Additive improvement induced by bezafibrate in patients with primary biliary cirrhosis showing refractory response to ursodeoxycholic acid.

BACKGROUND AND AIM: Ursodeoxycholic acid (UDCA) has been widely used in the treatment of patients with primary biliary cirrhosis (PBC). However, some patients are refractory to UDCA. The aim of this study is to clarify the additive improvement induced by bezafibrate in patients refractory to UDCA. METHODS: This study was a prospective analysis of 37 consecutive PBC patients. All patients were treated first for 6 months with UDCA, and then with bezafibrate, if their alkaline phosphatase (ALP) levels did not decrease more than 40% or within the normal range after 6 months' treatment with UDCA. Clinical parameters were monitored for the subsequent 24 months. RESULT: Twenty-two patients (59%) achieved improvement of ALP levels after the treatment with UDCA. Those patients (Group A) had significantly lower levels of ALP at diagnosis than those with abnormal ALP levels after 6 months' treatment with UDCA (Group B; P = 0.020). They continued UDCA monotherapy, and maintained normal ALP levels at subsequent follow ups. However, immunoglobulin M (IgM) levels remained abnormal in eight patients, whose IgM levels at the time of diagnosis were significantly higher than those whose IgM were normalized after 6 months' treatment with UDCA (P = 0.026). Those in Group B were treated additionally with bezafibrate, and 12 patients (80%) achieved normal ALP and IgM levels within 12 months of commencement of therapy. CONCLUSION: Higher ALP level at diagnosis is one of the predictors for UDCA failure. Combination treatment of bezafibrate in addition to UDCA may be an effective treatment for PBC patients refractory to UDCA.

Two cases of development of entecavir resistance during entecavir treatment for nucleoside-naive chronic hepatitis B.

BACKGROUND: Entecavir (ETV) is a potent nucleoside analogue against hepatitis B virus (HBV), and emergence of drug resistance is rare in nucleoside-naive patients because development of ETV resistance (ETVr) requires at least three amino acid substitutions in HBV reverse transcriptase. We observed two cases of genotypic ETVr with viral rebound and biochemical breakthrough during ETV treatment of nucleoside-naive patients with chronic hepatitis B (CHB). RESULTS: Case 1: A 44-year-old HBeAg-positive man received ETV 0.1 mg/day for 52 weeks and 0.5 mg/day for 96 weeks consecutively. HBV DNA was 10.0 log(10) copies/ml at baseline, declined to a nadir of 3.1 at week 100, and rebounded to 4.5 at week 124 and 6.7 at week 148. Alanine aminotransferase (ALT) level increased to 112 IU/l at week 148. Switching to a lamivudine (LVD)/adefovir-dipivoxil combination was effective in decreasing HBV DNA. Case 2: A 47-year-old HBeAg-positive man received ETV 0.5 mg/day for 188 weeks. HBV DNA was 8.2 log(10) copies/ml at baseline, declined to a nadir of 2.9 at week 124, and then rebounded to 4.7 at week 148 and 6.4 at week 160. ALT level increased to 72 IU/l at week 172. The ETVr-related substitution (S202G), along with LVD-resistance-related substitutions (L180M and M204V), was detected by sequence analysis at week 124 in both case 1 and case 2. CONCLUSIONS: ETVr emerged in two Japanese nucleoside-naive CHB patients after prolonged therapy and incomplete suppression and in one patient after <0.5 mg of dosing. ETV patients with detectable HBV DNA or breakthrough after extended therapy should be evaluated for compliance to therapy and potential emergence of resistance.

Clinical characteristics of drug-induced liver injury in the elderly.

AIM: The average age of Japanese patients with drug-induced liver injury (DILI) is expected to rise as the population ages. The aim of this study was to evaluate the clinical characteristics of DILI in elderly Japanese subjects. METHODS: A total of 142 hospitalized patients with DILI were divided into three groups by age (Group A, < 65 years; Group B, 65-74 years; Group C, >/= 75 years). Patients were examined retrospectively with regard to the number of concomitant drugs, duration of drug intake until onset of DILI, clinical types of DILI, treatment, prognosis, and scores on the diagnostic scale described by Digestive Disease Week, Japan 2004. RESULTS: Patients in Group C used more concomitant drugs (P = 0.0019) compared with patients in Group A. The elderly had a higher frequency of unclear duration of drug intake (P = 0.0020), were more likely to experience cholestatic type DILI (P = 0.033), and underwent more intensive treatment for DILI (P = 0.011). There were fewer cases diagnosed as a "high possibility" of DILI in Group C compared with Group A (P = 0.030) and Group B (P = 0.013). In cases diagnosed as "possible" or "low possibility" of DILI, unclear duration of drug intake was more frequently observed in the elderly (P = 0.0027). CONCLUSION: Elderly patients with DILI show different clinical features than younger patients with DILI. These features should be taken into account in the diagnosis and treatment of DILI in the elderly.

Efficient culture of CD8(+) T cells from the islets of NOD mice and their use for the study of autoreactive specificities.

During progression to type 1 diabetes (T1D), the pancreatic islets of humans and of the widely studied mouse model of T1D, the nonobese diabetic (NOD) mouse, are infiltrated by cells of the immune system. Here we report that infiltrated mouse islets ("translucent islets") can be identified visually. We demonstrate the use of an efficient method for the isolation and culture of the islet-infiltrating cells of NOD mice, which results in a high percentage of CD8(+) T cells after seven days, with minimal manipulation. We show that islet-infiltrating cells exit the islets soon after they are placed in culture and can be used in flow cytometric experiments several hours later. Importantly, we demonstrate that the cultured cells are antigen-responsive and that specificities present at the beginning of the culture are generally still present on day seven. In addition, some reactivities are undetected without culture, supporting the validity of the seven-day expansion period. This technique greatly facilitates investigations of the CD8(+) T cell reactivities that play a pivotal role in the demise of pancreatic beta cells leading to the development of T1D.

In vivo cytotoxicity of insulin-specific CD8+ T-cells in HLA-A*0201 transgenic NOD mice.

OBJECTIVE: CD8(+) T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8(+) T-cells will require the identification of disease-relevant epitopes. RESEARCH DESIGN AND METHODS: We used islet-infiltrating CD8(+) T-cells from HLA-A*0201 transgenic NOD mice in an interferon-gamma enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike. RESULTS: We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3-11, Ins1 B5-14, and Ins1/2 A2-10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5-14 and Ins1/2 A2-10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process. CONCLUSIONS: The human versions of B5-14 and A2-10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8(+) T-cell targets in HLA-A*0201-positive type 1 diabetic patients.

Improved outcomes in NOD mice treated with a novel Th2 cytokine-biasing NKT cell activator.

Activation of CD1d-restricted invariant NKT (iNKT) cells by alpha-galactosylceramide (alphaGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic alphaGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with alphaGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8(+) T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with alphaGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of alphaGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the alphaGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes.

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Individual nonobese diabetic mice exhibit unique patterns of CD8+ T cell reactivity to three islet antigens, including the newly identified widely expressed dystrophia myotonica kinase.

Spontaneous autoimmune diabetes development in NOD mice requires both CD8(+) and CD4(+) T cells. Three pathogenic CD8(+) T cell populations (represented by the G9C8, 8.3, and AI4 clones) have been described. Although the Ags for G9C8 and 8.3 are known to be insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein, respectively, only mimotope peptides had previously been identified for AI4. In this study, we used peptide/MHC tetramers to detect and quantify these three pathogenic populations among beta cell-reactive T cells cultured from islets of individual NOD mice. Even within age-matched groups, each individual mouse exhibited a unique distribution of beta cell-reactive CD8(+) T cells, both in terms of the number of tetramer-staining populations and the relative proportion of each population in the islet infiltrate. Thus, the inflammatory process in each individual follows its own distinctive course. Screening of a combinatorial peptide library in positional scanning format led to the identification of a peptide derived from dystrophia myotonica kinase (DMK) that is recognized by AI4-like T cells. Importantly, the antigenic peptide is naturally processed and presented by DMK-transfected cells. DMK is a widely expressed protein that is nonetheless the target of a beta cell-specific autoimmune response.

Requirement for both H-2Db and H-2Kd for the induction of diabetes by the promiscuous CD8+ T cell clonotype AI4.

The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8(+) T cells are essential for the destruction of the insulin-producing pancreatic beta cells characterizing this disease. AI4 is a pathogenic CD8(+) T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4(+) T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of beta cells by AI4 requires the beta cells to simultaneously express both H-2D(b) and H-2K(d) class I MHC molecules. The ability of the AI4 TCR to interact with both H-2D(b) and H-2K(d) was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2D(b)-restricted manner. Using ELISPOT and mimotope/H-2D(b) tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population.

Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes.

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.

During the early prediabetic period in NOD mice, the pathogenic CD8(+) T-cell population comprises multiple antigenic specificities.

In the NOD mouse model of type 1 diabetes, major histocompatibilitycomplex (MHC) class I-restricted CD8(+) T cells are essential for disease development. However, the extent of diversity of their antigenic specificities during early pathogenesis remains unclear. An insulin-derived peptide was recently identified as the epitope for the NOD-derived diabetogenic T-cell clone G9C8. To explore the possibility that the early pathogenic CD8(+) T-cell population comprises additional antigenic specificities, we employed the T-cell clones AI4 and NY8.3, both of which are pathogenic and represent specificities present in early insulitic lesions. The clones responded to distinct fractions of chromatographically separated class I MHC-bound peptides purified from NOD-derived NIT-1 beta cells, and neither clone recognized the insulin-derived peptide. NIT-1 cells represent an unlimited peptide source that will allow for the future isolation and sequencing of the novel multiple epitopes targeted early in the autoimmune response by pathogenic CD8(+) T cells.