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ObjectiveHepatitis B e antigen (HBeAg) seroconversion and hepatitis B surface antigen (HBsAg) loss are important clinical outcomes for patients with chronic hepatitis B (CHB) treated with antiviral therapy. To date, there have been few studies that have evaluated viral sequence markers predicting serological response to nucleos(t)ide analogue (NA) treatment.DesignWe used next-generation sequencing (NGS) and quantitative HBV serology (HBeAg and HBsAg) to identify viral sequence markers associated with serological response to long-term tenofovir disoproxil fumarate therapy among HBeAg-positive patients. In the GS-US-174-0103 study, approximately half the patients seroconverted to anti-HBe by week 192 and 11% of patients exhibited HBsAg loss, the closest outcome to functional cure. The frequency of HBV variants that have previously been associated with HBV clinical outcomes was evaluated. HBV viral diversity in baseline sequences generated by NGS was calculated using Shannon entropy.ResultsNGS analysis of HBV sequences from 157 patients infected with genotypes A to D showed the frequency of variants in the basal core promoter (BCP) and precore (PC) regions varied by genotype and that these mutations were associated with the absence of HBsAg loss. This was the case even when mutations were present at frequencies below the threshold of detection by population sequencing. Increased viral diversity across the HBV genome as determined by NGS was also associated with reduced likelihood of HBsAg loss.ConclusionPatients with detectable BCP and/or PC variants and higher viral diversity have a lower probability of HBsAg loss during long-term NA therapy. Strategies to achieve functional cure of HBV infection through combination therapy should consider using NGS to stratify patients according to BCP/PC sequence. Consideration should also be given to earlier initiation of therapy prior to the emergence of BCP/PC variants.Trial registration numberNCT00116805; Post result.

Background & Aims:Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms. Methods:Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli. Results:In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2and SLIGRL-NH2caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2and SLIGRL-NH2caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine. Conclusions:The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.

We studied the effect of ischaemia and reperfusion on vasoconstrictor and vasodilator mechanisms. Anaesthetized rabbits were subjected to 4-h abdominal aortic occlusion and 1-h reperfusion in vivo. Segments of the abdominal (ischaemic-reperfused) and thoracic (control) aorta were then removed for in vitro studies. Ischaemiareperfusion had no significant effect on relaxant responses to either acetylcholine (AChendothelium-dependent) or sodium nitroprusside (SNPendothelium-independent). The sensitivity of the aorta to contraction by phenylephrine was significantly increased in aortic rings with or without endothelium (by 2.2- and 3.7-fold, respectively), but was not different after 4-h ischaemia without reperfusion. In contrast, responses to methoxamine, serotonin, and U46619 were not affected by ischaemiareperfusion. Moreover, the relative increase in aortic sensitivity to phenylephrine was prevented by treatment of control and ischaemic-reperfused aortic rings with the neuronal uptake inhibitor cocaine (10−5M). These results suggest that after 4-h ischaemia, reperfusion damages sympathetic neuronal uptake mechanisms in rabbit aorta. As a result, phenylephrine, an agonist normally susceptible to neuronal uptake, may exert more potent contractile effects. Endothelium-dependent and endothelium-independent relaxant mechanisms in the aorta appear to be resistant to acute ischaemia and reperfusion.