Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

RING 4.0: faster residue interaction networks with novel interaction types across over 35,000 different chemical structures.

Residue interaction networks (RINs) are a valuable approach for representing contacts in protein structures. RINs have been widely used in various research areas, including the analysis of mutation effects, domain-domain communication, catalytic activity, and molecular dynamics simulations. The RING server is a powerful tool to calculate non-covalent molecular interactions based on geometrical parameters, providing high-quality and reliable results. Here, we introduce RING 4.0, which includes significant enhancements for identifying both covalent and non-covalent bonds in protein structures. It now encompasses seven different interaction types, with the addition of π-hydrogen, halogen bonds and metal ion coordination sites. The definitions of all available bond types have also been refined and RING can now process the complete PDB chemical component dictionary (over 35000 different molecules) which provides atom names and covalent connectivity information for all known ligands. Optimization of the software has improved execution time by an order of magnitude. The RING web server has been redesigned to provide a more engaging and interactive user experience, incorporating new visualization tools. Users can now visualize all types of interactions simultaneously in the structure viewer and network component. The web server, including extensive help and tutorials, is available from URL: https://ring.biocomputingup.it/.

HBcrAg values may predict virological and immunological responses to pegIFN-α in NUC-suppressed HBeAg-negative chronic hepatitis B.

OBJECTIVE: Selected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess the immunomodulatory effect of pegIFN-α on T and natural killer (NK) cell responses in NUC-suppressed patients to identify cellular and/or serological parameters to predict better T cell-restoring effect and better control of infection in response to pegIFN-α for a tailored application of IFN-α add-on. DESIGN: 53 HBeAg-negative NUC-treated patients with CHB were randomised at a 1:1 ratio to receive pegIFN-α-2a for 48 weeks, or to continue NUC therapy and then followed up for at least 6 months maintaining NUCs. Serum hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels as well as peripheral blood NK cell phenotype and function and HBV-specific T cell responses upon in vitro stimulation with overlapping HBV peptides were measured longitudinally before, during and after pegIFN-α therapy. RESULTS: Two cohorts of pegIFN-α treated patients were identified according to HBsAg decline greater or less than 0.5 log at week 24 post-treatment. PegIFN-α add-on did not significantly improve HBV-specific T cell responses during therapy but elicited a significant multispecific and polyfunctional T cell improvement at week 24 post-pegIFN-α treatment compared with baseline. This improvement was maximal in patients who had a higher drop in serum HBsAg levels and a lower basal HBcrAg values. CONCLUSIONS: PegIFN-α treatment can induce greater functional T cell improvement and HBsAg decline in patients with lower baseline HBcrAg levels. Thus, HBcrAg may represent an easily and reliably applicable parameter to select patients who are more likely to achieve better response to pegIFN-α add-on to virally suppressed patients.

Endoscopic vacuum therapy (eVAC) combined with continuous perianastomotic irrigation for prevention of anastomotic leak after surgical ampullectomy.

PURPOSE: Transduodenal surgical ampullectomy (tAMP) with papillary reimplantation is a valid alternative to pancreaticoduodenectomy for lesions of the periampullary region not amenable to endoscopic resection. As tAMP is burdened by high rates of biliopancreatic-enteric anastomotic leak, we tested preventive endoluminal vacuum therapy (eVAC) combined with post-operative continuous perianastomotic irrigation (CPI) to reduce such anastomotic leak. METHODS: Between 10/2013 and 09/2023, 37 patients undergoing laparotomic tAMP (with or without jejunal transposition) and papillary reimplantation at Hirslanden Klinik Zurich were retrospectively analysed; of these, 16 received prophylactic eVAC combined with CPI, while the remaining represented the historical cohort. RESULTS: The eVAC-CPI-group and the historical-cohort were homogeneous in demographic characteristics. Surgery in the prophylactic eVAC-CPI-group lasted about 30 min longer due to eVAC application (p = 0.008). The biliopancreatico-enteric anastomotic leak rates were 6.2% in the eVAC-CIP-group vs. 19.0% in the historical-cohort (p = 0.266). Along, a strong trend of less severe post-operative complications in general (p = 0.073), and borderline-significantly less cases of acute pancreatitis (p = 0.057) and tAMP-related re-operations or re-interventions (p = 0.057) in particular, were observed in the eVAC-CPI-group. The only anastomotic leak in the eVAC-CPI-group was successfully managed through repeated cycles of eVAC. The device was well tolerated by all patients; no vacuum/irrigation-related complications or malfunctioning occurred. CONCLUSION: Our study is the first to provide some technical insights demonstrating the safety and feasibility of a prophylactic approach with eVAC and perianastomotic irrigation to reduce anastomotic leak after tAMP. Increasing the number of subjects will confirm the benefit of our promising results.

Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Deregulated intracellular pathways define novel molecular targets for HBV-specific CD8 T cell reconstitution in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, elevated reactive oxygen species levels derived from dysfunctional mitochondria can cause increased protein oxidation and DNA damage in exhausted virus-specific CD8 T cells. The aim of this study was to understand how these defects are mechanistically interconnected to further elucidate T cell exhaustion pathogenesis and, doing so, to devise novel T cell-based therapies. METHODS: DNA damage and repair mechanisms, including parylation, CD38 expression, and telomere length were studied in HBV-specific CD8 T cells from chronic HBV patients. Correction of intracellular signalling alterations and improvement of antiviral T cell functions by the NAD precursor nicotinamide mononucleotide and by CD38 inhibition was assessed. RESULTS: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, and mitochondrial and proteostasis functions by NAD supplementation, which could also improve the HBV-specific antiviral CD8 T cell function. CONCLUSIONS: Our study delineates a model of CD8 T cell exhaustion whereby multiple interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore antiviral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. IMPACT AND IMPLICATIONS: Correction of HBV-specific CD8 T cell dysfunction is believed to represent a rational strategy to cure chronic HBV infection, which however requires a deep understanding of HBV immune pathogenesis to identify the most important targets for functional T cell reconstitution strategies. This study identifies a central role played by NAD depletion in the intracellular vicious circle that maintains CD8 T cell exhaustion, showing that its replenishment can correct impaired intracellular mechanisms and reconstitute efficient antiviral CD8 T cell function, with implications for the design of novel immune anti-HBV therapies. As these intracellular defects are likely shared with other chronic virus infections where CD8 exhaustion can affect virus clearance, these results can likely also be of pathogenetic relevance for other infection models.

Mortality risk factors in community-dwelling, subjectively healthy, Swiss older adults: update after 8-years follow-up.

BACKGROUND: Worldwide population is ageing, but little is known regarding risk factors associated with increased mortality in subjectively healthy, community-dwelling older adults. We present the updated results of the longest follow-up carried out on Swiss pensioners and we provide results on potential risk factors associated with mortality before the onset of the COVID-19 pandemic. MATERIALS AND METHODS: Within the SENIORLAB study, we collected demographic data, anthropometric measures, medical history, and laboratory parameters of 1467 subjectively healthy, community-dwelling, Swiss adults aged ≥ 60 years over a median follow-up of 8.79 years. The variables considered in the multivariable Cox-proportional hazard model for mortality during follow-up were selected based on prior knowledge. Two separate models for males and females were calculated; moreover, we fitted the old model obtained in 2018 to the complete follow-up data to highlight differences and similarities. RESULTS: The population sample included 680 males and 787 females. Age of participants ranged between 60 and 99 years. We experienced 208 deaths throughout the entire follow-up period; no patients were lost at follow-up. The Cox-proportional hazard regression model included female gender, age, albumin levels, smoking status, hypertension, osteoporosis and history of cancer within predictors of mortality over the follow-up period. Consistent findings were obtained also after gender stratification. After fitting the old model, female gender, hypertension, and osteoporosis still showed statistically significant independent associations with all-cause mortality. CONCLUSIONS: Understanding the predictors of a healthy survival can improve the overall quality of life of the ageing population and simultaneously reduce their global economic burden. TRIAL REGISTRATION: The present study was registered in the International Standard Randomized Controlled Trial Number registry: https://www.isrctn.com/ISRCTN53778569 (registration date: 27/05/2015).

Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

Hierarchical representation for PPI sites prediction.

BACKGROUND: Protein-protein interactions have pivotal roles in life processes, and aberrant interactions are associated with various disorders. Interaction site identification is key for understanding disease mechanisms and design new drugs. Effective and efficient computational methods for the PPI prediction are of great value due to the overall cost of experimental methods. Promising results have been obtained using machine learning methods and deep learning techniques, but their effectiveness depends on protein representation and feature selection. RESULTS: We define a new abstraction of the protein structure, called hierarchical representations, considering and quantifying spatial and sequential neighboring among amino acids. We also investigate the effect of molecular abstractions using the Graph Convolutional Networks technique to classify amino acids as interface and no-interface ones. Our study takes into account three abstractions, hierarchical representations, contact map, and the residue sequence, and considers the eight functional classes of proteins extracted from the Protein-Protein Docking Benchmark 5.0. The performance of our method, evaluated using standard metrics, is compared to the ones obtained with some state-of-the-art protein interface predictors. The analysis of the performance values shows that our method outperforms the considered competitors when the considered molecules are structurally similar. CONCLUSIONS: The hierarchical representation can capture the structural properties that promote the interactions and can be used to represent proteins with unknown structures by codifying only their sequential neighboring. Analyzing the results, we conclude that classes should be arranged according to their architectures rather than functions.

DNA Methylation and Immune Cell Markers Demonstrate Evidence of Accelerated Aging in Patients with Chronic Hepatitis B Virus or Hepatitis C Virus, with or without Human Immunodeficienct Virus Co-infection.

BACKGROUND: Several chronic diseases accelerate biological aging. We investigated age acceleration and the association between peripheral blood DNA methylation (DNAm) and immune cell markers in patients chronically infected with the hepatitis B virus (HBV) or the hepatitis C virus (HCV) with and without human immunodeficiency virus (HIV) co-infection. METHODS: Age acceleration was measured as the difference between epigenetic age (Horvath clock) and chronological age. The immune marker model of age acceleration was developed using Elastic Net regression to select both the immune markers and their associated weights in the final linear model. RESULTS: Patients with chronic HBV (n = 51) had a significantly higher median epigenetic age compared to chronological age (age accelerated) (P < .001). In patients with chronic HCV infection (n = 63), age acceleration was associated with liver fibrosis as assessed by histology (P < .05), or presence of HIV co-infection (P < .05), but not HCV mono-infection. Age acceleration defined by immune markers was concordant with age acceleration by DNA methylation (correlation coefficient = .59 in HBV; P = .0025). One-year treatment of HBV patients with nucleoside therapy was associated with a modest reduction in age acceleration, as measured using the immune marker model (-.65 years, P = .018). CONCLUSION: Our findings suggest that patients with chronic viral hepatitis have accelerated epigenetic aging, that immune markers define biological age, and have the potential to assess the effects of therapeutic intervention on age acceleration.

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS: Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS: HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS: Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY: In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.

RNA Interference Therapy With ARC-520 Results in Prolonged Hepatitis B Surface Antigen Response in Patients With Chronic Hepatitis B Infection.

BACKGROUND AND AIMS: ARC-520, the first an RNA interference (RNAi) therapeutic, was designed to reduce all RNA transcripts derived from covalently closed circular DNA, leading to a reduction in viral antigens and hepatitis B virus (HBV) DNA. APPROACH AND RESULTS: We aimed to evaluate the depth of hepatitis B surface antigen (HBsAg) decline in response to multiple doses of ARC-520 compared to placebo (PBO) in two randomized, multicenter studies in nucleoside/nucleotide analogue reverse-transcriptase inhibitor (NUC)-experienced patients with hepatitis B early antigen (HBeAg)-negative (E-neg) or HBeAg-positive (E-pos) disease. A total of 58 E-neg and 32 E-pos patients were enrolled and received four monthly doses of PBO (n = 20 E-neg, 11 E-pos), 1 mg/kg ARC-520 (n = 17 E-neg, 10 E-pos), or 2 mg/kg ARC-520 (n = 21 E-neg, 11 E-pos) concomitantly with NUC. HBsAg change from baseline to 30 days after the last ARC-520 dose were compared to PBO. Both E-neg and E-pos high-dose groups significantly reduced HBsAg compared to PBO, with mean reductions of 0.38 and 0.54 log IU/mL, respectively. HBsAg reductions persisted for approximately 85 days and >85 days after the last dose in E-neg and E-pos patients, respectively. The low-dose groups did not reach statistical significance in either study. E-pos patients showed a dose-dependent reduction in HBeAg from baseline. Mean maximum reduction was 0.23 and 0.69 log Paul Ehrlich IUs/mL in the low-dose and high dose ARC-520 groups respectively. ARC-520 was well tolerated, with only two serious adverse events of pyrexia possibly related to study drug observed. CONCLUSIONS: ARC-520 was active in both E-neg and E-pos, NUC-experienced HBV patients; but absolute HBsAg reductions were moderate, possibly due to expression of HBsAg from integrated HBV DNA, indicating the need for RNAi therapeutics that can target viral transcripts regardless of origin.

Coupling Epidemiological Models with Social Dynamics.

In this work we study a Susceptible-Infected-Susceptible model coupled with a continuous opinion dynamics model. We assume that each individual can take measures to reduce the probability of contagion, and the level of effort each agent applies can change due to social interactions. We propose simple rules to model the propagation of behaviors that modify the level of effort, and analyze their impact on the dynamics of the disease. We derive a two dimensional set of ordinary differential equations describing the dynamic of the proportion of the number of infected individuals and the mean value of the effort parameter, and analyze the equilibria of the system. The stability of the endemic phase and disease free equilibria depends only on the mean value of the levels of efforts, and not on the initial distribution of levels of effort.

COVID-19 outbreak and acute cholecystitis in a Hub Hospital in Milan: wider indications for percutaneous cholecystostomy.

BACKGROUND: COVID-19 pandemic has impacted the Italian National Health Care system at many different levels, causing a complete reorganization of surgical wards. In this context, our study retrospectively analysed the management strategy for patients with acute cholecystitis. METHODS: We analysed all patients admitted to our Emergency Department for acute cholecystitis between February and April 2020 and we graded each case according to 2018 Tokyo Guidelines. All patients were tested for positivity to SARS-CoV-2 and received an initial conservative treatment. We focused on patients submitted to cholecystostomy during the acute phase of pandemic and their subsequent disease evolution. RESULTS: Thirty-seven patients were admitted for acute cholecystitis (13 grade I, 16 grade II, 8 grade III). According to Tokyo Guidelines (2018), patients were successfully treated with antibiotic only, bedside percutaneous transhepatic gallbladder drainage (PC) and laparoscopic cholecystectomy (LC) in 29.7%, 21.6% and 48.7% of cases respectively. Therapeutic strategy of three out of 8 cases, otherwise fit for surgery, submitted to bedside percutaneous transhepatic gallbladder drainage (37.5%), were directly modified by COVID-19 pandemic: one due to the SARS-CoV-2 positivity, while two others due to unavailability of operating room and intensive care unit for post-operative monitoring respectively. Overall success rate of percutaneous cholecystostomy was of 87.5%. The mean post-procedural hospitalization length was 9 days, and no related adverse events were observed apart from transient parietal bleeding, conservatively treated. Once discharged, two patients required readmission because of acute biliary symptoms. Median time of drainage removal was 43 days and only 50% patients thereafter underwent cholecystectomy. CONCLUSIONS: Percutaneous cholecystostomy has shown to be an effective and safe treatment thus acquiring an increased relevance in the first phase of the pandemic. Nowadays, considering we are forced to live with the SARS-CoV-2 virus, PC should be considered as a virtuous, alternative tool for potentially all COVID-19 positive patients and selectively for negative cases unresponsive to conservative therapy and unfit for surgery.

Combined GS-4774 and Tenofovir Therapy Can Improve HBV-Specific T-Cell Responses in Patients With Chronic Hepatitis.

BACKGROUND & AIMS: One strategy to treat chronic hepatitis B virus (HBV) infection could be to increase the functions of virus-specific T cells. We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, given with tenofovir disoproxil fumarate (TDF) to untreated patients with chronic HBV infection. METHODS: We performed an open-label study at 34 sites in Canada, Italy, New Zealand, Romania, South Korea, and United States from July 2014 to August 2016. Adults who were positive for HB surface antigen (HBsAg) > 6 months and levels of HBV DNA ≥2000 IU/mL who had not received antiviral treatment for HBV within 3 months of screening were randomly assigned (1:2:2:2) to groups given oral TDF 300 mg daily alone (n = 27; controls) or with 2, 10, or 40 yeast units GS-4774 (n = 168), administered subcutaneously every 4 weeks until week 20 for a total of 6 doses. Blood samples were collected and analyzed and patients received regular physical examinations. Efficacy was measured by decrease in HBsAg from baseline to week 24. Specific responses to HBV (production of interferon gamma [IFNG], tumor necrosis factor [TNF], interleukin 2 [IL2], and degranulation) were measured in T cells derived from 12 HBeAg-negative patients with genotype D infections, after overnight or 10 days of stimulation of peripheral blood mononuclear cells with peptides from the entire HBV proteome. T-regulatory cells were analyzed for frequency and phenotype. Data from studies of immune cells were compared with data on reductions in HBsAg, HBV DNA, and alanine aminotransferase in blood samples from patients. RESULTS: GS-4774 was safe and well tolerated but did not produce significant decreases in levels of HBsAg. Production of IFNG, TNF, and IL2 increased significantly at weeks 24 and 48, compared with baseline, in HBV-specific CD8+ T cells from patients given GS-4774 but not from controls. GS-4774 had greater effects on CD8+ than CD4+ T cells, which were not affected at all or very weakly by TDF with or without GS-4774. GS-4774 did not affect responses of T cells to other viruses tested. HBV core peptides induced the greatest production of IFNG by T cells following overnight stimulation, whereas HBV envelope antigens did not induce a response. Following 10 days of stimulation, production of IFNG and TNF increased with time of exposure to GS-4774; the greatest levels of responses were to HBV envelope antigens followed by core and polymerase peptides. We observed a correlation in patients given GS-4774 between increased T-cell functions and reductions in numbers of T-regulatory cells. CONCLUSIONS: In a phase 2 study of patients with chronic HBV infection given TDF with or without GS-4774, we found that vaccination can increase production of IFNG, TNF, and IL2 by CD8+ T cells exposed to antigenic peptides, with little effect on CD4+ T cells. Although GS-4774 did not reduce levels of HBsAg in patients, its strong immune stimulatory effect on CD8+ T cells might be used in combination with other antiviral agents to boost the antivirus immune response. Clinicaltrials.gov no: NCT02174276.

Energy metabolism and cell motility defect in NK-cells from patients with hepatocellular carcinoma.

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients' and controls' plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-ß as possible mediator of this dysfunction. Blocking TGF-ß could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-ß rich HCC patients' plasma, suggesting that TGF-ß among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.

Antibody interface prediction with 3D Zernike descriptors and SVM.

MOTIVATION: Antibodies are a class of proteins capable of specifically recognizing and binding to a virtually infinite number of antigens. This binding malleability makes them the most valuable category of biopharmaceuticals for both diagnostic and therapeutic applications. The correct identification of the antigen-binding residues in the antibody is crucial for all antibody design and engineering techniques and could also help to understand the complex antigen binding mechanisms. However, the antibody-binding interface prediction field appears to be still rather underdeveloped. RESULTS: We present a novel method for antibody interface prediction from their experimentally solved structures based on 3D Zernike Descriptors. Roto-translationally invariant descriptors are computed from circular patches of the antibody surface enriched with a chosen subset of physico-chemical properties from the AAindex1 amino acid index set, and are used as samples for a binary classification problem. An SVM classifier is used to distinguish interface surface patches from non-interface ones. The proposed method was shown to outperform other antigen-binding interface prediction software. AVAILABILITY AND IMPLEMENTATION: Linux binaries and Python scripts are available at https://github.com/sebastiandaberdaku/AntibodyInterfacePrediction. The datasets generated and/or analyzed during the current study are available at https://doi.org/10.6084/m9.figshare.5442229. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Early Red Flags Associated with Delayed Discharge in Patients Undergoing Gastrectomy: Analysis of Perioperative Variables and ERAS Protocol Items.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) perioperative pathways are safe and effective for patients undergoing gastrectomy. However, adherence to these protocols varies and is generally underreported. This retrospective study aimed to assess whether perioperative variables or deviation from ERAS items is associated with delayed discharge after gastrectomy. METHODS: All patients undergoing gastrectomy at our institution were managed with a standardised perioperative pathway according to ERAS principles. The target length of stay was set as the ninth post-operative day (POD). All significant variables were derived from a bivariate analysis and were entered into a logistic regression to confirm their statistical value. RESULTS: The study included 180 patients. Multivariate regression analysis revealed that incomplete immunonutrition, failure to extubate the patient at the end of surgery, intraoperative crystalloids >2150 ml and blood transfusion >268 ml, surgery duration >195 min, and failure to mobilise patients within 24 h from surgery were associated with delayed discharge. The logistic regression model was statistically significant (p < 0.001) and correctly classified 73.6% of cases. Sensitivity and specificity were 74.1% and 73.2%, respectively. CONCLUSIONS: These results seem clinically significant and consistent with those of previous studies. The reported perioperative variables showed a strong relationship with the length of hospital stay.

Irrigation and passive drainage of pancreatic stump after distal pancreatectomy in high-risk patients: an innovative approach to reduce pancreatic fistula.

INTRODUCTION: Postoperative pancreatic fistula (POPF) represents the most common form of morbidity after distal pancreatectomy (DP). The aim of this study was to illustrate an innovative technique of irrigation and passive drainage to reduce clinically relevant POPF (CR-POPF) incidence in high-risk patients undergoing DP. MATERIAL AND METHODS: Twelve consecutive high-risk patients received irrigation and passive drainage of the pancreatic stump with a Salem sump drainage after DP. The Salem sump was irrigated with 100 ml/h of Ringer solution for 2 postoperative days (POD). In the case of low-drain amylase and lipase levels on POD 3, the irrigation was reduced to 50 ml/h. Persistence of low-drain pancreatic enzymes on POD 4 allowed for interruption of irrigation and subsequent removal of drainage from POD 7 onward in the absence of evidence of any pancreatic fistula. RESULTS: Overall, 16.6% of the patients experienced a grade 3 or higher surgical complication. We experienced only one case of POPF: the fistula was classified as grade B and it was managed with radiologic drainage of the fluid collection. We did not experience any case of re-operation nor in-hospital mortality. CONCLUSIONS: Irrigation with passive drainage of the pancreatic stump after DP is an interesting approach for CR-POPF prevention in high-risk patients.

Performance of computational methods for the evaluation of pericentriolar material 1 missense variants in CAGI-5.

The CAGI-5 pericentriolar material 1 (PCM1) challenge aimed to predict the effect of 38 transgenic human missense mutations in the PCM1 protein implicated in schizophrenia. Participants were provided with 16 benign variants (negative controls), 10 hypomorphic, and 12 loss of function variants. Six groups participated and were asked to predict the probability of effect and standard deviation associated to each mutation. Here, we present the challenge assessment. Prediction performance was evaluated using different measures to conclude in a final ranking which highlights the strengths and weaknesses of each group. The results show a great variety of predictions where some methods performed significantly better than others. Benign variants played an important role as negative controls, highlighting predictors biased to identify disease phenotypes. The best predictor, Bromberg lab, used a neural-network-based method able to discriminate between neutral and non-neutral single nucleotide polymorphisms. The CAGI-5 PCM1 challenge allowed us to evaluate the state of the art techniques for interpreting the effect of novel variants for a difficult target protein.