Therapeutic Effects of Treating COVID-19 Vaccine-Induced Anti-TIF1-γ-Positive Dermatomyositis.

An increase in skin-related autoimmune disorders has been reported as an adverse effect of coronavirus disease 2019 (COVID-19) vaccines. We present the case of a 90-year-old Taiwanese female who was newly diagnosed with anti-transcription intermediary factor 1-gamma (anti-TIF1-γ)-positive dermatomyositis (DM) after receiving a second dose of the AstraZeneca COVID-19 vaccine. Under treatment with prednisolone and monoclonal antibody therapy of abatacept, her skin lesions improved, and her muscle power increased. The serum creatinine phosphokinase level decreased from 4858 to 220 U/L, and the anti-TIF1-γ antibody titer decreased from 202 to 99. Flow cytometry data showed an increase in T cells, while NK cells, B cells (CD19), and plasma blasts all decreased. These findings suggest that standard DM treatment might be beneficial to patients with COVID-19 vaccine-induced DM.

Impact of PNPLA3 p.I148M and Hepatic Steatosis on Long-Term Outcomes for Hepatocellular Carcinoma and HBsAg Seroclearance in Chronic Hepatitis B.

Background: Coexistence of hepatitis B and nonalcoholic fatty liver disease is common; however, little is known about the impact of hepatic steatosis and its major genetic determinants on the natural history of HBV infection. We aimed to study the effects of hepatic steatosis and PNPLA3 variant p.I148M on the risk of hepatocellular carcinoma (HCC) and the lifetime probability of HBsAg seroclearance, which is associated with functional remission and improved long-term outcome of HBV infection. Methods: We conducted a cohort study of 2385 male, HBsAg-positive Taiwanese civil servants recruited in 1989-1992, and followed up until 2019. Cox regression with competing-risk models was used to estimate sub-distribution hazard ratios (sHRs) and 95% confidence intervals (CIs). Results: Of 2385 participants, 628 experienced HBsAg seroclearance and 217 developed HCC. Hepatic steatosis, excess body-mass index, and the PNPLA3-148M variant were significantly associated with higher HBsAg seroclearance rate. However, multivariate analyses accounting for HBsAg seroclearance and various HCC risk factors showed that, while steatosis was associated with decreased HCC risk (sHR [95% CI]: 0.49 [0.36-0.66]), carriage of the PNPLA3-148M variant allele (vs II homozygotes: 1.64 [1.20-2.25] for MI heterozygotes; 1.83 [1.20-2.78] for MM homozygotes) and obesity (1.51 [1.07-2.13]) were associated with increased risk. The inverse hepatic steatosis-HCC association persisted after additional adjustment for other viral factors or using different follow-up time cut-offs to account for reverse causality. Moreover, the PNPLA3 MM genotype was positively associated with elevations of ALT and AST and liver cirrhosis, while hepatic steatosis was positively associated with ALT but inversely associated with AST and liver cirrhosis. Conclusion: Hepatic steatosis and PNPLA3-148M variant appeared to have distinct impacts on the development of HBV-related progressive liver disease and HCC. PNPLA3 p.I148M, but not a diagnosis of hepatic steatosis, can help to identify HBV carriers with high-risk fatty liver disease in the progression to HCC.

TLC599 in patients with osteoarthritis of the knee: a phase IIa, randomized, placebo-controlled, dose-finding study.

BACKGROUND: Corticosteroid injection for knee osteoarthritis is limited by its modest duration of treatment effect. The liposome formulation of dexamethasone sodium phosphate (TLC599) was developed for the sustained relief of osteoarthritis pain. This clinical study was conducted to evaluate the efficacy and safety of TLC599 at two dose levels in patients with knee osteoarthritis. METHODS: A randomized, double-blinded, placebo-controlled study was conducted in 75 patients with osteoarthritis of the knee from 13 study centers. Patients were randomized and administered a single intra-articular injection of TLC599 or placebo and assessed for efficacy and safety for 24 weeks. Patient-reported outcomes included the Western Ontario and McMaster Universities Arthritis (WOMAC) Index for pain and function and visual analog scale for pain. RESULTS: TLC599 at 12 mg demonstrated significantly greater reduction in WOMAC pain through 12 weeks (least squares (LS) mean difference = - 0.37, p = 0.0027) and through 24 weeks (LS mean difference = - 0.35, p = 0.0037) when compared to placebo. TLC599 12 mg also exhibited significantly greater improvement in function when compared to placebo at 24 weeks (LS mean difference = - 0.26, p = 0.0457). TLC599 18 mg did not significantly improve pain or function in comparison with placebo. The use of acetaminophen during the study was less in both TLC599 groups in comparison with placebo. No major or unexpected safety issues were reported. CONCLUSIONS: In participants with symptomatic knee osteoarthritis, TLC599 is a well-tolerated treatment that reduces pain and improves function for up to 24 weeks, a longer duration than that reported for existing IA treatments. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03005873 . Registered on 29 December 2016.

Modular evolution of secretion systems and virulence plasmids in a bacterial species complex.

BACKGROUND: Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. RESULTS: We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. CONCLUSIONS: We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

Hepatic steatosis and development of type 2 diabetes: Impact of chronic hepatitis B and viral specific factors.

BACKGROUND: Chronic hepatitis B (CHB) was associated with a lower prevalence of nonalcoholic fatty liver disease (NAFLD). The impact of CHB on the link between NAFLD and type 2 diabetes (T2D) and related virological implications remain unclear. METHODS: We recruited 2255 middle-to older-aged individuals who were examined serially for hepatic steatosis by ultrasonography and blood biochemistry as part of a population-based hepatocellular-carcinoma cohort study. In CHB patients, hepatitis B surface antigen (HBsAg) seroclearance and variation in viral load trajectory were also evaluated. RESULTS: During the average follow-up of 6 years, 168 participants developed T2D. CHB, as compared with uninfected subjects, was associated with lower risks for both new development and persistence of hepatic steatosis. Furthermore, the risk of steatosis decreased with higher levels of past viral load trajectories (p for trend = 0.0002). However, concomitant steatosis at baseline in CHB patients was still significantly associated with a 1.98-fold increased risk for T2D after multivariate adjustment including age, impaired fasting glucose, cirrhosis, and time-varying body mass index, although CHB reduced the propensity of hepatic steatosis to develop diabetes, especially for patients with high levels of past viral-load trajectory. In CHB, the functional cure of HBV infection, as indicated by HBsAg seroclearance, was associated with a 1.41-fold (95% CI 1.12-1.79) increased risk of steatosis. In addition, the increased risk for progressive impairment of glucose metabolism due to steatosis was especially prominent after HBsAg seroclearance. CONCLUSION: The data showed that HBV interferes with fatty liver disease and modulates its related T2D risk, offering additional insight into the interplay between NAFLD and CHB.

Diversification of the Type VI Secretion System in Agrobacteria.

The type VI secretion system (T6SS) is used by many Gram-negative bacteria to deploy toxic effectors for interbacterial competition. This system provides a competitive advantage in planta to agrobacteria, a diverse group with phytopathogenic members capable of genetically transforming plants. To inform on the ecology and evolution of agrobacteria, we revealed processes that diversify their effector gene collections. From genome sequences of diverse strains, we identified T6SS loci, functionally validated associated effector genes for toxicity, and predicted genes homologous to those that encode proteins known to interact with effectors. The gene loci were analyzed in a phylogenetic framework, and results show that strains of some species-level groups have different patterns of T6SS expression and are enriched in specific sets of T6SS loci. Findings also demonstrate that the modularity of T6SS loci and their associated genes engenders dynamicity, promoting reshuffling of entire loci, fragments therein, and domains to swap toxic effector genes across species. However, diversification is constrained by the need to maintain specific combinations of gene subtypes, congruent with observations that certain genes function together to regulate T6SS loading and activation. Data are consistent with a scenario where species can acquire unique T6SS loci that are then reshuffled across the genus in a restricted manner to generate new combinations of effector genes. IMPORTANCE The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bacteria reside. We uncovered the diversity of T6SS loci across a genus of plant-associated bacteria and show that diversification is driven by the acquisition of new loci and reshuffling among species. However, linkages between specific subtypes of genes need to be maintained to ensure that proteins whose interactions are necessary to activate the T6SS remain together. Results reveal how organization of gene loci and domain structure of genes provides flexibility to diversify under the constraints imposed by the system. Findings inform on the evolution of a mechanism that influences bacterial communities.

Longitudinal change of metabolite profile and its relation to multiple risk factors for the risk of developing hepatitis B-related hepatocellular carcinoma.

Despite considerable knowledge of viral pathogenesis, the pathophysiological changes related to the multifactorial, multistep process of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) development remains unclear. Longitudinal metabolomics study can reveal biological process for disease progression. We performed metabolite profiling with longitudinal prediagnostic plasma samples from two nested case-control studies of hepatitis B surface antigen carriers participating in ultrasound screening for HCC, one within a government employee cohort (870 samples from 109 HCC cases and 107 controls) and the other within a hospital-based cohort (266 samples from 63 HCC cases and 114 controls). Of the 34 measured metabolites, tyrosine, isoleucine, and glutamine were consistently associated with HCC. In analyses combining longitudinal data, a high metabolic risk score based on the three amino acids was robustly associated with increased risk of HCC (OR = 3.71, 95% confidence interval: 2.53-5.42), even after adjustment for clinical factors, or when assessed for different times up to ≥8 years before diagnosis. Similar association was observed in an independent, prospective analysis comprising 633 randomly selected individuals of the government employee cohort. More importantly, this metabolite signature was longitudinally influenced by HBV-infection phase and involved in gradual progression to liver fibrosis and cirrhosis. Furthermore, mediation analysis showed that the score mediated substantial proportions of the associations of key viral factors, insulin resistance, and diabetes status with HCC risk. Our results suggest that an amino-acid dysregulation metabotype may play a role in HBV-related HCC development, and may also be linked to common pathways that mediate increased HCC risks.

Effector loading onto the VgrG carrier activates type VI secretion system assembly.

The type VI secretion system (T6SS) is used by many bacteria to engage in social behavior and can affect the health of its host plant or animal. Because activities associated with T6SSs are often costly, T6SSs must be tightly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Using the plant pathogen Agrobacterium tumefaciens, we show that effectors are not just passengers but also impact on T6SS assembly. The A. tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. Here, we demonstrate that loading of Tde effectors onto their cognate carriers, the VgrG spikes, is required for active T6SS secretion. The assembly of the TssBC contractile sheath occurs only in the presence of Tde effectors. The requirement of effector loading for efficient T6SS secretion was also validated in other A. tumefaciens strains. We propose that such a mechanism is used by bacteria as a strategy for efficacious T6SS firing and to ensure that effectors are loaded onto the T6SS prior to completing its assembly.

Plant-Pathogenic Agrobacterium tumefaciens Strains Have Diverse Type VI Effector-Immunity Pairs and Vary in In-Planta Competitiveness.

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli. The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens. Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens, but genetic features have a significant role on the efficacy of interbacterial antagonism.

Redundancy and Specificity of Type VI Secretion vgrG Loci in Antibacterial Activity of Agrobacterium tumefaciens 1D1609 Strain.

Type VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found ∼70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e., vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609's antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58's VgrG2 shares 99% amino acid sequence identity with 1D1609's VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition.

Smoking and Hepatitis B Virus-Related Hepatocellular Carcinoma Risk: The Mediating Roles of Viral Load and Alanine Aminotransferase.

Smoking interacts with hepatitis B virus (HBV) to increase the risk of hepatocellular carcinoma (HCC), which might be explained by its role in antiviral immunity. We evaluated the potential mediating role of viral load and/or alanine aminotransferase (ALT) in the relation of smoking with HBV-associated HCC risk. Using multiple mediation analyses to analyze data from 209 HCC cases and 1,256 controls nested within a cohort of 4,841 male HBV carriers, we found that the effect of smoking on the risk of subsequent HCC was substantially mediated through viral load (percent mediated, 31.7%; P = 0.0054), and a significant mediation effect by both viral load and ALT was also evidenced. Among the 1,143 subjects with repeated measures of viral load and ALT over periods of up to 16 years, we further observed that a higher number of pack-years of smoking was associated with higher viral load, maintenance of a high viral load (>4.39 log copies/mL), more severe hepatotoxicity grade, and increased likelihood of ALT ≥80 U/L (odds ratio, 3.14; 95% confidence interval, 1.03-9.64; odds ratio, 6.06; 95% confidence interval, 1.10-33.25, respectively, for 10-19 and ≥20 pack-years versus nonsmokers) during follow-up. Furthermore, plasma interferon-γ levels were reduced in smokers compared with nonsmokers (interferon-γ-positive rate, 14.9% versus 28.7%; P < 0.0001) at baseline. Smoking was also associated with a reduced natural killer (NK) cell frequency in peripheral blood, characterized by reduced NK function through a systems immunology approach, after long-term follow-up in a subsample (n = 171). The combination of smoking and reduced NK cell frequency further increased viral load and the likelihood of ALT ≥80 U/L. Conclusion: The data highlight a role of smoking in HBV viral load, underlining the importance of smoking prevention and cessation in hepatitis B management.

Qualitative study for betel quid cessation among oral cancer patients.

The psychoactive effects of using areca nut and its potential for dependence have been observed. However, the factors that create barriers to or promote chewing cessation are not well understood. This study aims to explore the behavioral changes of betel quid chewers who have been diagnosed with oral cancer within a transtheoretical model framework. Thirty oral cancer patients with betel quid chewing history were chosen for in-depth interviews. Qualitative content analysis was used to analyze the data and identify themes that described the behavioral changes of betel quid cessation. Our research showed that betel quid chewers with oral cancer typically experience four significant stages of behavior: pre-contemplation, contemplation, action, and maintenance. Each stage change was marked by specific characteristics. At first, chewers showed positive attitudes toward the psychoactive or social effects of betel quid. They then realized the negative effects of betel quid, such as dental or other physical problems. Some also realized that they were addicted to betel quid. When they decided to quit, most chewers reported going "cold turkey." Some chewers successfully quit betel quid and attributed it to willpower. Those quitting because of the loss of oral functions were unable to chew anymore, though some chewers had experienced a relapse. In the maintenance stage, ex-chewers reported overcoming their addiction; however, relapse was possible. In this study, those who quit betel quid because of oral cancer usually quit tobacco and alcohol as well, with a lesser chance of recurrence. As the maintenance of chewing betel quid is multifactorial, this study provides information for betel quid cessation and oral cancer prevention.

The Agrobacterium Type VI Secretion System: A Contractile Nanomachine for Interbacterial Competition.

The bacterial type VI secretion system (T6SS) is a contractile nanomachine dedicated to delivering molecules out of bacterial cells. T6SS-encoding loci are in the genome sequences of many Gram-negative bacteria, and T6SS has been implicated in a plethora of roles. In the majority of cases, the T6SSs deliver effector proteins in a contact-dependent manner to antagonize other bacteria. Current models suggest that the effectors are deployed to influence social interactions in microbial communities. In this chapter, we describe the structure, function, and regulation of the T6SS and its effectors. We provide focus on the T6SS of Agrobacterium tumefaciens, the causative agent of crown gall disease, and relate the role of the T6SS to the ecology of A. tumefaciens.

Genome variations between rhizosphere and bulk soil ecotypes of a Pseudomonas koreensis population.

Bulk soil and rhizosphere are soil compartments selecting different microbial communities. However, it is unknown whether this selection also can change the genome content of specific bacterial taxa, splitting a population in distinct ecotypes. To answer this question we compared the genome sequences of 53 isolates obtained from sugarcane rhizosphere (28) and bulk soil (25). These isolates were previously classified in the Pseudomonas koreensis subgroup of the P. fluorescens complex. Phylogenomics showed a trend of separation between bulk soil and rhizosphere isolates. Discriminant analysis of principal components (DAPC) identified differences in the accessory genome of rhizosphere and bulk soil sub-populations. We found significant changes in gene frequencies distinguishing rhizosphere from bulk soil ecotypes, for example, enrichment of phosphatases and xylose utilization (xut) genes, respectively. Phenotypic assays and deletion of xutA gene indicated that accumulation of xut genes in the bulk soil sub-population provided a higher growth capacity in a d-xylose medium, supporting the corresponding genomic differences. Despite the clear differences distinguishing the two ecotypes, all 53 isolates were classified in a single 16S rRNA gene OTU. Collectively, our results revealed that the gene pool and ecological behavior of a bacterial population can be different for ecotypes living in neighbouring soil habitats.

Influence of Metabolic Risk Factors on Risk of Hepatocellular Carcinoma and Liver-Related Death in Men With Chronic Hepatitis B: A Large Cohort Study.

BACKGROUND & AIMS: Little is known about the absolute risk of hepatocellular carcinoma (HCC) and liver-disease related death, in association with metabolic risk factors, for patients with hepatitis B virus (HBV) infection. METHODS: We collected data from 5373 male Taiwanese civil servants who visited Taiwan's Government Employees' Central Clinics and received routine free physical examinations from 1989 through 1992. We obtained information on liver-related morbidity and mortality in HBV carriers, 40-65 years of age (n=1690), with different metabolic risk factors. We compared their medical histories with those of study participants without HBV or HCV infection in the same age range (n=1289). We used patients' baseline data on obesity, diabetes, hypertriglyceridemia, and high blood pressure to assign them to metabolic risk categories. We then performed a case-cohort analysis of the effects of hepatitis B viral factors on risk for HCC, based on metabolic factors and insulin resistance. RESULTS: Over a median follow-up period of 19 years, 158 of the 1690 HBV carriers developed HCC and 126 died from liver-related diseases. Among participants without HBV or HCV infection, only 6 developed HCC or died from liver-related disease. HBV carriers with different metabolic risk factors had significant differences in cumulative incidence of HCC and liver-related death. Patients with 3 or more metabolic risk factors had a substantially higher risk for HCC (10-year cumulative incidence, 13.60%) than patients with a low metabolic risk profile (10-year cumulative incidence, 4.83%; adjusted-hazard ratio, 2.32; 95% CI, 1.18-4.54). Smoking had a significant effect on this association (Pinteraction = .0044). Having 3 or more metabolic risk factors, compared with no factors, significantly increased the risk of HCC (adjusted-hazard ratio, 5.06; 95% CI, 2.23-11.47) and 10-year cumulative incidence of HCC (25.0% in smokers with 3 or more metabolic risk factors vs 3.87% in smokers with none; P < .0001) in smokers, but did not increase risk of HCC in nonsmokers. Metabolic risk factors and insulin resistance had the largest effect on HCC risk in patients with levels of HBV-DNA <10,000 copies/mL. CONCLUSIONS: In a study of men with chronic HBV infection ages 40-65 years in Taiwan, we associated a high burden of metabolic risk factors with increased risk of HCC; smoking has a significant effect on this association.

Complete response of huge buccal malignant melanoma in an octogenarian patient to arterial chemotherapy.

BACKGROUND: Primary oral mucosal melanoma is uncommon. However, it is an aggressive entity, and the absence of a standardized treatment protocol makes for an extremely poor prognosis. METHODS: We described the clinical course and treatment by arterial chemotherapy of an 87-year-old patient with nonresectable huge buccal malignant melanoma. Continuous intra-arterial infusion of fluorouracil (50 mg/24 hours) and 1 intermittent weekly 10-mg shot of cisplatin were given. RESULTS: The patient with oral melanoma presented with a roughly 6- × 4-cm exophytic mass that was noticed on the right buccal mucosa. The buccal tumor regressed dramatically until complete disappearance of the tumor mass was achieved at 2.5 months after intra-arterial chemotherapy was initiated. In total, 2880 mg of fluorouracil and 80 mg of cisplatin were administrated. The side effects of intra-arterial chemotherapy were mild and tolerable. CONCLUSION: Our data demonstrate that intra-arterial chemotherapy could be an alternative treatment for nonresectable buccal malignant melanoma.

Circulating programmed death-1 as a marker for sustained high hepatitis B viral load and risk of hepatocellular carcinoma.

OBJECTIVE: Recent evidence indicates a crucial role of the immunoinhibitory receptor programmed death-1 (PD-1) in enforcing T-cell dysfunction during chronic viral infection and cancer. We assessed the impact of circulating soluble PD-1 (sPD-1) levels on long-term dynamics of hepatitis B virus (HBV) load and hepatocellular carcinoma (HCC) risk. METHODS: In a case-cohort study on longitudinal analysis of viral load within a cohort of 2903 men chronically infected with HBV, followed up from baseline (1989-1992) through 2010, we determined sPD-1 levels in baseline plasma with enzyme-linked immunosorbent assay from 126 men who subsequently developed HCC and 1155 men who did not develop HCC. To evaluate whether patients' characteristics involved the use of sPD-1 as a biomarker, sPD-1 was also tested in 614 newly-diagnosed patients with HBV-related HCC recruited from a multicenter study for comparison with the 1155 noncases in the case-cohort study. RESULTS: Plasma quartile levels of sPD-1 were positively associated with HCC risk for men in the case-cohort analysis (vs. quartile 1: adjusted odds ratios [95% confidence intervals] for quartile 2-quartile 4 were 1.51 [0.75-3.03], 2.15 [1.12-4.13], and 2.29 [1.20-4.38], respectively), and in the case-control study regardless of age-of-onset and clinical stage. Furthermore, we found longitudinal effect of elevated sPD-1 levels to maintain higher viral load for 4 or more years, with greater and more prolonged effect among HBV genotype C- vs. non-C-infected participants. High levels of viral load and sPD-1 (vs. absence of both) was associated with a 6.29-fold increase in risk of HCC, and combining both conditions with HBV genotype C yielded an odds ratio of 30.47 with significant additive interaction (relative excess risk due to interaction: 27.08 [95% confidence interval = 8.76-45.41]). CONCLUSIONS: Our data suggest plasma sPD-1 as an important immune-related marker for assessment of HBV activity and HCC risk.

Impact of genetic heterogeneity in polymerase of hepatitis B virus on dynamics of viral load and hepatitis B progression.

OBJECTIVE: The hepatitis B virus (HBV)-polymerase region overlaps pre-S/S genes with high epitope density and plays an essential role in viral replication. We investigated whether genetic variation in the polymerase region determined long-term dynamics of viral load and the risk of hepatitis B progression in a population-based cohort study. METHODS: We sequenced the HBV-polymerase region using baseline plasma from treatment-naïve individuals with HBV-DNA levels≥1000 copies/mL in a longitudinal viral-load study of participants with chronic HBV infection followed-up for 17 years, and obtained sequences from 575 participants (80% with HBV genotype Ba and 17% with Ce). RESULTS: Patterns of viral sequence diversity across phases (i.e., immune-tolerant, immune-clearance, non/low replicative, and hepatitis B e antigen (HBeAg)-negative hepatitis phases) of HBV-infection, which were associated with viral and clinical features at baseline and during follow-up, were similar between HBV genotypes, despite greater diversity for genotype Ce vs. Ba. Irrespective of genotypes, however, HBeAg-negative participants had 1.5-to-2-fold higher levels of sequence diversity than HBeAg-positive participants (P<0.0001). Furthermore, levels of viral genetic divergence from the population consensus sequence, estimated by numbers of nucleotide substitutions, were inversely associated with long-term viral load even in HBeAg-negative participants. A mixed model developed through analysis of the entire HBV-polymerase region identified 153 viral load-associated single nucleotide polymorphisms in overall and 136 in HBeAg-negative participants, with distinct profiles between HBV genotypes. These polymorphisms were most evident at sites within or flanking T-cell epitopes. Seven polymorphisms revealed associations with both enhanced viral load and a more than 4-fold increased risk of hepatocellular carcinoma and/or liver cirrhosis. CONCLUSIONS: The data highlight a role of viral genetic divergence in the natural course of HBV-infection. Interindividual differences in the long-term dynamics of viral load is not only associated with accumulation of mutations in HBV-polymerase region, but differences in specific viral polymorphisms which differ between genotypes.

One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

Alpha-crystallin-type small heat shock proteins (sHsps) are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS). HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence-induced condition and its more efficient VirB8 chaperone activity as compared to other sHsps.

Acid-induced type VI secretion system is regulated by ExoR-ChvG/ChvI signaling cascade in Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.