Immune effectors required for hepatitis B virus clearance.

To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4(+) and CD8(+) T cells, NK cells, Fas, IFN-gamma (IFN-gamma), IFN-alpha/beta receptor (IFN-alpha/betaR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8(+) T cell response in these animals depended on the presence of CD4(+) T cells. These results are consistent with a model in which CD4(+) T cells serve as master regulators of the adaptive immune response to HBV; CD8(+) T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-gamma, TNFR1, and IFN-alpha/betaR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.

Combination therapy with amylin and peptide YY[3-36] in obese rodents: anorexigenic synergy and weight loss additivity.

Circulating levels of the pancreatic beta-cell peptide hormone amylin and the gut peptide PYY[3-36] increase after nutrient ingestion. Both have been implicated as short-term signals of meal termination with anorexigenic and weight-reducing effects. However, their combined effects are unknown. We report that the combination of amylin and PYY[3-36] elicited greater anorexigenic and weight-reducing effects than either peptide alone. In high-fat-fed rats, a single ip injection of amylin (10 microg/kg) plus PYY[3-36] (1000 microg/kg) reduced food intake for 24 h (P < 0.05 vs. vehicle), whereas the anorexigenic effects of either PYY[3-36] or amylin alone began to diminish 6 h after injection. These anorexigenic effects were dissociable from changes in locomotor activity. Subcutaneous infusion of amylin plus PYY[3-36] for 14 d suppressed food intake and body weight to a greater extent than either agent alone in both rat and mouse diet-induced obesity (DIO) models (P < 0.05). In DIO-prone rats, 24-h metabolic rate was maintained despite weight loss, and amylin plus PYY[3-36] (but not monotherapy) increased 24-h fat oxidation (P < 0.05 vs. vehicle). Finally, a 4 x 3 factorial design was used to formally describe the interaction between amylin and PYY[3-36]. DIO-prone rats were treated with amylin (0, 4, 20, and 100 microg/kg.d) and PYY[3-36] (0, 200, 400 microg/kg.d) alone and in combination for 14 d. Statistical analyses revealed that food intake suppression with amylin plus PYY[3-36] treatment was synergistic, whereas body weight reduction was additive. Collectively, these observations highlight the importance of studying peptide hormones in combination and suggest that integrated neurohormonal approaches may hold promise as treatments for obesity.

Activation of the early B-cell-specific mb-1 (Ig-alpha) gene by Pax-5 is dependent on an unmethylated Ets binding site.

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5'CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions.

Requirements for selective recruitment of Ets proteins and activation of mb-1/Ig-alpha gene transcription by Pax-5 (BSAP).

Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-alpha. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPalpha (with GABPbeta1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the beta-hairpin/beta-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPalpha (with GABPbeta1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.

Weight loss with naltrexone SR/bupropion SR combination therapy as an adjunct to behavior modification: the COR-BMOD trial.

This 56-week, randomized, placebo-controlled trial examined the efficacy and safety of naltrexone plus bupropion as an adjunct to intensive behavior modification (BMOD). A total of 793 participants (BMI = 36.5 ± 4.2 kg/m²) was randomly assigned in a 1:3 ratio to: (i) placebo + BMOD (N = 202); or (ii) naltrexone sustained-release (SR, 32 mg/day), combined with bupropion SR (360 mg/day) plus BMOD (i.e., NB32 + BMOD; N = 591). Both groups were prescribed an energy-reduced diet and 28 group BMOD sessions. Co-primary end points were percentage change in weight and the proportion of participants who lost ≥5% weight at week 56. Efficacy analyses were performed on a modified intent-to-treat population (ITT; i.e., participants with ≥1 postbaseline weight while taking study drug (placebo + BMOD, N = 193; NB32 + BMOD, N = 482)). Missing data were replaced with the last observation obtained on study drug. At week 56, weight loss was 5.1 ± 0.6% with placebo + BMOD vs. 9.3 ± 0.4% with NB32 + BMOD (P < 0.001). A completers analysis revealed weight losses of 7.3 ± 0.9% (N = 106) vs. 11.5 ± 0.6% (N = 301), respectively (P < 0.001). A third analysis, which included all randomized participants, yielded losses of 4.9 ± 0.6 vs. 7.8 ± 0.4%, respectively (P < 0.001). Significantly more NB32 + BMOD- vs. placebo + BMOD-treated participants lost ≥5 and ≥10% of initial weight, and the former had significantly greater improvements in markers of cardiometabolic disease risk. NB32 + BMOD was generally well tolerated, although associated with more reports of nausea than placebo + BMOD. The present findings support the efficacy of combined naltrexone/bupropion therapy as an adjunct to intensive BMOD for obesity.

Implications of amylin receptor agonism: integrated neurohormonal mechanisms and therapeutic applications.

Amylin receptor agonism is emerging as part of an integrated neurohormonal therapeutic approach for managing diabetes mellitus (DM) and body weight. Pramlintide acetate, an analogue of the pancreatic hormone amylin, has been studied in the United States as an antihyperglycemic agent in patients with type 1 or type 2 DM treated with mealtime insulin(1). Further clinical testing of pramlintide in subjects with obesity demonstrated that pramlintide monotherapy induced significant, sustained, and dose-dependent weight loss(2). Recent clinical observations point to its compatibility as a combination therapy with the hormone leptin, eliciting double-digit weight loss in patients with overweight and obesity(3). Herein, we link amylin activation of central neural circuits to these therapeutic effects, and we speculate on other potential therapeutic applications of amylin receptor agonism.

Enhanced weight loss with pramlintide/metreleptin: an integrated neurohormonal approach to obesity pharmacotherapy.

The neurohormonal control of body weight involves a complex interplay between long-term adiposity signals (e.g., leptin), and short-term satiation signals (e.g., amylin). In diet-induced obese (DIO) rodents, amylin/leptin combination treatment led to marked, synergistic, fat-specific weight loss. To evaluate the weight-lowering effect of combined amylin/leptin agonism (with pramlintide/metreleptin) in human obesity, a 24-week, randomized, double-blind, active-drug-controlled, proof-of-concept study was conducted in obese or overweight subjects (N = 177; 63% female; 39 +/- 8 years; BMI 32.0 +/- 2.1 kg/m(2); 93.3 +/- 13.2 kg; mean +/- s.d.). After a 4-week lead-in period with pramlintide (180 microg b.i.d. for 2 weeks, 360 microg b.i.d. thereafter) and diet (40% calorie deficit), subjects achieving 2-8% weight loss were randomized 1:2:2 to 20 weeks of treatment with metreleptin (5 mg b.i.d.), pramlintide (360 microg b.i.d.), or pramlintide/metreleptin (360 microg/5 mg b.i.d.). Combination treatment with pramlintide/metreleptin led to significantly greater weight loss from enrollment to week 20 (-12.7 +/- 0.9%; least squares mean +/- s.e.) than treatment with pramlintide (-8.4 +/- 0.9%; P < 0.001) or metreleptin (-8.2 +/- 1.3%; P < 0.01) alone (evaluable, N = 93). The greater reduction in body weight was significant as early as week 4, and weight loss continued throughout the study, without evidence of a plateau. The most common adverse events with pramlintide/metreleptin were injection site events and nausea, which were mostly mild to moderate and decreased over time. These results support further development of pramlintide/metreleptin as a novel, integrated neurohormonal approach to obesity pharmacotherapy.

Pramlintide in the treatment of diabetes mellitus.

Pramlintide, the first member of a new class of drugs for the treatment of insulin-using patients with type 2 or type 1 diabetes mellitus, is an analog of the peptide hormone amylin. Amylin is co-secreted with insulin from pancreatic beta cells and acts centrally to slow gastric emptying, suppress postprandial glucagon secretion, and decrease food intake. These actions complement those of insulin to regulate blood glucose concentrations. Amylin is relatively deficient in patients with type 2 diabetes, depending on the severity of beta-cell secretory failure, and is essentially absent in patients with type 1 diabetes. Through mechanisms similar to those of amylin, pramlintide improves overall glycemic control, reduces postprandial glucose levels, and reduces bodyweight in patients with diabetes using mealtime insulin. Reductions in postprandial glucose and bodyweight are important, since postprandial hyperglycemia is associated with an increased risk of microvascular and macrovascular complications, and increased weight is an independent risk factor for cardiovascular disease. Pramlintide is generally well tolerated, with the most frequent treatment-emergent adverse event being mild to moderate nausea, which decreases over time. Pramlintide treatment is also associated with improvements in markers of oxidative stress and cardiovascular risk and improved patient-reported treatment satisfaction. These factors make pramlintide an attractive option for the treatment of postprandial hyperglycemia in patients with diabetes using mealtime insulin.

Effect of pramlintide as an adjunct to basal insulin on markers of cardiovascular risk in patients with type 2 diabetes.

OBJECTIVE: Intensification of insulin therapy in patients with type 2 diabetes, while improving glycemic control, often leads to an increase in body weight and other markers of cardiovascular risk. The effects of pramlintide as an adjunct to basal insulin titration (without mealtime insulin) on glycemia and cardiovascular risk markers were examined. RESEARCH DESIGN AND METHODS: This was a post hoc analysis of a 16-week, double-blind, placebo-controlled study in patients with type 2 diabetes (N = 211) using insulin glargine (without mealtime insulin) +/- oral agents. Patients were randomized to treatment with placebo or pramlintide (60 or 120 mug with major meals), and insulin glargine was titrated to target a fasting plasma glucose concentration of > or = 70 to < 100 mg/dL. MAIN OUTCOME MEASURES: Endpoints included the change from baseline to Week 16 in body weight, high sensitivity C-reactive protein (hsCRP), triglycerides, HDL, LDL, and blood pressure. RESULTS: Pramlintide-treated patients lost weight and placebo-treated patients gained weight during 16 weeks of treatment (-1.6 +/- 0.3 kg vs. +0.7 +/- 0.3 kg, p < 0.001; mean +/- SE). hsCRP was reduced in pramlintide-treated versus placebo-treated patients (-0.8 +/- 0.2 mg/L vs. 0.1 +/- 0.2 mg/L, p < 0.01; mean +/- SE). Patients with baseline hsCRP > 3 mg/L (high cardiovascular risk) demonstrated greater hsCRP reductions with pramlintide versus placebo treatment at Week 16 (p < 0.05). Patients with baseline triglycerides > or = 150 mg/dL or > or = 200 mg/dL (high cardiovascular risk) showed significant reductions from baseline in triglyceride concentrations with pramlintide (-43 +/- 14 mg/dL or -59 +/- 19 mg/dL; p < 0.05; mean +/- SE) but not with placebo (1 +/- 29 mg/dL or -3 +/- 54 mg/dL; mean +/- SE). No significant differences between pramlintide and placebo were observed for changes in HDL, LDL, or blood pressure. Pramlintide treatment was generally well tolerated. The most frequent adverse event related to pramlintide was mild-to-moderate nausea (31% pramlintide vs. 10% placebo). Pramlintide added to basal insulin did not increase the incidence of hypoglycemia. A limitation of the study was its relatively short duration. CONCLUSIONS: Pramlintide, as an adjunct to basal insulin, was associated with improvements in several cardiovascular risk markers, warranting long-term clinical studies to determine its potential effects on cardiovascular risk.

PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver.

Mechanisms contributing to the development of chronic viral infections, including chronic hepatitis B virus (HBV) infections, are not well understood. We have shown recently that production of IFN-gamma, an important antiviral cytokine, by HBV-specific CTLs is rapidly induced when they enter the liver of HBV transgenic mice, and then rapidly suppressed, despite the continued presence of Ag. Suppression of IFN-gamma production by the CTLs coincides with the up-regulation of programmed cell death (PD)-1, a cell surface signaling molecule known to inhibit T cell function. To determine whether PD-1 plays a role in the functional suppression of IFN-gamma secretion by CTLs, we treated HBV transgenic mice with blocking Abs specific for PD ligand (PD-L)1, the most widely expressed PD-1 ligand, and adoptively transferred HBV-specific CTLs. Treatment with anti-PD-L1 Abs resulted in a delay in the suppression of IFN-gamma-producing CTLs and a concomitant increase in the absolute number of IFN-gamma-producing CTLs in the liver. These results indicate that PD-1:PD-L1 interactions contribute to the suppression of IFN-gamma secretion observed following Ag recognition in the liver. Blockade of inhibitory pathways such as PD-1:PD-L1 may reverse viral persistence and chronic infection in cases in which the CTL response is suppressed by this mechanism.

Pramlintide improved glycemic control and reduced weight in patients with type 2 diabetes using basal insulin.

OBJECTIVE: To assess the efficacy and safety of pramlintide in patients with type 2 diabetes suboptimally controlled with basal insulin. RESEARCH DESIGN AND METHODS: In a 16-week, double-blind, placebo-controlled study, 212 patients using insulin glargine with or without oral antidiabetes agents (OAs) were randomized to addition of pramlintide (60 or 120 microg b.i.d./t.i.d.) or placebo. Insulin glargine was adjusted to target a fasting plasma glucose concentration of 70-100 mg/dl. One coprimary end point was the change in A1C at week 16. The other coprimary end point was a composite measure of overall diabetes control comprising A1C < or = 7.0% or reduction > or = 0.5%, mean daily postprandial glucose (PPG) increments < or = 40 mg/dl, no increase in body weight, and no severe hypoglycemia. Patients meeting all four conditions at week 16 achieved this end point. RESULTS: More pramlintide- than placebo-treated patients achieved the composite end point (25 vs. 7%; P < 0.001). Reductions (means +/- SE) in A1C (-0.70 +/- 0.11% vs. -0.36 +/- 0.08%; P < 0.05) and PPG increments (-24.4 +/- 3.6 mg/dl vs. -0.4 +/- 3.0 mg/dl; P < 0.0001) were greater in pramlintide- versus placebo-treated patients, respectively. Glycemic improvements were accompanied by progressive weight loss with pramlintide and weight gain with placebo (-1.6 +/- 0.3 kg vs. +0.7 +/- 0.3 kg; P < 0.0001). No treatment-related severe hypoglycemia occurred. CONCLUSIONS: Pramlintide improved multiple glycemic parameters and reduced weight with no increase in hypoglycemia in patients with type 2 diabetes who were not achieving glycemic targets with basal insulin with or without OAs.

Effects of prior or concurrent food restriction on amylin-induced changes in body weight and body composition in high-fat-fed female rats.

Amylin infusion reduces food intake and slows body weight gain in rodents. In obese male rats, amylin (but not pair feeding) caused a preferential reduction of fat mass with protein preservation despite equal body weight loss in amylin-treated (fed ad libitum) and pair-fed rats. In the present study, the effect of prior or concurrent food restriction on the ability of amylin to cause weight loss was evaluated. Retired female breeder rats were maintained on a high-fat diet (40% fat) for 9 wk. Prior to drug treatment, rats were either fed ad libitum or food restricted for 10 days to lose 5% of their starting body weight. They were then subdivided into treatment groups that received either vehicle or amylin (100 microgxkg(-1)xday(-1) via subcutaneous minipump) and placed under either a restricted or ad libitum feeding schedule (for a total of 8 treatment arms). Amylin 1) significantly reduced body weight compared with vehicle under all treatment conditions, except in always restricted animals, 2) significantly decreased percent body fat in all groups, and 3) preserved lean mass in all groups. These results indicate that amylin's anorexigenic and fat-specific weight loss properties can be extended to a variety of nutritive states in female rats.

Roles of EBF and Pax-5 in B lineage commitment and development.

B lymphocyte development is regulated by the nuclear proteins Early B cell factor (EBF) and Pax-5. EBF and Pax-5 work separately and in concert to activate genes required for B cell differentiation. Recent studies have defined mechanisms by which these two factors control transcription, including chromatin remodeling activities and recruitment of partner proteins. This review addresses the structures, functions, and roles of these proteins in early B cell commitment and development, as well as in later stages of B cell differentiation.

Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription.

Cd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cells but became progressively unmethylated as B cell development proceeded. The transcription factor Pax5 activated endogenous mb-1 transcription in a plasmacytoma cell line, but could not when the promoter was methylated. In this context, early B cell factor (EBF), a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by Pax5. EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function was enhanced by interaction with the transcription factor Runx1. These data suggest a molecular basis for the hierarchical dependence of Pax5 function on EBF and E2A in B lymphocyte development.