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Depending on age of acquisition, hepatitis B virus (HBV) can induce a cell-mediated immune response that results in either cure or progressive liver injury. In adult-acquired infection, HBV antigens are usually cleared, whereas in infancy-acquired infection, they persist. Individuals infected during infancy therefore represent the majority of patients chronically infected with HBV (CHB). A therapy that can promote viral antigen clearance in most CHB patients has not been developed and would represent a major health care advance and cost mitigator. Using an age-dependent mouse model of HBV clearance and persistence in conjunction with human blood and liver tissue, we studied mechanisms of viral clearance to identify new therapeutic targets. We demonstrate that age-dependent expression of the costimulatory molecule OX40 ligand (OX40L) by hepatic innate immune cells is pivotal in determining HBV immunity, and that treatment with OX40 agonists leads to improved HBV antigen clearance in young mice, as well as increased strength of T cell responses in young mice and adult mice that were exposed to HBV when they were young and developed a CHB serological profile. Similarly, in humans, we show that hepatic OX40L transcript expression is age-dependent and that increased OX40 expression on peripheral CD4+ T cells in adults is associated with HBV clearance. These findings provide new mechanistic understanding of the immune pathways and cells necessary for HBV immunity and identify potential therapeutic targets for resolving CHB.

Killer cell immunoglobulin-like receptors (KIR) are encoded by highly polymorphic genes that regulate the activation of natural killer (NK) cells and other lymphocyte subsets and likely play key roles in innate and adaptive immunity. Association studies increasingly implicate KIR in disease predisposition and outcome but could be confounded by unknown KIR genetic structure in heterogeneous populations. To examine this, we characterized the diversity of 16 KIR genes in 712 Northern Californians (NC) stratified by self-assigned ethnicities and compared the profiles of KIR polymorphism with other US and global populations using a reference database. Sixty-eight distinct KIR genotypes were characterized: 58 in 457 Caucasians (NCC), 17 in 47 African Americans (NCAA), 21 in 80 Asians (NCA), 20 in 74 Hispanics (NCH), and 18 in 54 "other" ethnicities (NCO). KIR genotype patterns and frequencies in the 4 defined ethnicities were compared with each other and with 34 global populations by phylogenetic analysis. Although there were no population-specific genotypes, the KIR genotype frequency patterns faithfully traced the ancestry of NCC, NCAA, and NCA but not of NCH whose ancestries are known to be more heterogeneous. KIR genotype frequencies can therefore track ethnic ancestries in modern urban populations. Our data emphasize the importance of selecting ethnically matched controls in KIR-based studies to avert spurious associations.

Background & Aims: The immunosuppressive treatment for autoimmune hepatitis (AIH) patients is prednisone and azathioprine. Ten percent to 20% of patients do not respond or are intolerant of standard treatment. The aim of this study was to assess the biochemical, histologic, and hematologic parameters during mycophenolate mofetil (MMF) treatment in AIH patients who did not respond to or were intolerant of prednisone and/or azathioprine. Methods: A retrospective study was performed of 15 AIH patients who received MMF either as monotherapy or in combination with prednisone after failure or intolerance of the initial regimen. Records were reviewed as to initial therapy, reasons why MMF was initiated, liver enzyme levels, histology on MMF, and complications. Results: The mean age was 60 ± 15 years. All patients were started on MMF at 1 gram twice a day, 3 on MMF monotherapy, and 12 on prednisone and MMF. The average MMF treatment duration was 41 months. Alanine aminotransferase levels decreased significantly from 91.73 ± 88.69 to 60.87 ± 71.2 (P = .03) on MMF treatment. Inflammatory scores (2.59 ± 0.97 to 1.14 ± 1.21, P = .02) and Ishak fibrosis scores (4.10 ± 1.37 to 2.5 ± 1.51, P = .02) also decreased. No significant hematologic complications were noted during MMF treatment. Conclusions: Administration of MMF, either as monotherapy or in combination with prednisone, results in biochemical and histologic improvement in AIH patients who are prednisone and/or azathioprine intolerant or resistant without the development of significant complications. MMF should be studied prospectively as an alternative agent in the treatment of autoimmune liver disease.

This prospective, multicenter, community-based and academic-based, open-label, investigator-initiated, U.S. study evaluated efficacy and safety of pegylated interferon (PEG-IFN) alfa-2b plus a flat or weight-based dose of ribavirin (RBV) in adults with chronic hepatitis C. Patients (n = 5027) were randomly assigned to receive PEG-IFN alfa-2b 1.5 μg/kg/week plus flat-dose (800 mg/day) or weight-based (800-1400 mg/day) RBV for 48 weeks (patients with genotype 1, 4, 5, or 6) and for 24 or 48 weeks (genotype 2/3 patients). Primary end point was sustained virologic response (undetectable [

SummaryTo monitor IL-4 expression at the single-cell level, we generated mice with insertions of different reporter genes into both copies of the Il4 gene that permitted the simultaneous analysis of IL-4 transcripts via GFP and IL-4 protein secretion by use of huCD2. Innate and adaptive cells competent for IL-4 production were marked by GFP, while cells that presently or recently secreted IL-4 additionally displayed huCD2. After challenge with the strictly enteric helminth, Heligmosomoides polygyrus, GFP-positive innate and adaptive cells disseminated widely, but IL-4 secretion was predominantly mediated by CD4+ T cells in the intestines and draining lymphoid organs. IL-4-competent cells persisted in cured animals, and memory responses reflected rapid cytokine production at the site of rechallenge. These data reveal a two-step process for cytokine production: the first generating poised cells that disseminate systemically and the second inducing the rapid production of the cytokine in response to local stimulation.

Organ transplant recipients have an increased tumor incidence owing to their imumunocompromised state. The origin of such tumors, whether donor or recipient, will have a clinical impact on decision-making concerning immunosuppressive therapy, retransplantation, and for recipients of other organs from the same donors. We report molecular cytogenetic determination of donor origin in 2 cases of small-cell neuroendocrine carcinoma developing in sex-mismatched transplant recipients (kidney and fiver). Fluorescence in situ hybridization (FISH) analysis was performed on liver core needle biopsy material from the fiver transplant patient and on fiver fine needle aspiration cytopreparations from the kidney transplant patient. The results for the liver transplant patient were confirmed with microsatellite allefic analysis and with comparative genomic hybridization. In both cases, FISH showed the presence of only X chromosomes within the tumor cells, indicating the donor origin of the neoplasms. FISH is an excellent method to determine neoplastic origin in sex-mismatched transplant patients.

The diverse response of individuals within populations to infectious pathogens remains poorly understood, although genetic determinants undoubtedly contribute in substantial ways to the outcome of infection. In a mouse model of infection with the intramacrophage protozoan Leishmania major, susceptibility correlates both with aberrant helper T cell differentiation biased towards the production of interleukin 4 and with the presence of an endogenous CD4 T cell repertoire that recognizes an immunodominant parasite antigen with high frequency. In the setting of the particular ecological niche occupied by Leishmania, this combination of otherwise unrelated factors synergizes to result in exquisite susceptibility to this single pathogen, without seemingly compromising host defenses against other agents. Similar paradigms could underlie susceptibility to other pathogenic organisms.

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Hepatitis B virus (HBV) is a major human pathogen that causes immune-mediated hepatitis. Successful immunity to HBV is age dependent: viral clearance occurs in most adults, whereas neonates and young children usually develop chronic infection. Using a mouse model of HBV infection, we sought mechanisms underpinning the age-dependent outcome of HBV and demonstrated that hepatic macrophages facilitate lymphoid organization and immune priming within the adult liver and promote successful immunity. In contrast, lymphoid organization and immune priming was greatly diminished in the livers of young mice, and of macrophage-depleted adult mice, leading to abrogated HBV immunity. Furthermore, we found that CXCL13, which is involved in B lymphocyte trafficking and lymphoid architecture and development, is expressed in an age-dependent manner in both adult mouse and human hepatic macrophages and plays an integral role in facilitating an effective immune response against HBV. Taken together, these results identify some of the immunological mechanisms necessary for effective control of HBV.

Protection against Leishmania major infection among inbred strains of mice is dependent upon successful expansion and activation of type 1 CD4+ effector (Th1) cells, a process that is aberrant in highly susceptible BALB strains. We sought to establish whether vaccination strategies using whole parasite lysates or a characterized immunodominant antigen, the Leishmania homolog of mammalian receptor for activated protein kinase C (LACK), would be capable of protecting subsequently infected BALB mice if given within a cytokine milieu capable of biasing the immune response toward Th1 cells. When given with neutralizing antibody to IL-4, but not when given alone, subcutaneously administered soluble Leishmania antigens mediated substantial protection to BALB/c mice against subsequent infection with parasites as assessed by size of the local lesion, enhanced Th1-type immune responses, and decreased parasite burdens. Similarly, when given with recombinant IL-12, LACK conferred substantial protection to cohorts of BALB.B, BALB/c, and BALB.K mice that was associated with reduction in serum IgE levels, consistent with effects on IL-4 production. Thus altering the cytokine milieu during administration of vaccine antigens by neutralizing IL-4 induced powerful Th1 recall responses during infection that were capable of mediating substantial levels of protection.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the α chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell–deficient mice by an IL-4 receptor α chain–dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Leishmania major infection has proven an exceptional model for CD4+ subset development in inbred mice. Most strains contain infection coincident with the appearance of T helper 1 (Th1) cells that produce gamma-interferon (IFN-gamma) required for macrophage activation. In contrast, mice on the BALB background are unable to control infection due to the development of Th2 cells that produce counter-regulatory cytokines, particularly interleukin 4 (IL-4), capable of abrogating the effects of IFN-gamma. Selective gene disruption studies in mice have illustrated critical components of the host response to L. major. Mice deficient in beta 2 microglobulin, which have no major histocompatibility complex (MHC) class I or CD8+ T cells, control infection as well as wild-type mice, whereas mice deficient in MHC class II (and CD4+ T cells) suffer fatal infection. Mice with disruption of the gene coding IFN-gamma are also incapable of containing infection, reflecting absolute requirements for this cytokine. A number of interventions have been demonstrated to abrogate Th2 cell development in BALB mice, enabling these mice to control infection. Each of these--IL-12, anti-IL-4, anti-IL-2, anti-CD4 and CTLA4-Ig--has in common the capacity to make IL-4 rate limiting at the time of CD4+ cell priming.

Background/Aims: Chronic hepatitis C (HCV) patients who have failed previous treatment have low sustained viral response (SVR) rates with repeat treatment. We evaluated whether interferon (IFN) induction during retreatment improves response rates. Methods: Two randomized, controlled trials were conducted in chronic HCV patients who failed IFN. In Study 1, patients received IFN 3 MU daily plus ribavirin (RBV) 1000 mg/day for 4 weeks, followed by IFN 3 MU TIW plus RBV 1000 mg/day for 44 weeks (induction; n=232), or IFN 3 MU TIW plus RBV 1000 mg/day for 48 weeks (non-induction; n=237). In Study 2, patients received IFN 5 MU B.I.D. plus RBV 1000–1200 mg/day for 2 weeks, followed by pegylated IFN (PEG-IFN) 75–150 μg weekly plus RBV 1000–1200 mg/day for 46 weeks (induction; n=201), or PEG-IFN 75–150 μg weekly plus RBV 1000–1200 mg/day for 48 weeks (non-induction; n=206). The primary end point for both trials was SVR. Results: Induction did not increase SVR compared with non-induction, but did increase the on-treatment response among genotype non-1 patients in Study 2. By intention-to-treat (ITT) analysis, SVR in Study 1 was 13% for induction vs. 9% for non-induction (P=NS). In Study 2 (ITT), SVR was 20% for induction vs. 24% for non-induction (P=NS). However, by non-ITT analysis of Study 2, genotype non-1-previous non-responders showed significantly higher response rates with induction than non-induction. Conclusion: For chronic HCV patients who have failed IFN, induction with retreatment does not improve SVR, but may be beneficial for patients with genotype non-1 HCV.

Antivirals for hepatitis B virus (HBV) reduce viral load and improve liver histology, however, their effect on covalently closed circular DNA (cccDNA), the HBV transcriptional template, has not been extensively examined. This study evaluated a newly designed LightCycler based quantitative cccDNA PCR assay. A linear range of 2.5 x 10(1) to 1 x 10(9) copies/assay using primers specific for HBV cccDNA and 2.5 x 10(1) to 2.5 x 10(9) copies/assay using primers specific for total HBV DNA (tDNA) was established. beta-Globin was used to estimate the number of cells in each PCR reaction. Enzymatic digestion with an ATP-dependent DNase improved the analytic specificity to a greater than 1:10000 ratio of cccDNA:RC DNA (relaxed circular DNA). One-tenth of the extracted DNA from 1mg of liver biopsy, was analyzed from six patients, three HBV-infected and three uninfected individuals, under blinded conditions; three were found positive and three negative for cccDNA and tDNA. Approximately 6 x 10(3) copies of cccDNA/mg of tissue were detected in a pre-transplant biopsy from an HBV-infected patient treated with lamivudine. Sequential post-transplant liver biopsies were negative for both HBV cccDNA and tDNA. An HBV-infected patient with cirrhosis who was antiviral therapy naive had 3.7 x 10(4) copies of cccDNA/mg of liver tissue. Another treatment-naive patient with a history of high HBV viral load had 1 x 10(5) copies of cccDNA/mg of tissue (4 x 10 (6) copies of tDNA/mg of tissue). Further studies are warranted but the high level of sensitivity, specificity, rapidity and accuracy provided by this novel assay with the LightCycler system indicate that it could be useful for monitoring antiviral therapy.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly recognized clinico-pathologic entity typically associated with obesity, type II diabetes and hyperlipidemia. It has been noted to recur after orthotopic liver transplantation (OLT). We report four patients who developed de novo NAFLD within 3 months of OLT without the typical predisposing factors of diabetes mellitus or obesity. Three of the four patients underwent OLT for hepatitis C-related cirrhosis, and the other for alcoholic cirrhosis. Examination of the liver explants revealed no evidence of steatosis. No surreptitious alcohol use or a drug-induced process could be identified in these patients. Treatment of recurrent hepatitis C infection in one patient with interferon and ribavirin led to sustained suppression of the viral RNA to undetectable levels, but no improvement in histology or liver enzymes. All four patients had histologic evidence of preservation injury on the initial post-OLT biopsies, but the significance of this finding in relationship to the development of NAFLD is unknown. NAFLD can develop without any of the known predisposing conditions after transplantation, and this raises further questions about the pathogenesis of this condition.

Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a “fingerprint” pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5′ noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5′ NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.

The mechanisms by which naive helper T cells differentiate into potent cytokine-expressing effectors remain critical to understanding both successful and aberrant immune responses. Studies using Leishmania major infection of mice have revealed genetic contributions to factors that influence this differentiative process. Further, antigen recognition at the level of the T cell repertoire can also profoundly affect the outcome of disease and the appearance of discrete T cell subsets. It is likely that such mechanisms also underpin genetic susceptibility to diverse other infectious and autoimmune diseases.

Hepatitis B virus (HBV) is a non-cytopathic hepadnavirus and major human pathogen that causes immune-mediated acute and chronic hepatitis. The immune response to HBV antigens can result in viral clearance by some individuals, or lead to chronic infection and liver disease in others. The age of the individual at the time of infection dictates the likelihood of HBV clearance. We sought mechanisms underpinning the age-dependent outcome of HBV. We now present an animal model that recapitulates key differences in viral clearance during early life and adulthood, and show an unexpected role for T Follicular Helper T cells and IL-21 in determining the effectiveness of the primary hepatic immune response to HBV. Furthermore, we provide evidence that IL-21 is part of an effective immune response to HBV in patients acutely infected with HBV who clear the virus. In addition, we now demonstrate that hepatic macrophages facilitate lymphoid organization and immune priming within the adult liver and promote successful immunity. In contrast, lymphoid organization and immune priming is greatly diminished in the livers of young mice, and of macrophage-depleted adult mice, leading to abrogated HBV immunity. These observations provide a new mechanism to explain the dichotomous, age-dependent outcome of HBV infection in humans, and carry implications for therapeutic augmentation of immune responses to HBV and potentially other persistent liver viruses.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.