Myxoma Virus Serp2 Is a Weak Inhibitor of Granzyme B and Interleukin-1β-Converting Enzyme In Vitro and Unlike CrmA Cannot Block Apoptosis in Cowpox Virus-Infected Cells

The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter, and S. Bertagnoli, J. Virol. 72:7830-7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1beta-converting enzyme (ICE, caspase-1) (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Attempts to covalently cross-link ICE-serpin inhibitory complexes were successful with CrmA, but no complex between ICE and Serp2 was visible after cross-linking. Purified His10-tagged Serp2 protein was a relatively poor inhibitor of ICE, with a Ki of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (Ki = 420 nM) than CrmA (Ki = 100 nM). Finally, Serp2 was tested for the ability to replace CrmA and inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant deleted for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apoptosis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediated cleavage of DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]. These results indicate that Serp2 is unable to functionally substitute for CrmA within the context of CPV and that the inhibition spectra for Serp2 and CrmA are distinct.     

Viral Cross-Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.     

Gene therapy for acute hepatitis using CrmA gene transduction

Fas-Fas ligand interactions between adenovirus-infected hepatocytes and cytotoxic T lymphocytes (CTLs) play a major role in killing of vector-transduced hepatocytes. Cytokine response modifier A (CrmA) is known to protect lymphoid cells from Fas-mediated apoptosis by inhibiting caspase 8. In this study, we generated an E1-deleted adenovirus expressing CrmA, and investigated the effect of exogenous CrmA expression on the inhibition of Fas-mediated apoptosis in murine hepatocytes in vitro and on the prolongation of the transgene expression in adenovirus-transduced hepatocytes in vivo. Agonistic anti-Fas antibody induced massive apoptosis into hepatocytes, however, most of the cells expressing CrmA were escaped from apoptosis and survived. This result showed that anti-apoptic function was obtained in murine hepatocytes by expressing CrmA. The prolongation of the transgene expression was studied using mice with congenital deficiency of lysosomal beta-glucuronidase (GUSB). The serum GUSB activity from the mice injected only an adenovirus expressing human beta-glucuronidase disappeared within 70 days, however, significant GUSB activity was observed for more than 130 days in the mice co-transduced with adenoviruses expressing both GUSB and CrmA. Moreover, histochemical analysis showed GUSB expressions in the liver even at 130 days after the viral administration. These observations demonstrate that the prolongation of the transgene expression can be achieved in rodent liver by CrmA co-expression using adenoviral gene transfer.     

Lethal hepatitis after gene transfer of IL-4 in the liver is independent of immune responses and dependent on apoptosis of hepatocytes: a rodent model of IL-4-induced hepatitis

The putative role of IL-4 in human and animal models of hepatitis has not yet been directly determined. We now report that direct expression of IL-4 in the liver of rats or mice using recombinant adenoviruses coding for rat or mouse IL-4 (AdrIL-4 and AdmIL-4, respectively) results in a lethal, dose-dependent hepatitis. The hepatitis induced by IL-4 was characterized by hepatocyte apoptosis and a massive monocyte/macrophage infiltrate. IL-4-induced hepatitis was independent of T cell-mediated immune responses. Hepatitis occurred even after gene transfer of IL-4 into nude rats, CD8-depleted rats, cyclosporine A-treated rats, or recombinase-activating gene 2(-/-) immunodeficient mice. Peripheral depletion of leukocytes using high doses of cyclophosphamide, and/or the specific depletion of liver macrophages with liposome-encapsulated dichloromethylene diphosphonate in rats did not block lethal IL-4-induced hepatitis. Direct transduction of hepatocytes with adenoviruses was not essential, since injection of AdrIL-4 into the hind limb induced an identical hepatitis. Finally, primary rat hepatocytes in culture also showed apoptosis when cultured in the presence of rIL-4. IL-4-dependent hepatitis was associated with increases in the intrahepatic levels of IFN-gamma, TNF-alpha, and Fas ligand. Administration of AdmIL-4 to IFN-gamma, TNF-alpha receptor type I, or TNF-alpha receptor type II knockout mice also resulted in lethal hepatitis, whereas a moderate protection was observed in Fas-deficient lpr mice. IL-4-dependent hepatocyte apoptosis could be abolished by treatment with caspase inhibitory peptides. Our results thus demonstrate that IL-4 causes hepatocyte apoptosis, which is only partially dependent on the activation of Apo-1-Fas signaling and is largely independent of any immune cells in the liver.     

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.     

CrmA gene transfer rescued CsA-induced renal cell apoptosis in graft kidney

Cyclosporine A(CsA) causes significant nephrotoxicity that contribute to kidney graft loss in the long-term, it can induce cell apoptosis in renal cortex and medulla, reduce kidney function. The mechanisms are complex and involved in six apoptosis pathways including classical pathway, mitochondrial pathway, endoplasmic reticulum pathway, angiotensin II pathway, NO- and hypertonicity-related pathway. All these pathways may converge to a single way by activated Caspase-3, -6, -8, -9 and -12 that may become a potential new target for intervention. CrmA protein belonging to one of serine protease inhibitor family members, it widely inhibits the inflammatory Caspases and apoptotic Caspases and has a strong function on inhibiting apoptosis induced by many chemical inducers through effectively blocking Caspase-1, -3, -4, -5, -8, -9, and -10 enzymes. It is not reported if CrmA is resistant to apoptosis induced by immunosuppressant so far. Therefore we speculate CrmA can block down CsA-induced renal cell apoptosis through inhibiting the Caspase-3, -6, -8, -9 and -12 activated and further to eliminate CRD induced by CsA.     

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in target cells

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.     

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.     

Adenoviral gene transfer to the neonatal rat pulmonary circulation

BACKGROUND: Gene delivery to the pulmonary circulation has been studied in adult animals, but has not been extensively investigated in neonates. METHODS: We tested the ability of recombinant, replication-defective adenovirus to transduce the pulmonary circulation when delivered by percutaneous ventricular puncture. Five-day-old rat pups were injected with 10(7) to 10(10) particles (approximately 10(5) to 10(8) pfu) in 30 micro l total volume. RESULTS: Using RT-PCR, we detected transgene expression in both lung and liver at all dosages. However, whereas only 1/6 pups injected with 10(7) particles had detectable expression, 8/9 pups in the two highest dose groups had detectable expression. In the highest dose group expression was approximately 5-fold greater in lung than liver, though in the lower dose groups no difference between lung and liver was found. Expression decreased by only 25% from day 4 through the last time point at day 28 in lung, whereas liver expression was undetectable in 7 of 9 samples on day 28. Histopathological examination demonstrated expression both within the media of large arteries and in small, peripheral arteries and capillaries, with a concentration of expression in the most distal areas of both the lungs and liver. No evidence of inflammation was seen. CONCLUSIONS: We conclude that the neonatal pulmonary circulation can be effectively transduced using systemic adenoviral vector injection, has more sustained expression than liver, and may be a target for therapeutic gene delivery.     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

Inhibitory effect of recombinant iNOS gene expression on vasomotor function of canine basilar artery

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37 degrees C) to an adenoviral vector [10(7), 10(8), or 10(9) plaque-forming units (pfu)/ml] encoding either the iNOS gene or the beta-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (10(9) pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10(-9)-10(-3) M) and diethylaminodiazen-1-ium-1,2-dioate (10(-10)-10(-5) M) were significantly shifted to the right in iNOS gene (10(9) pfu/ml)-transduced rings compared with control and beta-galactosidase-transduced rings (P < 0.05, n = 5-6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings (P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O(2)(-).) production were elevated in iNOS-transduced rings (P < 0.05, n = 7 for cGMP; P < 0.01, n = 6-9 for O(2)(-). production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.     

Selective Regulation of Apoptosis: the Cytotoxic Lymphocyte Serpin Proteinase Inhibitor 9 Protects against Granzyme B-Mediated Apoptosis without Perturbing the Fas Cell Death Pathway

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P₁ mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P₁(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.     

Differential inhibitory effects of CrmA, P35, IAP and three mammalian IAP homologues on apoptosis in NIH3T3 cells following various death stimuli

We have investigated the effects of expression of the viral proteins CrmA, P35 and IAP, and the three mammalian IAP homologues (MIHA, MIHB and MIHC), on the regulation of apoptosis induced by either the overexpression of caspases (ICE, CPP32 and Nedd2), by serum-deprivation, or by gamma-irradiation in NIH3T3 fibroblasts. As previously shown, CrmA strongly inhibited ICE-induced apoptosis but was ineffective against Nedd2- or CPP32-mediated apoptosis. P35, IAP and MIHA protected cells from apoptosis induced by the three caspases to varying extents but MIHB and MIHC were largely ineffective. NIH3T3 cells expressing P35 and MIHA, but not IAP, CrmA, MIHB and MIHC, showed enhanced cell survival under serum-deprived conditions. In addition, P35, CrmA and MIHA could provide substantial protection against death induced by gamma-irradiation. These results suggest the presence of multiple apoptotic pathways with differential sensitivity to various naturally occurring apoptosis inhibitors.     

Antinecrotic and antiapoptotic effects of hepatocyte growth factor on cholestatic hepatitis in a mouse model of bile-obstructive diseases

Cholestasis, an impairment of bile outflux, frequently occurs in liver diseases. In this process, an overaccumulation of bile acids causes hepatocyte necrosis and apoptosis, leading to advanced hepatitis. Hepatocyte growth factor (HGF) is mitogenic toward hepatocytes, but it is still unclear whether HGF has physiological and therapeutic functions during the progression of cholestasis. Using anti-HGF IgG or recombinant HGF in mice that had undergone bile duct ligation (BDL), we investigated the involvement of HGF in cholestasis-induced hepatitis. After the BDL surgery, HGF and c-Met mRNA levels transiently increased in livers during the progression of cholestatic hepatitis. When c-Met tyrosine phosphorylation was blocked in the livers of BDL-treated mice by anti-HGF IgG, hepatic dysfunction became evident, associated with the acceleration of hepatocyte necrosis and apoptosis. Inversely, administration of recombinant HGF into the mice led to the prevention of cholestasis-induced inflammation: HGF suppressed the hepatic expression of intracellular adhesion molecule-1 and neutrophil infiltration in BDL-treated mice. As a result, parenchymal necrosis was suppressed in the HGF-injected BDL mice. In addition, HGF supplement therapy reduced the number of apoptotic hepatocytes in cholestatic mice, associated with the early induction of Bcl-xL. The administration of HGF enhanced hepatic repair, via accelerating G1/S progression in hepatocytes. Our study showed that 1) upregulation of HGF production is required for protective mechanisms against cholestatic hepatitis and 2) enhancement of the intrinsic defense system by adding HGF may be a reasonable strategy to attenuate hepatic inflammation, necrosis, and apoptosis under bile-congestive conditions.     

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-alpha production by Kupffer cells

The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.     

Blockade of PD-1/B7-H1 interaction restores effector CD8+ T cell responses in a hepatitis C virus core murine model

The impaired function of CD8(+) T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8(+) T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8(+) T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8(+) T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-gamma, TNF-alpha, and granzyme B production by CD8(+) T cells. In addition, the impaired CD8(+) T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8(+) T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8(+) T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection.     

Ex vivo hypothermic recirculatory adenoviral gene transfer to the transplanted pig heart

BACKGROUND: To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. METHODS: Experimental animals were assigned into three groups; controls, 1x10(8) plaque-forming units (pfu)/ml group and 1x10(9) pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4 degrees C and the perfusion pressure was adjusted at 50 mmHg. RESULTS: Cardiac myocyte transduction efficiencies in the 1x10(8) pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1x10(9) pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1x10(9) pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. CONCLUSIONS: This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation.     

IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1

We have previously shown that IFN-gamma/STAT1 plays an essential role in concanavalin A (ConA)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we demonstrate that IFN-gamma/STAT1 also plays a crucial role in leukocyte infiltration into the liver in T cell hepatitis. After injection of ConA, leukocytes were significantly infiltrated into the liver, which was suppressed in IFN-gamma(-/-) and STAT1(-/-) mice. Disruption of the IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-gamma/STAT1, abolished ConA-induced liver injury and suppressed leukocyte infiltration into the liver. Additionally, ConA injection induced expression of a wide variety of chemokines and adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, monokine induced by IFN-gamma (Mig), CC chemokine ligand-20, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IFN-inducible T cell-alpha chemoattractant (I-TAC), and IFN-inducible protein-10 (IP-10) was markedly attenuated in IFN-gamma(-/-), STAT1(-/-), and IRF-1(-/-) mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro treatment with IFN-gamma activated STAT1, STAT3, and IRF-1, and induced expression of VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these chemokines and adhesion molecules was markedly diminished in STAT1(-/-) and IRF-1(-/-) hepatic cells compared with wild-type hepatic cells. These findings suggest that in addition to induction of apoptosis, previously well documented, IFN-gamma also stimulated hepatocytes, sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent mechanism to produce multiple chemokines and adhesive molecules responsible for promoting infiltration of leukocytes and, ultimately, resulting in hepatitis.