Intrahepatic Lymphocytes Research Articles

Influence of B cells in liver fibrosis associated with hepatitis B virus harboring basal core promoter mutations

The development of the liver disease in chronic hepatitis B with common viral variants can be determined through the interaction between the virus and the host immune response. B cells constitute half of the intrahepatic lymphocyte population with an impact on fibrosis. A proliferation-inducing ligand (APRIL) has been shown to have a co-stimulatory activity on B cells. For this study HBV DNA was amplified and then sequenced to show the presence of the basal core promoter (BCP) mutations in the serum from 57 patients with chronic hepatitis B. The range of IgD-positive B cells was detected by immunohistochemistry in liver biopsies; and patients serum was assayed for APRIL levels by enzyme immunoassay. Twenty-seven patients (47.4%) harbored the A1762T-G1764A BCP mutations. Coefficients of logistic regression showed that the effect of increasing IgD-positive B cells in rising odds of the liver disease is the same in the patients with BCP mutation A1762T-G1764A and in the patients without mutation, nevertheless the effect of APRIL is not similar in these two groups of patients. Logistic regression in patients with BCP A1762T-G1764A mutations demonstrated that increasing one score of APRIL decreased the odds of fibrosis stage about 8%. These results suggest that in infection with viral variants of hepatitis B virus, the population of IgD-positive B cells may play a decisive role in later stages of the liver disease which is reduced by APRIL in chronic hepatitis patients with BCP mutations.

Analysis of Notch and TGF-β Signaling Expression in Different Stages of Disease Progression During Hepatitis B Virus Infection

OBJECTIVES:CD4+ regulatory T cells (Tregs) seem to have a key role in persistence of hepatitis B virus (HBV) infection. Notch and transforming growth factor (TGF-β) signaling independently help in the differentiation and regulation of CD4+T cells, including T-helper (TH) 1, TH2, and Tregs. Whether, the two pathways have modulatory role on different stages of HBV infection and severity of liver disease is not clear. We investigated Notch and TGF-β families' gene expression in peripheral blood and intrahepatic lymphocytes in patients with different stages of chronic HBV (CHB) infection.METHODS:Peripheral blood mononuclear cells (PBMCs), CD4+, and CD8+ T cells were isolated from patients with acute HBV (AVH-B, n=15), CHB (n=16), and controls (HC, n=10). In addition to PBMCs, intrahepatic lymphocytes were obtained from liver biopsies from CHB (n=12), cirrhosis (n=12), hepatocellular carcinoma (HCC, n=5), and healthy livers (n=5). Notch family (Notch1–4, Hes1, Jag1, and NF-kβ) and TGF-β family gene expressions were studied by real-time PCR, flow cytometry, and immunohistochemistry.RESULTS:Relative expression of Notch signaling target genes, Hes1 and NF-kβ, was higher in the total PBMCs of AVH-B and CHB patients than that in HC patients (Log relative quantification (RQ); 1.1 AVH-B vs. 0.3 HC, 1.3 CHB vs. 0.3 HC; P=0.02). CD8+ T cells showed upregulated expression of Hes1 and Notch1 (P=0.02 and 0.01, respectively) in AVH-B than in CHB patients. Also, in AVH-B patients, HBV-specific CD8+ T-cell proliferation (5.74% vs. 2.7%) and TGF-β signaling activity were higher. All Notch receptors and ligands were upregulated in the PBMCs in CHB infection (CHB vs. cirrhosis, P=0.001; CHB vs. HCC, P=0.023; and cirrhosis vs. HCC, P=NS). Intrahepatic expression of Notch1 and FoxP3 were significantly higher in cirrhotics and HCCs, and further blockage of Notch signaling reduced the FoxP3 expression. Array data of TGF-β family showed increased TGF-β3, TGF-α, SMAD3, SMAD4, SMAD6, and GDF9 expression on intrahepatic lymphocytes in cirrhotic and HCC patients compared with CHB.CONCLUSIONS:Our findings suggest that there is a complementary association between Notch1 and Hes1 in CD8+T cells during AVH-B infection. On development of CHB infection, repression of the Notch receptors mediates the regulation of immune response in patients, who progress to cirrhosis and HCC. Finally, HBV infection drives increased Notch1, TGF-β, and FoxP3 expression on intrahepatic T cells in cirrhosis, resulting in fibrogenesis and disease progression.

Differential regulation of pathogenesis and immune reactions in C57BL/6 and CBA/N mice infected with Clonorchis sinensis (164.22)

Abstract The current knowledge of immunological response to clonorchiasis, a major Asian endemic helminthic disease, is insufficient. To develop a mouse model of clonorchiasis, CBA/N and C57BL/6 mice were infected with Clonorchis sinensis metacercariae and analyzed host responses at 2, 4, and 8 weeks post infection. CBA/N mice infected with C. sinensis developed more severe gross and histopathological changes than C57BL/6 mice. In addition, more pronounced inflammatory cell infiltration and hepatocyte necrosis were noticed in CBA/N mice, especially at 2 weeks post-infection. Serum biomarkers of liver injury (ALT and AST) were increased significantly in CBA/N mice after C. sinensis infection as compared with those of C57BL/6 mice. We also analyzed the proliferation of splenocytes stimulated with crude C. sinensis antigen and cytokine production in the culture supernatant of activated splenocytes. We found that splenocytes from CBA/N mice were highly proliferative and the IL-17 level was increased. Additionally, an increased IL-4 level was sustained until 8 weeks post-infection in CBA/N mice. The serum levels of C. sinensis-specific IgG1 and IgG2a were increased in CBA/N mice, whereas IgE was increased in C57BL/6 mice. Furthermore, the level of IL-17 was increased in intrahepatic lymphocytes in CBA/N infected groups. Present study suggest that CBA/N could be a appropriate mouse model for clonorchiasis and might be useful for studying early immune responses in liver fluke infection.

Effect of cadmium chloride and ascorbic acid exposure on the vital organs of freshwater Cyprinid, Labeo rohita

The present study was carried out to evaluate the impact of sub lethal concentrations of heavy metal, cadmium chloride (CdCl 2 .H 2 O), ascorbic acid (vitamin C) and their combination on Labeo rohita . The effect was investigated on the basis of histopathological examinations of control and experimental groups exposed to heavy metal. The acute semi statistical toxicity test for L. rohita revealed 96 h LC 50 value 22.92 mg L -1 for cadmium chloride (CdCl 2 .H 2 O). 215 fingerlings of L. rohita were exposed to three different experimental conditions for 96 h: 11.46 mg L -1 CdCl 2 .H 2 O, or 450 mg Kg -1 ascorbic acid or combination of both these doses. No alterations were observed in gill sections upon comparison between treated and untreated groups. Congestion in sinusoids, fatty change, an increase in Kupffer cells and intrahepatic lymphocytes was observed in CdCl 2 .H 2 O treated group. In kidney sections of heavy metal treated group, degeneration of the glomerular tissue, occlusion in tubular lumen and necrosis were observed. Similar changes but in less severe form, as described above, were observed in the fish exposed to combination of CdCl 2 .H 2 O and ascorbic indicating that ascorbic acid do detoxify the effect of heavy metal to some extant. Our results indicate that cadmium chloride (CdCl 2 .H 2 O), if present in fresh water bodies, may act as strong toxic agent for L. rohita . Keywords: Labeo rohita , cadmium chloride (CdCl 2 .H 2 O), ascorbic acid, histopathology

Consequences of TCDD treatment on intra-hepatic lymphocytes during liver regeneration

Increasing evidence demonstrates a physiological role for the aryl hydrocarbon receptor (AhR) in regulating hepatocyte cell cycle progression. Previous studies have used a murine model of liver regeneration to show that exposure to the potent exogenous AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppresses hepatocyte proliferation in vivo. Based on recent reports that natural killer (NK) cells negatively regulate liver regeneration, coupled with the well-established immunomodulatory effects of TCDD, it was hypothesized that alterations in lymphocyte activation contribute to the suppression of liver regeneration in TCDD-treated mice. To test this, mice were treated with TCDD (20 μg/kg) 1 day prior to 70% partial hepatectomy (PH), in which two-thirds of the liver was surgically resected. Lymphocytes were collected from the remnant liver and analyzed by flow cytometry. Whereas exposure to TCDD did not alter the number of NK cells or CD3+ T-cells recovered from the regenerating liver, it reduced the percentage and number of intra-hepatic NKT cells 42 h after PH. With regard to lymphocyte activation, TCDD treatment transiently increased CD69 expression on NK and NKT cells 12 h after PH, but had no effect on intracellular levels of IFNγ in NK, NKT, or CD3+ T-cells. To determine the relevance of NK cells to the suppression of liver regeneration by TCDD, mice were treated with anti-Asialo GM-1 (ASGM-1) antibody to deplete NK cells prior to TCDD treatment and PH, and hepatocyte proliferation was measured using bromodeoxyuridine incorporation. Exposure to TCDD was found to inhibit hepatocyte proliferation in the regenerating liver of NK cell-depleted mice and control mice to the same extent. Hence, it is unlikely that enhanced numbers or increased activation of NK cells contribute to the suppression of liver regeneration in TCDD-treated mice.

Interleukin (IL)-17/IL-22-Producing T cells Enriched Within the Liver of Patients with Chronic Hepatitis C Viral (HCV) Infection

Effector CD4+ helper T cells have historically been classified into T helper 1 (Th1) and Th2 based on the production of signature cytokines. The recently identified interleukin (IL)-17 cytokine family plays important roles in host immunity against intracellular pathogens and in chronic inflammatory conditions; data have implicated IL-17 in autoimmune and viral liver disease. This study used three patient groups with HCV infection: acute HCV who either cleared spontaneously or became chronically infected (n = 12), endstage liver disease from whom both peripheral and intrahepatic lymphocytes were studied directly ex vivo (n = 11), and 134 patients with different stages of HCV-related fibrosis from whom serum was collected concurrently with liver biopsy. Normal healthy subjects (n = 41) served as controls. Acute HCV was not associated with expansion of either CD4+ or CD8+ T cells producing IL-17 (Th17, Tc17) or IL-22, and frequencies did not differ in the blood of patients who cleared versus became persistently infected. The hepatic compartment of chronic HCV patients demonstrated statistically more CD4+ and CD8+ that produced IL-17, IL-22 or both as compared to peripheral blood. These T cells displayed a distinct phenotypic profile, high expression of the homing receptor CD161 and low levels of inhibitory receptors, mucin-domain-containing-molecule-3 (Tim-3) and programmed-death 1. Using a sensitive ELISA, we found no significant differences in serum levels of IL-17 according to HCV-related fibrosis. In chronic HCV, T cells producing IL-17/IL-22 may home to the liver; however, circulating levels of IL-17 do not correlate with fibrosis.

Immunophenotypic Profile of Intrahepatic and Circulating Lymphocytes in Chronic Hepatitis B Patients

Persistent hepatitis B virus (HBV) infection is associated with particular deficiencies in the host immune system. To gain insight into the role of lymphocyte subsets involved in viral clearance and hepatic injury. The immunophenotype of peripheral blood and biopsied liver tissues in hepatitis B patients were examined. Among lymphocyte subsets analyzed, CD45RA+CD62L+ subsets were significantly lower in HBV-infected livers than in healthy controls. Intrahepatic naive lymphocytes was negatively correlated with serum viral load (r =-0.47, p<0.05) and liver injury measured by serum alanine aminotransferase (ALT) (r=-0.36, p<0.05). Serum HBV DNA was also negatively associated with intrahepatic CD8+CD95+ (r=-0.49, p<0.01), circulating CD4+HLA-DR+ (r=-0.43, p<0.05) and circulating CD3+CD(16+56)+ (r =-0.35, p<0.05). CD3+CD8+ subsets were positively correlated with serum ALT and HBV DNA (r=0.56, 0.74, p<0.01), respectively. These data suggest a key role for the exhaustion of intrahepatic naive lymphocyte reservoir in the development of a weak antiviral immune response and the inability to control viral replication in chronic hepatitis B patients. While cellular immunity is critical to clear the viral load, over-activated cytotoxic lymphocytes may also be involved in hepatic injury.

Preliminary study on immunologic mechanism of chemokine receptor CXCR3 and its ligands involved in mouse fulminant hepatitis

Objective To investigate the role of the chemokine receptor CXCR3 and its ligands in the migration of lymphocytes and acute hepatic failure. Methods BALB/cJ mice (6-8 weeks, female) were intraperitoneally injected with 100 PFU mouse hepatitis virus-3(MHV-3). The proportions and numbers of T cells and NK cells in liver, spleen, and blood as well as the expression of CXCR3 in T cells, and NK cells post MHV-3 infection was analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines(CXCL9 and CXCL10) was detected by real-time PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes and CXCL10 on the splenic lymphocytes. Results Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. Conclusion Interactions between CXCR3 and its ligands (CXCL9 and CXCL10),especially CXCL10 may play a key role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and acute hepatic failure in MHV-3 infection. Key words: Chemokine; CXCR3; CXCL10; Fulminant hepatitis; Natural killer cell

Parenchymal expression of CD40 exacerbates adenovirus-induced hepatitis in mice

The healthy adult human liver expresses low levels of major histocompatibility complex class II (MHC II) and undetectable levels of immune costimulatory molecules. However, high levels of MHC II, CD40, and B7 family molecules are expressed in the activated Kupffer cells and hepatocytes of patients with viral hepatitis. The precise role of these molecules in viral clearance and immune-mediated liver injury is not well understood. We hypothesized that parenchymal CD40 expression enhances T cell recruitment and effector functions, which may facilitate viral clearance and alleviate liver injury. To test this hypothesis, we generated novel liver-specific, conditional CD40 transgenic mice, and we challenged them intravenously with a recombinant replication-deficient adenovirus carrying Cre recombinase (AdCre). Wild-type mice infected with AdCre developed a relatively mild course of viral hepatitis and recovered spontaneously. CD40 expression in the livers of transgenic animals, however, resulted in CD80 and CD86 expression. The dysregulation of population dynamics and effector functions of intrahepatic lymphocytes (IHLs) resulted in severe lymphocytic infiltration, apoptosis, necroinflammation, and serum alanine aminotransferase elevations in a dose-dependent fashion. To our surprise, an early expansion and subsequent contraction of IHLs (especially CD8(+) and natural killer cells), accompanied by increased granzyme B and interferon-γ production, did not lead to faster viral clearance in CD40 transgenic mice. Our results demonstrate that hepatic CD40 expression does not accelerate adenoviral clearance but rather exacerbates liver injury. This study unveils a previously unknown deleterious effect of hepatic CD40 on adenovirus-induced liver inflammation.

Prevalence and Clinical Significance of Küpffer Cell Hyperplasia With Hemophagocytosis in Liver Biopsies

Hemophagocytic syndrome (HS) is a rare life-threatening condition due to uncontrolled macrophagic activation. Liver involvement is constant in HS, characterized by Küpffer cell hyperplasia with hemophagocytosis. Conversely, the specificity, frequency, and clinical significance of this histologic lesion remain poorly investigated. We aimed to evaluate the prevalence of this elementary lesion in liver biopsies (LB) to attempt to identify its clinical significance and to investigate its potential association with perforin expression deficiency. Küpffer cell hyperplasia with hemophagocytosis has been systematically searched for in consecutive LBs in a 6-year period. In positive cases, clinical, biological, and outcome characteristics have been retrospectively recorded. The ratio of perforin to CD3(+) lymphocytes was assessed on immunostained LB sections. This histologic lesion was detected in LB of 69 of 5194 patients (1.3%). It was not associated with hepatotropic viral infection, alcohol-related chronic liver disease, or autoimmune chronic liver disease. Although only 36% of patients with this histologic lesion had a complete HS (association of fever, splenomegaly, bicytopenia, hypertriglyceridemia, hyperferritinemia, and/or hypofibrinogenemia), almost all patients had similar underlying diseases (human immunodeficiency virus infection, malignant hemopathy, and autoimmune disease) and/or acute ongoing infections (tuberculosis, cytomegalovirus, and Epstein-Barr virus). A decrease of the perforin to CD3(+) lymphocytes ratio was specifically associated with this lesion. Küpffer cell hyperplasia with hemophagocytosis in LB is a rare finding; although it does not necessarily denote a complete HS, it is associated with the same underlying disease and/or infection, with a decrease in intrahepatic perforin-positive lymphocytes.

789 PRECLINICAL CHARACTERIZATION OF NOVEL CYCLOPHILIN INHIBITORS BASED ON THE POLYKETIDE, SANGLIFEHRIN

Introduction: Progressive hepatic fibrosis is a common feature of chronic hepatitis C. Natural Killer (NK) cells represent a major component of intra-hepatic lymphocytes and have been shown to be important for the early control and natural course of HCV infection. Moreover, in mouse models it has been shown that natural killer cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSC) in a NKG2D and TRAIL dependent manner. Here, we analyzed the interactions of human NK cells from HCVpositive patients with activated primary HSC and the potential effects of interferon-a (IFN-a) treatment. Material and methods: NK cells from untreated HCV-RNA(+) patients (n = 11), interferon-a (IFN-a) treated patients (n = 10) and healthy controls (n = 12) were co-incubated with activated primary HSC (ScienCell). NK cells from healthy persons were incubated in the presence or absence of IFN-a (25 IU/ml). Cytotoxic activity of NK cells was studied using the CD107a assay. In addition, INF-g and TNFa production of NK cells was measured by FACS analysis. Induction of HSC apoptosis (active Caspase-3) was analyzed flowcytometrically. Results: Analyzing cytolytic activity of NK cells following coincubation with HSC only discrete CD107a expression could be observed on both NK cells from HCV(+) patients and healthy controls ([mean±SEM: 5.7±0.7% vs. 4.7±0.8%; p =n.s.). Furthermore, only negligible secretion of IFN-g and TNF-a could be observed. However, NK cells from untreated HCV-infected patients were significantly more effective in induction of HSC apoptosis (20.8±2.1%) than NK cells from healthy controls (6.2±0.6%). Of note, NK cells from IFN-a/RBV treated HCV(+) patients displayed an even stronger capability to kill HSC (26.9±4.3%). In vitro stimulation of NK cells with IFN-a resulted in a significant up-regulation of TRAIL and was associated with increased killing of HSC as compared to un-stimulated NK cells. This effect could be blocked with both NKG2Dand TRAIL-specific antibodies. Conclusion: NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated hepatic stellate cells. This function of NK cell is increased following IFN-a treatment. Thus, NK cells may play an important anti-fibrotic role in chronic hepatitis C.

CD8 T Cells Mediate Direct Biliary Ductule Damage in Nonobese Diabetic Autoimmune Biliary Disease

We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.

Intrahepatic natural killer T cell populations are increased in human hepatic steatosis

To determine if natural killer T cell (NKT) populations are affected in nonalcoholic fatty liver disease (NAFLD). Patients undergoing bariatric surgery underwent liver biopsy and blood sampling during surgery. The biopsy was assessed for steatosis and immunocyte infiltration. Intrahepatic lymphocytes (IHLs) were isolated from the remainder of the liver biopsy, and peripheral blood mononuclear cells (PBMCs) were isolated from the blood. Expression of surface proteins on both IHLs and PBMCs were quantified using flow cytometry. Twenty-seven subjects participated in this study. Subjects with moderate or severe steatosis had a higher percentage of intrahepatic CD3+/CD56+ NKT cells (38.6%) than did patients with mild steatosis (24.1%, P = 0.05) or those without steatosis (21.5%, P = 0.03). Patients with moderate to severe steatosis also had a higher percentage of NKT cells in the blood (12.3%) as compared to patients with mild steatosis (2.5% P = 0.02) and those without steatosis (5.1%, P = 0.05). NKT cells are significantly increased in the liver and blood of patients with moderate to severe steatosis and support the role of NKT cells in NAFLD.

Blockade of Immunosuppressive Cytokines Restores NK Cell Antiviral Function in Chronic Hepatitis B Virus Infection

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56bright NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/− TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/− TGF-β blockade.

Tumor Necrosis Factor Is Critical for Cytolytic T Cell Activity Against Allospecific Hepatocytes and Splenic Targets in Major Histocompatibility Complex Class I Disparate Graft Versus Host Disease

The present studies determined the role of tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) interactions on cytolytic (CTL) activity of splenic and intrahepatic lymphocytes (IHL) isolated from mice undergoing graft versus host disease, induced by transfer of B6 T cells to major histocompatibility complex (MHC) class I disparate bm1 × B6 F1 mice. Allospecific killing of anti-H-2(bm1) splenic and hepatocyte targets was assessed by 4-h (51)Cr release and 16-h DNA lysis assays, respectively, utilizing spleen cells (SpC) and IHL isolated (1) from sublethally irradiated bm1 × B6 F1 who had received B6 spleen and bone marrow cells, and a control adenovirus (Adv-βgal) or a TNF inhibitor expressing adenovirus (Adv-TNFi), or (2) from bm1 × B6 F1 recipients of B6, B6.129-Tnfrsf1a(tm1Mak)/J (TNFR1(-/-)), B6.129S2-Tnfrsf1b(tm1Mwm)/J (TNFR2(-/-)), or B6.129S-Tnfrsf1a(tm1Imx) Tnfrsf1b(tm1Imx)/J (TNFR(-/-)) SpC and bone marrow cells, or (3) from in vitro-activated SpC. Splenic and IHL from bone marrow transplant recipients who had received Adv-TNFi at the time of transplant displayed lower allospecific CTL activity than controls. Addition of TNFR-Ig or a TNF antibody before the CTL activity assay further reduced allospecific killing against bm1 SpC blast targets. Both TNF/TNFR1 and TNF/TNFR2 interactions were critical for the development of optimal CTL activity against allospecific hepatocyte targets. Further, TNFR1- and TNFR2-deficient SpC from MHC class I disparate mixed lymphocyte cultures displayed lower CTL activity and expression of effector molecules than control B6 SpC. TNF/TNFR interactions were critical for the development of optimal CTL activity of IHL and splenic cytotoxic T cells against MHC class I disparate SpC blast and hepatocyte targets in MHC class I disparate graft versus host disease.

Ability of IDO To Attenuate Liver Injury in α-Galactosylceramide–Induced Hepatitis Model

IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in α-galactosylceramide (α-GalCer)-induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered α-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after α-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-D-tryptophan also exacerbated liver injury in this murine hepatitis model. In α-GalCer-induced hepatitis models, TNF-α is critical in the development of liver injury. The mRNA expression and protein level of TNF-α in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with α-GalCer were analyzed by flow cytometry, and the numbers of CD49b(+) and CD11b(+) cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with α-GalCer, TNF-α secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in α-GalCer-induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-α-producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the α-GalCer-induced hepatitis model.

OP20 Hepatocytes and cholangiocytes do not have significant telomere shortening with increasing chronological age in normal livers

IntroductionTelomeres, which cap and protect chromosomal DNA, shorten with each cell division reaching a critical point eventually when the cell is arrested in G1 phase and enters a state of...

Hepatic AAV Gene Transfer and the Immune System: Friends or Foes?

Two ongoing clinical trials utilize different adeno-associated viral (AAV) vectors for liver-directed factor IX (F.IX) gene transfer with the goal of sustained therapy in patients with severe hemophilia B. Although preclinical studies have documented immune tolerance and long-term expression of F.IX in animals, the single prior clinical trial of this approach achieved only transient therapeutic gene expression and exposed preexisting immunity to the AAV vector as a major obstacle for therapy.1 Although accumulating preclinical data continue to fuel a debate over the potential impact of immune responses on hepatic AAV gene transfer, these data do not allow us to predict whether the human immune system will reject or tolerate therapy. This commentary dissects the relevant human and animal data, some of which are contradictory or allow us to draw only limited conclusions. More clinical trial data are critically needed and should ultimately help us develop better protocols.

Possible Recruitment of Peripheral Blood CXCR3+CD27+CD19+B Cells to the Liver of Chronic Hepatitis C Patients

It has been suggested that hepatitis C virus (HCV) infects not only hepatocytes but also immune cells, including B cells. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma. To clarify the effects of chronic HCV infection on B-cell dynamics, peripheral B cells from chronic hepatitis C patients (CHC) were characterized. We found that the frequency of CD27(+) B cells, that is memory phenotype, was significantly reduced in the peripheral blood of CHC. At the same time, the amount of IFN-gamma-inducible protein-10 (IP-10), a CXCR3 ligand, was markedly elevated in the plasma of CHC. Furthermore, the CD27(+) B-cell population was found to highly express CXCR3 in CHC, thus suggesting that the CD27(+) B-cell population was recruited from peripheral blood to the inflammatory site of the liver of CHC, where IP-10 is produced. Immunohistochemical analyses of intrahepatic lymphocytes indicated that CXCR3(+) B cells were infiltrated in the liver of CHC. Our results thus offer new insight into the role of memory B cells in the HCV pathogenesis.

Heterogeneous Inflammatory Changes in Liver Graft Recipients With Normal Biochemistry

Patients with established liver grafts may receive excessive immune suppression. Liver biopsies were analyzed in those with normal liver biochemistry to identify parameters that might identify such cases. Patients with established grafts (>3 years from engraftment) and normal liver biochemistry (normal alanine transaminase, alkaline phosphatase, and bilirubin) were invited to undergo liver biopsy. Liver tissue was assessed by routine histopathology, a modified Ishak score, and immunohistochemistry for lymphocyte and cell-cycle markers. Circulating and intrahepatic lymphocytes were subjected to flow cytometry. Data were subjected to principal component analysis. Two hundred twenty-five (40%) patients under regular review had an established graft with normal liver biochemistry; liver tissue was obtained in 55. Liver histology was normal in eight cases (14.5%). The most common abnormalities were mild nonspecific hepatitis in 25 (45.4%) and disease recurrence in 14 (25.4%). Principal component analysis identified a cluster of variables that accounted for a significant degree of variation within the dataset. These were lobular inflammation, portal inflammation, interface hepatitis, and fibrosis. Inflammation persisted in established grafted livers in most patients with normal liver biochemistry. Systematic histological and lymphocyte phenotype analysis generated an index that distinguished patient groups. Those with least inflammation and the lowest alanine transaminase may have a reduced requirement for immune suppression.