anti-Fas Antibody Research Articles

Anti-Fas Antibody Conjugated Nanoparticles Enhancing the Antitumor Effect of Camptothecin by Activating the Fas-FasL Apoptotic Pathway.

Emerging evidence suggest that the introduction of Fas ligand (FasL) can enhance the Fas-dependent apoptosis and induce durable immune responses against tumor. However, selective triggering of apoptosis in tumor cells while sparing normal cells remains a great challenge for the application of FasL-based therapeutic strategies. Herein, smart nanoparticles (NPs) with a sandwich structure were fabricated. These NPs consist of a matrix metalloproteinase (MMP) cleavable PEG outer layer, an anti-Fas antibody middle layer, and a camptothecin (CPT)-loaded inner core. They could accumulate at a tumor site by the enhanced permeability and retention (EPR) effect. The removable PEG layer protects the cytotoxic anti-Fas antibody from premature contact with normal tissues, thus avoiding the unexpected lethal side effect before they reach the tumor site. Due to the high level of MMP expressed by tumor cells inside the tumor tissue, these NPs would shed their PEG layers, resulting in the exposure of anti-Fas antibody to bind the Fas receptor and triggering the apoptosis of tumor cells. Results of Western blot confirmed that these NPs could mimic the function of activated cytotoxic lymphocyte (CTL) to activate the Fas-FasL apoptosis pathway of tumor cells. With the aid of CPT payload, these anti-Fas antibody conjugated NPs achieved a high tumor inhibition in the B16 allograft tumor animal model. The design of these NPs provides a method for delivering cytotoxic ligand to targeting tissue, which may be valuable in cancer therapy.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

Microsomal prostaglandin E synthase-1 protects against Fas-induced liver injury.

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme for the synthesis of prostaglandin E2 (PGE2), a proproliferative and antiapoptotic lipid molecule important for tissue regeneration and injury repair. In this study, we developed transgenic (Tg) mice with targeted expression of mPGES-1 in the liver to assess Fas-induced hepatocyte apoptosis and acute liver injury. Compared with wild-type (WT) mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, less hepatic necrosis/apoptosis, and lower level of caspase cascade activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analysis revealed increased expression and activation of the serine/threonine kinase Akt and associated antiapoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling and suggest that induction of mPGES-1 or treatment with PGE2 may represent important therapeutic strategy for the prevention and treatment of Fas-associated liver injuries.

Disruption of Iron Regulation after Radiation and Donor Cell Infusion

Iron overload is common in patients undergoing hematopoietic cell transplantation (HCT). Peritransplant events, such as total body irradiation (TBI), and the effects of donor cell infusion may contribute to iron overload, in addition to disease-associated anemia and RBC transfusions. Using murine models we show complex time- and dose-dependent interactions of TBI and transplanted donor cells with expression patterns of iron regulatory genes in the liver. Infusion of allogeneic or syngeneic donor T lymphocytes increased serum iron, transiently up-regulated interleukin-6 (IL-6) and hepcidin (Hamp), and down-regulated ferroportin1 (Fpn1). After 7 to 14 days, however, changes were significant only with allogeneic cells. TBI (200 to 400 Gy) also induced IL-6 and Hamp expression but had little effect on Fpn1. TBI combined with allogeneic donor cell infusion resulted in modest early up-regulation of IL-6, followed by a decline in IL-6 levels and Hamp as well as Fpn1, and was accompanied by increased liver iron content. Injection of Fas ligand–deficient T lymphocytes from gld mice resulted in substantially lower alterations of gene expression than infusion of wild-type T cells. The agonistic anti-Fas antibody, JO2, triggered early up-regulation of Stat3 and IL-6, followed by an increase in Hamp and decreased expression of Fpn1 by 7 to 14 days, implicating Fas as a key modulator of gene expression in HCT. Minimal histologic changes were observed in mouse liver and duodenum. These data show profound and interacting effects of TBI and cell transplantation on the expression of iron regulatory genes in murine recipients. Alterations are largely related to induction of cytokines and Fas-dependent signals.

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl−D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

Corrigendum.

XenotransplantationVolume 23, Issue 2 p. 171-172 CorrigendumFree Access Corrigendum This article corrects the following: Expression of Fas but not Fas ligand on fetal pig β cells Lijun Bai, Kathrin Maedler, Marc Donath, Bernard E. Tuch, Volume 11Issue 5Xenotransplantation pages: 426-435 First Published online: August 10, 2004 First published: 22 April 2016 https://doi.org/10.1111/xen.12200AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Bai et al. 1, Figures 1 and 5 were published incorrectly, and the correct figures and their legends are illustrated below: The authors regret the error. Figure 1Open in figure viewerPowerPoint Fas expression on fetal pig pancreatic cells. Fetal pig ICCs stained with (A) anti-Fas polyclonal antibody. Positively stained cells are red. Bar = 20 μm. (B) Anti-insulin antibody. Positively stained cells are green. (C) The enriched single β-cells stained with Fas antibody. Positively stained cells are red. Bar = 20 μm. (D) The enriched single β-cells stained with Fas antibody and analyzed by flow cytometry. M1 is the gate for positive cells. The percentage of positive cells in this Figure is 73%. (E) Negative control for flow cytometry. Figure 5Open in figure viewerPowerPoint Expression of FLIP on fetal pig pancreas. (A) Double immunostaining for FLIP in red and insulin in green in a fetal pig pancreatic tissue section. (B) Immunoblotting of FLIPL, actin, caspase 8 and 3. Fetal pig ICCs cultured with or without IL-1β or anti-Fas antibody were analyzed after 24-, 72-, and 96-h incubation. Samples were loaded in parallel and anti-FLIP and anti-caspase 3 as well as anti-caspase 8 and anti-actin were blotted on the same membrane, respectively, after stripping. One representative out of four experiments is shown. Reference 1Bai LJ, Maedler K, Donath M, Tuch BE. Expression of Fas but not Fas ligand on fetal pig β cells. Xenotransplantation 2004; 11: 426– 435. Volume23, Issue2March/April 2016Pages 171-172 FiguresReferencesRelatedInformation

A biodegradable killer microparticle to selectively deplete antigen-specific T cells in vitro and in vivo.

The specific eradication of pathogenic T cells for the treatment of allograft rejections and autoimmune disorders without impairment of overall immune function is a fundamental goal. Here, cell-sized poly(lactic-co-glycolic acid) microparticles (PLGA MPs) were prepared as a scaffold to co-display the peptide/major histocompatibility complex (pMHC, target antigen) and anti-Fas monoclonal antibody (apoptosis-inducing molecule) for the generation of biodegradable killer MPs. Ovalbumin (OVA) antigen-targeted killer MPs significantly depleted OVA-specific CD8+ T cells in an antigen-specific manner, both in vitro and in OT-1 mice. After intravenous administration, the killer MPs predominantly accumulated in the liver, lungs, and gut of OT-1 mice with a retention time of up to 48 hours. The killing effects exerted by killer MPs persisted for 4 days after two injections. Moreover, the H-2Kb alloantigen-targeted killer MPs were able to eliminate low-frequency alloreactive T cells and prolong alloskin graft survival for 41.5 days in bm1 mice. Our data indicate that PLGA-based killer MPs are capable of specifically depleting pathogenic T cells, which highlights their therapeutic potential for treating allograft rejection and autoimmune disorders.

MiR-223 Deficiency Protects against Fas-Induced Hepatocyte Apoptosis and Liver Injury through Targeting Insulin-Like Growth Factor 1 Receptor

The biological functions and molecular mechanisms of miR-223 action in liver cells and liver diseases remain unclear. We therefore determined the effect and mechanism of action of miR-233 in Fas-induced hepatocyte apoptosis and liver injury. Wild-type (WT) and miR-223 knockout (KO) mice were treated i.p. with 0.5 μg/g body weight anti-Fas antibody Jo2, and the animals were monitored for survival and the extent of liver injury. Although WT mice died 4 to 6 hours after Jo2 injection (n = 6), all of the miR-223 KO mice (n = 6) survived. In comparison to WT mice, the miR-223 KO mice showed resistance to Fas-induced liver injury, as indicated by less tissue damage under histopathological examination, fewer apoptotic hepatocytes under caspase-3 immunostaining, and less elevation of serum transaminases. miR-223 KO livers showed less caspase-3, caspase-8, and caspase-9 activation and less poly (ADP-ribose) polymerase cleavage compared with WT livers (P < 0.05). Furthermore, tail vein injection of miR-223 lentiviral vector to miR-223 KO mice restored Jo2-induced liver injury. Transfection of miR-223 KO hepatocytes with miR-223 mimic enhanced Jo2-induced activation of caspase-3, caspase-8, and caspase-9, whereas transfection of WT hepatocytes with the miR-223 inhibitor attenuated Jo2-induced apoptosis. These findings demonstrate that miR-223 deficiency protects against Fas-induced hepatocyte apoptosis and liver injury. Further in vitro and in vivo data indicate that miR-223 regulates Fas-induced hepatocyte apoptosis and liver injury by targeting the insulin-like growth factor 1 receptor.

Protecting liver sinusoidal endothelial cells suppresses apoptosis in acute liver damage.

Apoptosis is associated with various types of hepatic disorders. We have developed a novel cell-transfer drug delivery system (DDS) using a multifunctional envelope-type nano device that targets liver sinusoidal endothelial cells (LSECs). The purpose of this study was to determine the efficacy of the novel DDS containing siRNA at suppressing apoptosis in LSECs. Bax siRNA was transfected into a sinusoidal endothelial cell line (M1) to suppress apoptosis induced by an anti-Fas antibody and staurosporine. C57BL/6J mice were divided into three groups: (i) a control group, only intravenous saline; (ii) a nonselective group, injections of siRNA sealed in the nonselective DDS; and (iii) an LSEC-transfer efficient group, injections of siRNA sealed in an LSEC-transfer efficient DDS. Hepatic cell apoptosis was induced by an anti-Fas antibody. Bax siRNA had an anti-apoptotic effect on M1 cells. Serum alanine aminotransferase was reduced in the LSEC-transfer efficient group, as were cleaved caspase-3 and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling positive hepatocytes. Silver impregnation staining indicated that the sinusoidal space was maintained in the LSEC-transfer efficient group but not in the other groups. Electron microscopy showed that the LSECs were slightly impaired, although the sinusoidal structure was maintained in the LSEC-transfer efficient group. Hepatocyte apoptosis was reduced by the efficient suppression of LSEC apoptosis with a novel DDS. Protecting the sinusoidal structure by suppressing LSEC damage will be an effective treatment for acute liver failure.

15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies.

15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.

Tumstatin induces apoptosis mediated by Fas signaling pathway in oral squamous cell carcinoma SCC-VII cells.

Oral squamous cell carcinoma is a cancer originating in the tissues lining the mouth and lips. The present study investigated the effects of recombinant tumstatin, an anti-angiogenic agent with distinct antitumor activity, on oral squamous cell carcinoma SCC-VII cells. Apoptosis was characterized by YO-PRO-1 staining, sub-G1 population, and DNA fragmentation analysis. Apoptotic mechanism of tumstatin was also investigated. The antitumor activity of tumstatin was further evaluated using an SCC-VII animal model. Recombinant tumstatin was found to decrease the viability of SCC-VII cells in a dose-dependent manner. The number of cells stained with the apoptotic marker YO-PRO-1, the sub-G1 cell population and the level of apoptotic DNA fragmentation increased in the SCC-VII cells following treatment with recombinant tumstatin. In addition, recombinant tumstatin treatment increased the expression of the Fas gene at the transcript and protein levels, and the inhibition of cell viability by recombinant tumstatin was suppressed by a neutralizing anti-Fas antibody. Furthermore, treatment with recombinant tumstatin decreased the volume and weight of tumors in C3H/HeJ mice implanted with SCC-VII cells. In conclusion, the results indicated that tumstatin induced apoptosis that is mediated by the Fas signaling pathway in SCC-VII cells and inhibited tumor growth in an SCC-VII animal model.

TRO40303, a mitochondrial-targeted cytoprotective compound, provides protection in hepatitis models.

TRO40303 is cytoprotective compound that was shown to reduce infarct size in preclinical models of myocardial infarction. It targets mitochondria, delays mitochondrial permeability transition pore (mPTP) opening and reduces oxidative stress in cardiomyocytes submitted to ischemia/reperfusion in vitro. Because the involvement of the mitochondria and the mPTP has been demonstrated in chronic as well as acute hepatitis, we investigated the potential of TRO40303 to prevent hepatocyte injury. A first set of in vitro studies showed that TRO40303 (from 0.3 to 3 μmol/L) protected HepG2 cells and primary mouse embryonic hepatocytes (PMEH) from palmitate intoxication, a model mimicking steatohepatitis. In PMEH, TRO40303 provided similar protection against cell death due to Jo2 anti-Fas antibody intoxication. Further studies were then preformed in a mouse model of Fas-induced fulminant hepatitis induced by injecting Jo2 anti-Fas antibody. When mice received a sublethal dose of Jo2 at 125 μg/kg, TRO40303 pretreatment prevented liver enzyme elevation in plasma in parallel with a decrease in cytochrome C release from mitochondria and caspase 3 and 7 activation in hepatic tissue. When higher, lethal doses of Jo2 were administered, TRO40303 (10 and 30 mg/kg) significantly reduced mortality by 65–90% when administered intraperitoneally (i.p.) 1 h before Jo2 injection, a time when TRO40303 plasma concentrations reached their peak. TRO40303 (30 mg/kg, i.p.) was also able to reduce mortality by 30–50% when administered 1 h postlethal Jo2 intoxication. These results suggest that TRO40303 could be a promising new therapy for the treatment or prevention of hepatitis.

Antigen-Specific Killer Polylactic-Co-Glycolic Acid (PLGA) Microspheres Can Prolong Alloskin Graft Survival in a Murine Model

Background: The strategy of specifically depleting antigen-specific T cells can potentially be used for the treatment of allograft rejection and autoimmunity because it does not suppress the overall immune systems.Methods: In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling major histocompatibility complex (MHC) class I antigens and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto PLGA microspheres. A modified double-emulsion method was used for the preparation of cell-sized PLGA microspheres. H-2Kb/peptide monomers were generated in-house and analyzed through flow cytometry. The killer PLGA microspheres were administered intravenously into BALB/c mice (H-2Kd) that had previously been grafted with skin squares from C57BL/6 mice (H-2Kb). Tumor cell challenge and third-party mixed lymphocyte culture were used to assess the general immune functions of host.Results: The alloskin graft survival was prolonged by 4 days. The killer PLGA microspheres could specifically deplete the H-2Kb alloantigen-reactive CD8+ T cells that infiltrated into the alloskin graft but not CD4+ T cells, without impairment of host overall immune function.Conclusions: Here, we initially report that PLGA microspheres, which have been widely used as medicine-delivering carriers, were used to prepare antigen-specific killer complexes and treat allograft rejection. Our data highlight the therapeutic potential of this biocompatible and biodegradable antigen-specific killer effector for the treatment of allograft rejection and autoimmune disease.

Antigen-specific killer polylactic-co-glycolic acid microspheres selectively deplete antigen-specific T cells in vitro and in vivo (TRAN1P.924)

Abstract The strategy of specifically depleting antigen-specific T cells can potentially be used to treat allograft rejection and autoimmunity because it does not suppress the overall immune systems. In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling H-2Kb-Ig and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto cell-sized PLGA microspheres. The Kb/OVA257-264-Killer PLGAs resulted in a dramatic reduction in the number of OVA257-264-specific CD8+ T cells in an antigen-specific manner in vitro and in vivo, by using a transgenic OT-1 mice model. The bioluminescence imaging revealed that Killer PLGAs dominantly distributed in lung, liver and gut, and was maintained for up to 48 hours in OT-1 mice and a longer time in OT-1 mice than in C57BL/6 mice. Moreover, the Kb-Killer PLGAs were administered intravenously into bm1 mice (H-2Kbm1) that had previously been grafted with ear skin from C57BL/6 mice (H-2Kb). The killer PLGAs selectively decreased the H-2Kb alloantigen-reactive CD8+ T cells infiltrating into the alloskin graft but not CD4+ T cells, and significantly prolonged allograft survival for 41.5 days without the impairment of host overall immune function. Thus, our data highlight the therapeutic potential of this biocompatible and biodegradable killer effectors for the treatment of allograft rejection and autoimmune disease. Keywords: Allograft rejection, PLGA, H-2Kb-Ig

Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.

Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1), and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the light of an already described influence on pregnancy disorders and therefore might provide a useful clinical tool in the future to prevent pregnancy complications provoked by inadequate implantation.

Deletion of Mir155 Prevents Fas-Induced Liver Injury through Up-Regulation of Mcl-1

Fas-induced apoptosis is involved in diverse liver diseases. Herein, we investigated the effect of Mir155 deletion on Fas-induced liver injury. Wild-type (WT) mice and Mir155 knockout (KO) mice were i.p. administered with the anti-Fas antibody (Jo2) to determine animal survival and the extent of liver injury. After Jo2 injection, the Mir155 KO mice exhibited prolonged survival versus the WT mice (P < 0.01). The Mir155 KO mice showed lower alanine aminotransferase and aspartate aminotransferase levels, less liver tissue damage, fewer apoptotic hepatocytes, and lower liver tissue caspase 3/7, 8, and 9 activities compared with the WT mice, indicating that Mir155 deletion prevents Fas-induced hepatocyte apoptosis and liver injury. Hepatocytes isolated from Mir155 KO mice also showed resistance to Fas-induced apoptosis, in vitro. Higher protein level of myeloid cell leukemia-1 (Mcl-1) was also observed in Mir155 KO hepatocytes compared to WT hepatocytes. A miR-155 binding site was identified in the 3'-untranslated region of Mcl-1 mRNA; Mcl1 was identified as a direct target of miR-155 in hepatocytes. Consistently, pretreatment with a siRNA specific for Mcl1 reversed Mir155 deletion-mediated protection against Jo2-induced liver tissue damage. Finally, restoration of Mir155 expression in Mir155 KO mice abolished the protection against Fas-induced hepatocyte apoptosis. Taken together, these findings demonstrate that deletion of Mir155 prevents Fas-induced hepatocyte apoptosis and liver injury through the up-regulation of Mcl1.

Hepatitis B virus core protein inhibits Fas-mediated apoptosis of hepatoma cells via regulation of mFas/FasL and sFas expression.

Hepatitis B virus core protein (HBc) has been implicated in hepatocarcinogenesis through several mechanisms. Resistance of hepatitis B virus (HBV)-infected hepatocytes to apoptosis is considered one of the major contributors to the progression of chronic hepatitis to cirrhosis and ultimately to hepatocellular carcinoma. The Fas receptor/ligand (Fas/FasL) system plays a prominent role in hepatocyte death during HBV infection. Here we report that HBc mediates resistance of hepatoma cells to agonistic anti-Fas antibody (CH11)-induced apoptosis. When HBc was introduced into human hepatoma cells, the cells became resistant to CH11 cytotoxicity in a p53-dependent manner. HBc significantly down-regulated the expression of p53, total Fas, and membrane-bound Fas at the mRNA and protein levels and reduced FasL mRNA expression. In contrast, HBc up-regulated the expression of soluble forms of Fas by increasing Fas alternative mRNA splicing. Mechanistically, HBc-mediated Fas alternative mRNA splicing was associated with up-regulation of polypyrimidine tract-binding protein 1 and down-regulation of Fas-activated serine/threonine kinase. These results indicated that HBc may prevent hepatocytes from Fas-induced apoptosis by the dual effects of reducing the expression of the proapoptotic form of Fas and enhancing the expression of the antiapoptotic form of the receptor, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.

Increased Survival despite Failure of Transplanted Human Hepatocyte Implantation into Liver Parenchyma of Nude Mice with Repeated Lethal Jo2-Induced Liver Deficiency

We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 µg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 µg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.

Distinct effects of soluble and membrane-bound fas ligand on fibroblast-like synoviocytes from rheumatoid arthritis patients.

Injection of agonistic anti-Fas antibody has been shown to decrease disease symptoms in mouse models of arthritis. Additionally, membrane-bound FasL (mFasL) has been shown to induce cell death in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. However, levels of soluble FasL (sFasL) are increased in the joints of RA patients and have been associated with disease severity, indicating that mFasL and sFasL play opposing roles in RA. The purpose of this study was to analyze the effects of FasL on RA FLS responses. The responses of FLS from RA and osteoarthritis (OA) patients to soluble and oligomeric FasL, the latter mimicking mFasL, were analyzed by fluorescence-activated cell sorting and proliferation assays, using 3 different FasL variants. The signaling pathways that trigger FasL responses were characterized by Western blotting. We found that mFasL and sFasL have distinct roles in RA FLS. Crosslinked FasL preferentially induced apoptosis, whereas sFasL stimulated proliferation. Moreover, sFasL activated several signaling pathways in RA FLS, such as ERK-1/2, phosphatidylinositol 3-kinase, caspase 8, and JNK, with a prominent role of JNK, since only the blockade of this pathway rendered FLS more susceptible to FasL-induced apoptosis. Crosslinked FasL induced apoptosis in FLS from OA patients, but sFasL failed to stimulate their proliferation. Our findings suggest that sFasL is a disease promoter in RA, a finding consistent with previous reports describing a tumor-promoting role of FasL. Therefore, blocking of sFasL could be a therapeutic strategy for RA.