Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B.

OBJECTIVE: The involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B 'e' antigen (HBeAg)-negative chronic hepatitis B (CHB). DESIGN: Liver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR. RESULTS: Patients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia. CONCLUSIONS: HBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome.

Effects of Hepatitis B Surface Antigen on Virus-Specific and Global T Cells in Patients With Chronic Hepatitis B Virus infection.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) infection is characterized by the presence of defective viral envelope proteins (hepatitis B surface antigen [HBsAg]) and the duration of infection-most patients acquire the infection at birth or during the first years of life. We investigated the effects of these factors on patients' lymphocyte and HBV-specific T-cell populations. METHODS: We collected blood samples and clinical data from 243 patients with HBV infection (3-75 years old) in the United Kingdom and China. We measured levels of HBV DNA, HBsAg, hepatitis B e antigen, and alanine aminotransferase; analyzed HBV genotypes; and isolated peripheral blood mononuclear cells (PBMCs). In PBMCs from 48 patients with varying levels of serum HBsAg, we measured 40 markers on nature killer and T cells by mass cytometry. PBMCs from 189 patients with chronic infection and 38 patients with resolved infections were incubated with HBV peptide libraries, and HBV-specific T cells were identified by interferon gamma enzyme-linked immune absorbent spot (ELISpot) assays or flow cytometry. We used multivariate linear regression and performed variable selection using the Akaike information criterion to identify covariates associated with HBV-specific responses of T cells. RESULTS: Although T- and natural killer cell phenotypes and functions did not change with level of serum HBsAg, numbers of HBs-specific T cells correlated with serum levels of HBsAg (r = 0.3367; P < .00001). After we performed the variable selection, the multivariate linear regression model identified patient age as the only factor significantly associated with numbers of HBs-specific T cells (P = .000115). In patients younger than 30 years, HBs-specific T cells constituted 28.26% of the total HBV-specific T cells; this value decreased to 7.14% in patients older than 30 years. CONCLUSIONS: In an analysis of immune cells from patients with chronic HBV infection, we found that the duration of HBsAg exposure, rather than the quantity of HBsAg, was associated with the level of anti-HBV immune response. Although the presence of HBs-specific T cells might not be required for the clearance of HBV infection in all patients, strategies to restore anti-HBV immune responses should be considered in patients younger than 30 years.

Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver.

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.

Hepatitis B virus-specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation.

BACKGROUND: The clinical management of chronic hepatitis B virus (HBV) patients is based exclusively on virological parameters that cannot independently determine in which patients nucleos(t)ide-analogue (NUC) therapy can be safely discontinued. NUCs efficiently suppress viral replication, but do not eliminate HBV. Thus, therapy discontinuation can be associated with virological and biochemical relapse and, consequently, therapy in the majority is life-long. METHODS: Since antiviral immunity is pivotal for HBV control, we investigated potential biomarkers for the safe discontinuation of NUCs within immune profiles of chronic HBV patients by utilizing traditional immunological assays (ELISPOT, flow cytometry) in conjunction with analyses of global non-antigen-specific immune populations (NanoString and CyTOF). Two distinct cohorts of 19 and 27 chronic HBV patients, respectively, were analyzed longitudinally prior to and after discontinuation of 2 different NUC therapy strategies. RESULTS: Absence of hepatic flares following discontinuation of NUC treatment correlated with the presence, during NUC viral suppression, of HBV core and polymerase-specific T cells that were contained within the ex vivo PD-1+ population. CONCLUSIONS: This study identifies the presence of functional HBV-specific T cells as a candidate immunological biomarker for safe therapy discontinuation in chronic HBV patients. Furthermore, the persistent and functional antiviral activity of PD-1+ HBV-specific T cells highlights the potential beneficial role of the expression of T cell exhaustion markers during human chronic viral infection. FUNDING: This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant.

IL-2high tissue-resident T cells in the human liver: Sentinels for hepatotropic infection.

The liver provides a tolerogenic immune niche exploited by several highly prevalent pathogens as well as by primary and metastatic tumors. We have sampled healthy and hepatitis B virus (HBV)-infected human livers to probe for a subset of T cells specialized to overcome local constraints and mediate immunity. We characterize a population of T-betloEomesloBlimp-1hiHobitlo T cells found within the intrahepatic but not the circulating memory CD8 T cell pool expressing liver-homing/retention markers (CD69+CD103+ CXCR6+CXCR3+). These tissue-resident memory T cells (TRM) are preferentially expanded in patients with partial immune control of HBV infection and can remain in the liver after the resolution of infection, including compartmentalized responses against epitopes within all major HBV proteins. Sequential IL-15 or antigen exposure followed by TGFß induces liver-adapted TRM, including their signature high expression of exhaustion markers PD-1 and CD39. We suggest that these inhibitory molecules, together with paradoxically robust, rapid, cell-autonomous IL-2 and IFNγ production, equip liver CD8 TRM to survive while exerting local noncytolytic hepatic immunosurveillance.

Distinct Metabolic Requirements of Exhausted and Functional Virus-Specific CD8 T Cells in the Same Host.

T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1(hi) T cell response against hepatitis B virus (HBV) had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1(hi) HBV-specific T cells were dependent on glucose supplies, unlike the more functional cytomegalovirus (CMV)-specific T cells that could utilize oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. Interleukin (IL)-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.

CXCR6 marks a novel subset of T-bet(lo)Eomes(hi) natural killer cells residing in human liver.

Natural killer cells (NK) are highly enriched in the human liver, where they can regulate immunity and immunopathology. We probed them for a liver-resident subset, distinct from conventional bone-marrow-derived NK. CXCR6+ NK were strikingly enriched in healthy and diseased liver compared to blood (p < 0.0001). Human hepatic CXCR6+ NK had an immature phenotype (predominantly CD56(bright)CD16-CD57-), and expressed the tissue-residency marker CD69. CXCR6+ NK produced fewer cytotoxic mediators and pro-inflammatory cytokines than the non-liver-specific CXCR6- fraction. Instead CXCR6+ NK could upregulate TRAIL, a key death ligand in hepatitis pathogenesis. CXCR6 demarcated liver NK into two transcriptionally distinct populations: T-bet(hi)Eomes(lo)(CXCR6-) and T-bet(lo)Eomes(hi)(CXCR6+); the latter was virtually absent in the periphery. The small circulating CXCR6+ subset was predominantly T-bet(hi)Eomes(lo), suggesting its lineage was closer to CXCR6- peripheral than CXCR6+ liver NK. These data reveal a large subset of human liver-resident T-bet(lo)Eomes(hi) NK, distinguished by their surface expression of CXCR6, adapted for hepatic tolerance and inducible anti-viral immunity.

Consensus best practice pathway of the UK scleroderma study group: gastrointestinal manifestations of systemic sclerosis.

Systemic sclerosis is an autoimmune connective tissue disorder, which can be progressive with multisystem involvement. Guidance on the management of complications is based on a limited data set and practice amongst clinicians can vary. The UK Scleroderma study group set up several working groups to agree some consensus pathways for the management of specific complications. Approximately nine out of ten patients with systemic sclerosis will have involvement of the gastrointestinal system and in this review article we explore the management of these complications in a symptom-based approach. The algorithms are a useful tool for clinicians, which we hope, will be a point of reference and highlight the need for further research in these areas.

Sodium bicarbonate supplements for treating acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is a common, serious, but potentially treatable condition. Because AKI is often associated with acidosis, it has become common practice to recommend administration of sodium bicarbonate to correct acid imbalance. OBJECTIVES: To assess the benefits and harms of the use of sodium bicarbonate for people with AKI. The primary outcome measure was all-cause mortality, and secondary outcome measures were patients' need for renal replacement therapy; return to baseline kidney function; and overall survival. SEARCH METHODS: In November 2011 we searched the Cochrane Renal Group's Specialised Register using keywords relevant to this review. The register is populated using searches of Ovid MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE and handsearching records from renal-related journals and conference proceedings. SELECTION CRITERIA: All randomised controlled trials (RCTs) that investigated the use of sodium bicarbonate supplements, administered by any route, for the treatment of adults with AKI were to be included. The search strategy did not restrict inclusion based on an upper age limit or publication language. We did not consider inclusion of studies that investigated use of sodium bicarbonate for AKI prevention. DATA COLLECTION AND ANALYSIS: All authors planned to independently assess and extracted information. Information was to be collected on methods, participants, interventions and outcomes. Results were to be expressed as risk ratios (RR) for dichotomous outcomes or as mean differences (MD) for continuous data with 95% confidence intervals (CI). MAIN RESULTS: Although our literature search identified four studies, none of these met our predetermined selection criteria. Hence, no suitable studies were identified for inclusion in this review. AUTHORS' CONCLUSIONS: We found no RCT evidence - supportive or otherwise - for the use of sodium bicarbonate for people with AKI. We concluded that there is an urgent need for well conducted RCTs in this area.

Lean thinking transformation of the unsedated upper gastrointestinal endoscopy pathway improves efficiency and is associated with high levels of patient satisfaction.

BACKGROUND: Upper gastrointestinal (UGI) endoscopy is a routine healthcare procedure with a defined patient pathway. The objective of this study was to redesign this pathway for unsedated patients using lean thinking transformation to focus on patient-derived value-adding steps, remove waste and create a more efficient process. This was to form the basis of a pathway template that was transferrable to other endoscopy units. METHODS: A literature search of patient expectations for UGI endoscopy identified patient-derived value. A value stream map was created of the current pathway. The minimum and maximum time per step, bottlenecks and staff-staff interactions were recorded. This information was used for service transformation using lean thinking. A patient pathway template was created and implemented into a secondary unit. Questionnaire studies were performed to assess patient satisfaction. RESULTS: In the primary unit the patient pathway reduced from 19 to 11 steps with a reduction in the maximum lead time from 375 to 80 min following lean thinking transformation. The minimum value/lead time ratio increased from 24% to 49%. The patient pathway was redesigned as a 'cellular' system with minimised patient and staff travelling distances, waiting times, paperwork and handoffs. Nursing staff requirements reduced by 25%. Patient-prioritised aspects of care were emphasised with increased patient-endoscopist interaction time. The template was successfully introduced into a second unit with an overall positive patient satisfaction rating of 95%. CONCLUSION: Lean thinking transformation of the unsedated UGI endoscopy pathway results in reduced waiting times, reduced staffing requirements and improved patient flow and can form the basis of a pathway template which may be successfully transferred into alternative endoscopy environments with high levels of patient satisfaction.

The influence of 10 min of the Johrei healing method on laboratory stress.

OBJECTIVE: Johrei has been shown to decrease exam stress responses but its immediate effects have not been assessed. DESIGN: In a randomised, blinded, counter-balanced design, 33 medical students were asked to calculate mental arithmetic in the Paced Auditory Serial Addition Task (PASAT), which served as an acute stressor prior to two conditions, 10 min of Johrei or a control resting condition involving 10 min without Johrei in a cross-over trial; after each, saliva was collected and mood tested. SETTING: University EEG laboratory. INTERVENTION: Johrei, a non-touch healing method. MAIN OUTCOME MEASURES: Profile of mood states (POMS-Bi); state anxiety (STAI); salivary variables: cortisol, DHEA, IgA. RESULTS: Mood scores on 5/6 of the POMS-Bi subscales were slightly but significantly more positive in the Johrei condition. State anxiety was similarly decreased. IgA levels were unchanged but cortisol levels were found to be slightly but non-significantly lower after Johrei than after the control condition and DHEA levels slightly but non-significantly raised, with a negative correlation between cortisol and DHEA levels. CONCLUSIONS: This study gives some indication that Johrei can reduce negative mood and increase positive mood states after the acute effects of a laboratory stressor in comparison to a resting control condition.