to 4 of 18 (22.2%). Highly activated liver NK cells were cytotoxic to MHV-3-infected hepatocytes and this effect was markedly inhibited by anti-Fas ligand (FasL) plus anti-NKG2D mAbs. Furthermore, the accumulation of hepatic NK cells and increased expression of FasL and natural cytotoxicity receptors (NKp30 and NKp46) on the peripheral NK cells from patients with HBV-ACLF were correlated with disease progression. These results indicate NK cells play a pivotal role in the pathogenesis of FHF and HBV-ACLF, in which process Fas/FasL and NKG2D/NKG2D ligand pathway contribute to the liver NK cell-mediated hepatocyte injury.

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.

Cytotoxic T-lymphocyte–mediated killing of human pancreatic islet cells in vitro

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

Blockade of the Fas/Fas ligand interaction suppresses hepatocyte apoptosis in ischemia-reperfusion rat liver

Hepatic ischemia-reperfusion injury remains a significant problem for liver surgery, including transplantation, and apoptosis has been implicated in this type of hepatic injury. Here we found that through the Fas/Fas ligand interaction apoptosis is involved in the late phase of hepatic ischemia-reperfusion injury. The appearance of apoptotic hepatocytes increases significantly after reperfusion, reaching a maximum 12 h after reperfusion. The transcription levels of Fas and Fas ligand are increased after reperfusion. Fas is expressed on hepatocytes, while Fas ligand is expressed on infiltrating immune cells. A close spatial and temporal association of Fas expression and apoptotic cells is demonstrated in the histological observation. These results suggest that infiltrating cells induce apoptosis of hepatocytes through the Fas/Fas ligand interaction, leading to hepatocyte injury. Furthermore, an injection of anti-Fas antibody or neutralizing anti-Fas ligand antibody results in a dramatic decrease in the occurrence of hepatocyte apoptosis and hepatic infiltration of macrophages and natural killer cells as well as liver injury. Our results suggest that blockage of the Fas/Fas ligand interaction is a promising strategy for suppression of hepatic ischemia-reperfusion injury.

CM1 ligation induces the apoptosis of human lung cancer cells via Fas/FasL interaction

CM1 (centrocyte/‐blast marker I) defined by a monoclonal antibody developed against concanavalin‐A activated PBMC (peripheral blood mononuclear cell),is expressed specifically on a subpopulation of centroblasts and centrocytes of human germinal center (GC) B cells. We observed the existence of CM1 in human lung cancer tissue. So we investigated the function of CM1 in lung cancer cell lines (A549). We observed that CM1 ligation with anti‐CM1 mAb enhanced the apoptosis of A549 cells. And CM1 ligation induced the expression of both FasL and Fas in theses cells. Furthermore, both anti‐Fas blocking antibody, ZB4, and anti‐Fas ligand antibody NOK‐1 blocked CM1‐mediated apoptosis effectively. Caspase‐8 inhibitor, z‐IETD effectively blocked CM1‐induced cell death. Western blot analysis showed down‐regulated expression of procaspase‐8 and PARP. The release of cytochrome c and translocation to nucleus of endoG and AIF were showed by confocal micoscopy. The findings suggested that Fas/FasL pathway was activated by CM1 ligation on human lung cancer cell line.

TNF Alpha and Fas Mediate Tissue Damage and Functional Outcome after Traumatic Brain Injury in Mice

Tumor necrosis factor-alpha (TNFalpha) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFalpha, Fas, or both (TNFalpha/Fas-/-), we hypothesized that TNFalpha and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFalpha/Fas-/- mice had improved motor performance from 1 to 4 days (P<0.05), improved spatial memory acquisition at 8 to 14 days (P<0.05), and decreased brain lesion size at 2 and 6 weeks after CCI (P<0.05). Protection in TNFalpha/Fas-/- mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFalpha before CCI, and TNFalpha-/- mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice (P<0.05). Tumor necrosis factor-alpha/Fas-/- mice had decreased the numbers of cortical cells with plasmalemma damage at 6 h (P<0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFalpha and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P<0.05 versus WT), suggesting that the beneficial effects of TNFalpha/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFalpha and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFalpha/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.

Membrane Type 1 Matrix Metalloproteinase (MT1-MMP/MMP-14) Cleaves and Releases a 22-kDa Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Fragment from Tumor Cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.

99mTc-HYNIC-annexin V SPECT imaging of acute stroke and its response to neuroprotective therapy with anti-Fas ligand antibody

The first aim of the study was to determine whether (99m)Tc-HYNIC-annexin V, a marker of cellular stress and apoptosis, can detect ischemic injury in patients with acute stroke. Secondly, we wished to test radiolabeled annexin's ability to monitor therapy in a rodent model of focal ischemic injury. SPECT imaging of patients was performed between 1 and 2 h after intravenous injection of 30 mCi (1,110 MBq) of tracer. Eight MFL4 (anti-FasL) antibody-treated (400 microg i.p. days 0 and 3) and 21 control adult male Sprague-Dawley rats underwent small animal SPECT imaging with 5-10 mCi (185-370 MBq) of tracer, 1 and 6 days after a 2-h intraluminal thread occlusion of the left middle cerebral artery. Two patients with acute stroke had regions of multifocal annexin uptake that correlated with sites of restricted diffusion on MRI. Anti-FasL antibody treatment significantly reduced annexin uptake by 92% with a 60% decrease in the number of caspase-8 staining (apoptotic) neurons on day 1. On day 6, treated animals had an 80% reduction in tracer uptake with a 75% decrease in infarct size as compared with controls. Annexin uptake in controls and treated animals (day 6) linearly correlated with infarct size (r (2)=0.603, p=0.0036) and the number of TUNEL-positive (apoptotic) nuclei (r (2)=0.728, p=0.00084). Annexin imaging shows foci of increased uptake at sites of ischemic injury in patients with acute stroke. Annexin imaging can assess the effects of therapy for ischemic cerebral injury in rats, suggesting its potential as a non-invasive indicator of drug efficacy in future clinical trials.