Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma.

Considerable progress has been made in understanding the roles of Helicobacter pylori in inflammation and gastric cancer; however, far less is known about the roles of enterohepatic Helicobacter species (EHS) in carcinogenesis and their zoonotic or pathogenic potential. We determined the prevalence of EHS infection in a cohort of geriatric rhesus monkeys in which intestinal adenocarcinoma (IAC) is common and investigated the association between EHS infection and IAC. The cohort consisted of 36 animals, 14 of which (age 26-35 years) had IAC. Of the 36 rhesus, 35 (97%) were positive for EHS using PCR or bacterial isolation from faeces, colonic or tumour tissues. Only a single rhesus, which had IAC, was negative for EHS by all detection methods. The EHS identified by 16S rRNA sequencing in this study were from three Helicobacter taxa: Helicobacter macacae (previously rhesus monkey taxon 1), Helicobacter sp. rhesus monkey taxon 2, previously described from strain MIT 99-5507, and Helicobacter sp. rhesus monkey taxon 4, related to Helicobacter fennelliae. Thirteen of 14 monkeys with IAC were positive for either H. macacae (7/13, 54%), EHS rhesus monkey taxon 4 (4/13, 31%) or a mixture of the two EHS (2/13, 15%). These results indicate that EHS are prevalent among aged rhesus macaques with IAC. Using Helicobacter genus-specific florescent in situ hybridization, EHS were detected on the surface of colonic epithelia of infected monkeys. All Helicobacter isolates, including H. macacae, effectively adhered to, invaded, and significantly induced proinflammatory genes, including IL-8, IL-6, TNF-α and iNOS, while downregulating genes involved in the function of inflammasomes, particularly IL-1ß, CASPASE-1, NRLP3, NLRP6 and NLRC4 in the human colonic T84 cell line (P<0.0001). These results suggest that EHS may represent an aetiological agent mediating diarrhoea, chronic inflammation, and possibly intestinal cancer in non-human primates, and may play a role in similar disease syndromes in humans. Downregulation of inflammasome function may represent an EHS strategy for long-term persistence in the host and play a role in inducing pathological changes in the host's lower bowel.

Antiviral efficacy upon administration of a HepDirect prodrug of 2'-C-methylcytidine to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide, and the current standard of care, a combination of pegylated interferon alpha and ribavirin, is efficacious in achieving sustained viral response in ~50% of treated patients. Novel therapies under investigation include the use of nucleoside analog inhibitors of the viral RNA-dependent RNA polymerase. NM283, a 3'-valyl ester prodrug of 2'-C-methylcytidine, has demonstrated antiviral efficacy in HCV-infected patients (N. Afdhal et al., J. Hepatol. 46[Suppl. 1]:S5, 2007; N. Afdhal et al., J. Hepatol. 44[Suppl. 2]:S19, 2006). One approach to increase the antiviral efficacy of 2'-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5'-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2'-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ~1.4 log(10) IU/ml and by >3.6 log(10) IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.

Isolation and characterization of a novel helicobacter species, "Helicobacter macacae," from rhesus monkeys with and without chronic idiopathic colitis.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, "Helicobacter macacae" (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

Effects of inhibition of alpha-CGRP receptors on cardiac and peripheral vascular dynamics in conscious dogs with chronic heart failure.

Whether endogenous calcitonin gene-related peptide (CGRP) plays a role in heart failure is unclear. Seven dogs were instrumented with left ventricular (LV) pressure gauges, pacers, coronary occluder and aortic, atrial, and coronary sinus catheters. Hemodynamic recordings and response to alpha-CGRP challenge were obtained for baseline in the conscious state. Rapid pacing (240 beats/min) was then initiated. The coronary artery was occluded for 90 minutes followed by reperfusion after 2 weeks of pacing. After 6 weeks of pacing, LV pressure (-11 +/- 6%), LV dP/dt (-53 +/- 5%), and mean arterial pressure (-15 +/- 4%) decreased (P < 0.01), while left atrial pressure (+19 +/- 3 mm Hg from 7 +/- 1 mm Hg) and heart rate (+53 +/- 16%) increased (P < 0.01). Infusion of the alpha-CGRP receptor antagonist alpha-CGRP[8-37] (30 microg/kg/min, i.v.), which blocked the exogenous alpha-CGRP challenge, did not affect any of these indices. Regional blood flow, as measured by the microsphere technique, in the nonischemic myocardium, as well as cerebral and renal vasculatures were unaltered during the infusion of alpha-CGRP[8-37]. Plasma concentrations of CGRP from both arterial and coronary sinus samples were unchanged after 6 weeks of pacing as compared with control. Thus, we conclude that endogenous alpha-CGRP does not appear to play a major role in the regulation of cardiac and peripheral vascular dynamics in the late stage of heart failure.

Novel Techniques for Testing of Esophageal Irritancy of Liquids and Tablets in Dogs.

Esophageal irritation is a potential side effect of oral medications in humans. However, this complication is rarely evaluated in animal studies, in which test compounds are usually delivered directly into the stomach via gavage. The toxicology unit at our institution required development of a model to test compounds in liquid and tablet form for esophageal irritancy. To deliver liquids, an endotracheal tube (internal diameter, 5.0 mm) was passed into the esophagus of anesthetized dogs, with the cuff inflated directly caudal to the larynx. A rubber catheter (size, 8 French) was then passed through the tube, extending caudally 1 in past the end of the tube. An infusion pump delivered the test liquid through the catheter over a 30-min period. The cranial portion of the dog was elevated, so that the liquid would flow aborally. The infusions were generally administered for 5 consecutive days, and the dogs euthanized on the fifth day for gross and histopathologic examination of the esophagus. This technique was performed successfully on 80 dogs, without serious complication. For delivery of compounds in tablet form, a clear tube was passed into the cranial third of the esophagus. The tablet, tied to a pre-measured length of silk suture, was then introduced into the esophagus through the tube by using endoscopic retrieval forceps. The tablets were left in place for 1 h, and the dogs were then euthanized and examined. These techniques allowed for accurate and reliable testing in dogs of the esophageal irritancy of compounds in either the liquid or tablet form.