Christopher L. Bowlus

Exenatide effects on diabetes, obesity, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes treated for at least 3 years.

BACKGROUND Exenatide, an incretin mimetic for adjunctive treatment of type 2 diabetes (T2DM), reduced hemoglobin A(1c) (A1C) and weight in clinical trials. The objective of this study was to evaluate the effects of > or = 3 years exenatide therapy on glycemic control, body weight, cardiometabolic markers, and safety. METHODS Patients from three placebo-controlled trials and their open-label extensions were enrolled into one open-ended, open-label clinical trial. Patients were randomized to twice daily (BID) placebo, 5 mug exenatide, or 10 mug exenatide for 30 weeks, followed by 5 mug exenatide BID for 4 weeks, then 10 mug exenatide BID for > or = 3 years of exenatide exposure. Patients continued metformin and/or sulfonylureas. RESULTS 217 patients (64% male, age 58 +/- 10 years, weight 99 +/- 18 kg, BMI 34 +/- 5 kg/m(2), A1C 8.2 +/- 1.0% [mean +/- SD]) completed 3 years of exenatide exposure. Reductions in A1C from baseline to week 12 (-1.1 +/- 0.1% [mean +/- SEM]) were sustained to 3 years (-1.0 +/- 0.1%; p < 0.0001), with 46% achieving A1C < or = 7%. Exenatide progressively reduced body weight from baseline (-5.3 +/- 0.4 kg at 3 years; p < 0.0001). Patients with elevated serum alanine aminotransferase (ALT) at baseline (n = 116) had reduced ALT (-10.4 +/- 1.5 IU/L; p < 0.0001) and 41% achieved normal ALT. Patients with elevated ALT at baseline tended to lose more weight than patients with normal ALT at baseline (-6.1 +/- 0.6 kg vs. -4.4 +/- 0.5 kg; p = 0.03), however weight change was minimally correlated with baseline ALT (r = -0.01) or ALT change (r = 0.31). Homeostasis Model Assessment B (HOMA-B), blood pressure, and aspartate aminotransferase (AST) all improved. A subset achieved 3.5 years of exenatide exposure and had serum lipids available for analysis (n = 151). Triglycerides decreased 12% (p = 0.0003), total cholesterol decreased 5% (p = 0.0007), LDL-C decreased 6% (p < 0.0001), and HDL-C increased 24% (p < 0.0001). Exenatide was generally well tolerated. The most frequent adverse event was mild-to-moderate nausea. The main limitation of this study is the open-label, uncontrolled nature of the study design which does not provide a placebo group for comparison. CONCLUSION Adjunctive exenatide treatment for > or = 3 years in T2DM patients resulted in sustained improvements in glycemic control, cardiovascular risk factors, and hepatic biomarkers, coupled with progressive weight reduction.

Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis

CD4+CD25high regulatory T cells (Tregs) play a critical role in self‐tolerance, as seen in murine autoimmunity. Studies on Tregs in human autoimmunity have focused primarily on peripheral blood samples. A study targeting diseased tissue should identify direct relationships between Tregs and autoimmunity. Peripheral blood samples were collected from 91 patients with primary biliary cirrhosis (PBC), 28 immediate relatives, and 41 healthy controls, and Treg frequencies were determined as a percentage of CD4+CD25high T cells in CD4+TCR‐αβ+ T cells. A tissue‐targeted determination of frequency and distribution of FoxP3+ Tregs was also performed on 90 different liver tissue specimens exhibiting PBC (n = 52), chronic hepatitis C (CHC) (n = 30), and autoimmune hepatitis (AIH) (n = 8). Treg suppression studies were performed on 50 PBC patients and 27 controls. Patients with PBC demonstrated a relative reduction of Tregs compared with controls (P < .0002). Interestingly, a deficiency in CD4+CD25+ Tregs was also found in the daughters and sisters of PBC patients compared with controls (P < .0007). However, functional studies did not reveal a global PBC Treg defect. The level of FoxP3‐expressing Tregs was markedly lower in affected PBC portal tracts compared with CHC and AIH (P < .001). In addition, the CD8+T cell/FoxP3+ Treg ratio was significantly higher in livers of late‐stage PBC compared with those of CHC (P < .001) and early‐stage AIH (P < .001). In conclusion, these data provide support for a genetic modulation of Treg frequency and illustrate the role Tregs play in the loss of tolerance in PBC. (HEPATOLOGY 2006;43:729–737.)

A Placebo-controlled trial of obeticholic acid in primary biliary cholangitis

BACKGROUND Primary biliary cholangitis (formerly called primary biliary cirrhosis) can progress to cirrhosis and death despite ursodiol therapy. Alkaline phosphatase and bilirubin levels correlate with the risk of liver transplantation or death. Obeticholic acid, a farnesoid X receptor agonist, has shown potential benefit in patients with this disease. METHODS In this 12-month, double-blind, placebo-controlled, phase 3 trial, we randomly assigned 217 patients who had an inadequate response to ursodiol or who found the side effects of ursodiol unacceptable to receive obeticholic acid at a dose of 10 mg (the 10-mg group), obeticholic acid at a dose of 5 mg with adjustment to 10 mg if applicable (the 5-10-mg group), or placebo. The primary end point was an alkaline phosphatase level of less than 1.67 times the upper limit of the normal range, with a reduction of at least 15% from baseline, and a normal total bilirubin level. RESULTS Of 216 patients who underwent randomization and received at least one dose of obeticholic acid or placebo, 93% received ursodiol as background therapy. The primary end point occurred in more patients in the 5-10-mg group (46%) and the 10-mg group (47%) than in the placebo group (10%; P<0.001 for both comparisons). Patients in the 5-10-mg group and those in the 10-mg group had greater decreases than those in the placebo group in the alkaline phosphatase level (least-squares mean, -113 and -130 U per liter, respectively, vs. -14 U per liter; P<0.001 for both comparisons) and total bilirubin level (-0.02 and -0.05 mg per deciliter [-0.3 and -0.9 μmol per liter], respectively, vs. 0.12 mg per deciliter [2.0 μmol per liter]; P<0.001 for both comparisons). Changes in noninvasive measures of liver fibrosis did not differ significantly between either treatment group and the placebo group at 12 months. Pruritus was more common with obeticholic acid than with placebo (56% of patients in the 5-10-mg group and 68% of those in the 10-mg group vs. 38% in the placebo group). The rate of serious adverse events was 16% in the 5-10-mg group, 11% in the 10-mg group, and 4% in the placebo group. CONCLUSIONS Obeticholic acid administered with ursodiol or as monotherapy for 12 months in patients with primary biliary cholangitis resulted in decreases from baseline in alkaline phosphatase and total bilirubin levels that differed significantly from the changes observed with placebo. There were more serious adverse events with obeticholic acid. (Funded by Intercept Pharmaceuticals; POISE ClinicalTrials.gov number, NCT01473524; Current Controlled Trials number, ISRCTN89514817.).

Primary biliary cirrhosis

Primary biliary cirrhosis is a chronic liver disease characterised by intrahepatic bile-duct destruction, cholestasis, and, in some cases, cirrhosis. Evidence supporting the autoimmune nature of this disorder includes the appearance of highly specific antimitochondrial antibodies (AMAs) and autoreactive T cells. Concordance rates in monozygotic twins, familial prevalence, and genetic associations underscore the importance of genetic factors, whereas findings of epidemiological studies and murine models suggest a possible role for exogenous chemicals and infectious agents through molecular mimicry. The incidence of primary biliary cirrhosis has increased over recent decades, possibly attributable to augmented testing of liver biochemistry rather than a rise in disease incidence. AMAs remain the hallmark of diagnosis in most cases and allow detection of asymptomatic patients. Symptomatic individuals usually present with either pruritus or fatigue and, more rarely, with either jaundice or complications of cirrhosis. The prognosis of primary biliary cirrhosis has improved because of early diagnosis and use of ursodeoxycholic acid, the only established medical treatment for this disorder. Although not a cure, treatment can slow disease progression and delay the need for liver transplantation. However, some patients do not respond adequately to ursodeoxycholic acid and might need alternative therapeutic approaches.

Biliary apotopes and anti‐mitochondrial antibodies activate innate immune responses in primary biliary cirrhosis

Our understanding of primary biliary cirrhosis (PBC) has been significantly enhanced by the rigorous dissection of the multilineage T and B cell response against the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC‐E2). PDC‐E2 is a ubiquitous protein present in mitochondria of nucleated cells. However, the damage of PBC is confined to small biliary epithelial cells (BECs). We have previously demonstrated that BECs translocate immunologically intact PDC‐E2 to apoptotic bodies and create an apotope. To define the significance of this observation, we have studied the ability of biliary or control epithelial apotopes to induce cytokine secretion from mature monocyte‐derived macrophages (MDMϕs) from either patients with PBC or controls in the presence or absence of anti‐mitochondrial antibodies (AMAs). We demonstrate that there is intense inflammatory cytokine production in the presence of the unique triad of BEC apotopes, macrophages from patients with PBC, and AMAs. The cytokine secretion is inhibited by anti‐CD16 and is not due to differences in apotope uptake. Moreover, MDMϕs from PBC patients cultured with BEC apoptotic bodies in the presence of AMAs markedly increase tumor necrosis factor–related apoptosis‐inducing ligand expression. Conclusion: These results provide a mechanism for the biliary specificity of PBC, the recurrence of disease after liver transplantation, and the success of ursodiol in treatment. They further emphasize the critical role of the innate immune system in the perpetuation of this autoimmune disease. (HEPATOLOGY 2010;)

DMT1 gene expression and cadmium absorption in human absorptive enterocytes

Divalent metal transporter 1 (DMT1) is a transmembrane, proton-coupled metal ion transporter that is upregulated in the duodenum of iron-deficient rodents and in hereditary hemochromatosis patients, suggesting that it may constitute a key factor in the uptake of dietary iron. Functional expression studies in Xenopus oocytes have shown that DMT1 not only mediates transport of iron but also other divalent metal ions, including the toxic metal cadmium. In the present study, the correlation between the cadmium absorption process and gene expression of DMT1 was investigated in an experimental model of human absorptive enterocytes. Fully differentiated Caco-2 cells were grown in monolayers and treated with iron supplemented medium or control medium for 1, 3 or 7 days. At each time point, cadmium transport experiments across the Caco-2 cell monolayers were performed and gene expression of DMT1 measured. Iron treatment for 3 and 7 days resulted in a 50% reduction in the cadmium uptake and a 75% reduction in the transport of the metal across the basolateral membrane. No effects were observed at 24 h. Gene expression of DMT1 in the iron-treated Caco-2 cells was reduced by about 50% at 3 and 7 days and thus, correlated well with the uptake of cadmium. In summary, our results indicate that the uptake of cadmium into human absorptive enterocytes may be mediated by DMT1.

De novo nonalcoholic fatty liver disease after liver transplantation

Hepatic steatosis is a recognized problem in patients after orthotopic liver transplant (OLT). However, de novo development of nonalcoholic fatty liver disease (NAFLD) has not been well described. The aim of this study was to determine the prevalence and predictors of de novo NAFLD after OLT. A retrospective analysis was performed on 68 OLT patients with donor liver biopsies and posttransplantation liver biopsies. Individual medical charts were reviewed for demographics, indication for OLT, serial histology reports, genotypes for hepatitis C, comorbid conditions, and medications. Liver biopsies were reviewed blindly and graded according to the Brunt Scoring System. Multivariate logistic regression analysis was used to study the risk factors for developing NAFLD. The interval time from OLT to subsequent follow‐up liver biopsy was 28 ± 18 months. A total of 12 patients (18%) developed de novo NAFLD, and 6 (9%) developed de novo NASH. The regression model indicated that the use of angiotensin‐converting enzyme inhibitors (ACE‐I) was associated with a reduced risk of developing NAFLD after OLT (odds ratio, 0.09; 95% confidence interval, 0.010‐0.92; P = 0.042). Increase in body mass index (BMI) of greater than 10% after OLT was associated with a higher risk of developing NAFLD (odds ratio, 19.38; 95% confidence interval, 3.50‐107.40; P = 0.001). In conclusion, de novo NAFLD is common in the post‐OLT setting, with a significant association with weight gain after transplant. The use of an ACE‐I may reduce the risk of developing post‐OLT NAFLD. Liver Transpl, 2006.

The geoepidemiology of autoimmune intestinal diseases

Inflammatory bowel diseases (IBD) are chronic diseases of the intestinal tract which principally are composed of ulcerative colitis (UC) and Crohns disease (CD). The prevalence and incidence of both forms of IBD have historically been higher in developed countries with decreasing North-South gradient. However, more recent evidence demonstrate changing demographics as countries become more developed and immigration increases from underdeveloped countries to developed countries. Typically these changes are marked by an increase in ulcerative colitis followed by an increase in CD. Thus, most if not all human populations appear to be susceptible to IBD under certain environmental influences. Several hypothesis have been advanced to explain these changing demographics including alterations in the bowel microflora, but direct experimental evidence is lacking in most cases. Celiac disease or gluten-sensitive enteropathy is a related inflammatory condition which is induced in susceptible individuals when exposed to gluten-containing foods. Similarly, the prevalence of celiac disease is increasing as the consumption of gluten-containing foods is increasing worldwide.

IL‐12/Th1 and IL‐23/Th17 biliary microenvironment in primary biliary cirrhosis: Implications for therapy

The interleukin (IL)‐12/IL‐23‐mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL‐12p70, IL‐12p35, interferon‐gamma (IFN‐γ), IL‐12RB2, IL‐23p40, IL‐23p19, IL‐17, and IL‐23R using liver from PBC (n = 51) and non‐PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL‐12/Th1 and IL‐23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL‐23p19 by inflamed hepatocytes around IL‐23R, IL‐12RB2, and IFN‐γ expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL‐23/Th17 pathway in the perpetuation of IL‐12/Th1‐mediated immunopathology in PBC. Furthermore, localized IL‐23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN‐γ production that contribute to PBC pathology. Conclusion: Our data emphasize the pathogenic relevance of IL‐12/Th1 and IL‐23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL‐23/Th17 pathway as a potential target for therapeutic intervention. (Hepatology 2014;59:1944–1953)

Biochemical and immunologic effects of rituximab in patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid.

The aim of this study was to determine the safety and potential efficacy of B‐cell depletion with the anti‐CD20 monoclonal antibody rituximab in patients with primary biliary cirrhosis (PBC) and an incomplete response to ursodeoxycholic acid (UDCA). This open‐label study enrolled six patients with PBC and incomplete responses to UDCA to be treated with 2 doses of 1000 mg rituximab separated by 2 weeks and followed for 52 weeks. The primary endpoints were safety and changes in B‐cell function. Two patients received only 1 dose of rituximab, one due to activation of latent varicella and the other due to a viral upper respiratory infection. Serum levels of total IgG, IgM, and IgA as well as anti‐mitochondrial autoantibodies (AMAs) IgA and IgM decreased significantly from baseline by 16 weeks and returned to baseline levels by 36 weeks. Stimulation of B cells with CpG produced significantly less IgM at 52 weeks after treatment compared with B cells at baseline. In addition, transient decreases in memory B‐cell and T‐cell frequencies and an increase in CD25high CD4+ T cells were observed after treatment. These changes were associated with significant increases in mRNA levels of FoxP3 and transforming growth factor‐β (TGF‐β) and a decrease in tumor necrosis factor‐α (TNF‐α) in CD4+ T cells. Notably, serum alkaline phosphatase levels were significantly reduced up to 36 weeks following rituximab treatment. Conclusion: These data suggest that depletion of B cells influences the induction, maintenance, and activation of both B and T cells and provides a potential mechanism for treatment of patients with PBC with an incomplete response to UDCA. (HEPATOLOGY 2012)