Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

Prospective Latin American cohort evaluating outcomes of patients with COVID-19 and abnormal liver tests on admission.

INTRODUCTION & OBJECTIVES: The independent effect of liver biochemistries as a prognostic factor in patients with COVID-19 has not been completely addressed. We aimed to evaluate the prognostic value of abnormal liver tests on admission of hospitalized patients with COVID-19. MATERIALS & METHODS: We performed a prospective cohort study including 1611 hospitalized patients with confirmed SARS-CoV-2 infection from April 15, 2020 through July 31, 2020 in 38 different Hospitals from 11 Latin American countries. We registered clinical and laboratory parameters, including liver function tests, on admission and during hospitalization. All patients were followed until discharge or death. We fit multivariable logistic regression models, further post-estimation effect through margins and inverse probability weighting. RESULTS: Overall, 57.8% of the patients were male with a mean age of 52.3 years, 8.5% had chronic liver disease and 3.4% had cirrhosis. Abnormal liver tests on admission were present on 45.2% (CI 42.7-47.7) of the cohort (n = 726). Overall, 15.1% (CI 13.4-16.9) of patients died (n = 244). Patients with abnormal liver tests on admission presented higher mortality 18.7% (CI 15.9-21.7), compared to those with normal liver biochemistries 12.2% (CI 10.1-14.6); P < .0001). After excluding patients with history of chronic liver disease, abnormal liver tests on admission were independently associated with death [OR 1.5 (CI 1.1-2.0); P = 0.01], and severe COVID-19 (2.6 [2.0-3.3], P < .0001), both adjusted by age, gender, diabetes, pneumonia and body mass index >30. CONCLUSIONS: The presence of abnormal liver tests on admission is independently associated with mortality and severe COVID-19 in hospitalized patients with COVID-19 infection and may be used as surrogate marker of inflammation. CLINICALTRIALS.GOV: NCT04358380.

Comparison of different prognostic scores for patients with cirrhosis hospitalized with SARS-CoV-2 infection.

INTRODUCTION AND OBJECTIVES: Viral infections have been described to increase the risk of decompensation in patients with cirrhosis. We aimed to determine the effect of SARS-CoV-2 infection on outcome of hospitalized patients with cirrhosis and to compare the performance of different prognostic models for predicting mortality. PATIENTS: We performed a prospective cohort study including 2211 hospitalized patients with confirmed SARS-CoV-2 infection from April 15, 2020 through October 1, 2020 in 38 Hospitals from 11 Latin American countries. We registered clinical and laboratory parameters of patients with and without cirrhosis. All patients were followed until discharge or death. We evaluated the prognostic performance of different scoring systems to predict mortality in patients with cirrhosis using ROC curves. RESULTS: Overall, 4.6% (CI 3.7-5.6) subjects had cirrhosis (n = 96). Baseline Child-Turcotte-Pugh (CTP) class was assessed: CTP-A (23%), CTP-B (45%) and CTP-C (32%); median MELD-Na score was 19 (IQR 14-25). Mortality was 47% in patients with cirrhosis and 16% in patients without cirrhosis (P < .0001). Cirrhosis was independently associated with death [OR 3.1 (CI 1.9-4.8); P < .0001], adjusted by age, gender, and body mass index >30. The areas under the ROC curves for performance evaluation in predicting 28-days mortality for Chronic Liver Failure Consortium (CLIF-C), North American Consortium for the Study of End-Stage Liver Disease (NACSELD), CTP score and MELD-Na were 0.85, 0.75, 0.69, 0.67; respectively (P < .0001). CONCLUSIONS: SARS-CoV-2 infection is associated with elevated mortality in patients with cirrhosis. CLIF-C had better performance in predicting mortality than NACSELD, CTP and MELD-Na in patients with cirrhosis and SARS-CoV-2 infection. Clinicaltrials.gov:NCT04358380.

Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy.

Gene therapy is a promising emerging therapeutic modality for the treatment of cardiovascular diseases and hereditary diseases that afflict the heart. Hence, there is a need to develop robust cardiac-specific expression modules that allow for stable expression of the gene of interest in cardiomyocytes. We therefore explored a new approach based on a genome-wide bioinformatics strategy that revealed novel cardiac-specific cis-acting regulatory modules (CS-CRMs). These transcriptional modules contained evolutionary-conserved clusters of putative transcription factor binding sites that correspond to a "molecular signature" associated with robust gene expression in the heart. We then validated these CS-CRMs in vivo using an adeno-associated viral vector serotype 9 that drives a reporter gene from a quintessential cardiac-specific α-myosin heavy chain promoter. Most de novo designed CS-CRMs resulted in a >10-fold increase in cardiac gene expression. The most robust CRMs enhanced cardiac-specific transcription 70- to 100-fold. Expression was sustained and restricted to cardiomyocytes. We then combined the most potent CS-CRM4 with a synthetic heart and muscle-specific promoter (SPc5-12) and obtained a significant 20-fold increase in cardiac gene expression compared to the cytomegalovirus promoter. This study underscores the potential of rational vector design to improve the robustness of cardiac gene therapy.

Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Nuclear localized Influenza nucleoprotein N-terminal deletion mutant is deficient in functional vRNP formation.

BACKGROUND: The influenza RNA dependent RNA polymerase synthesizes viral RNA in the nucleus as functional viral ribonucleoprotein (vRNP) complexes with RNA and nucleoprotein (NP). The N-terminus of NP contains an unconventional nuclear localization signal (NLS) important for initial vRNP nuclear localization but which also interacts with various host factors. METHODS: To study the role of the N-terminus of NP aside from NLS function, we generated an N-terminal NP deletion mutant, del20NLS-NP, encoding the conventional SV40 T-antigen NLS in place of the first 20 amino acids of NP. We characterized expression, location, and activity of del20NLS-NP compared to wild type NP using reconstituted vRNP assays, cellular fractionation, Western blotting, and reverse transcription-PCR. We assessed NP nucleotide binding with gel-shift assays and analyzed NP complexes using 1D blue native gel electrophoresis. RESULTS: del20NLS-NP is expressed, localized in the nucleus and cytoplasm, and maintains ability to bind nucleic acids. Despite this, del20NLS-NP exhibits a defect in viral RNA expression exacerbated by increasing vRNA template length. We find diminished del20NLS-NP high molecular weight complexes in protein extracts; evidence the defect is with functional vRNP formation. Interestingly, the shortest template, NS vRNA, exhibits a limited defect. However, this is not due to short template size, but rather activity of the NS protein(s). Expression of NS1 rescues the gene expression defect primarily at the protein level, a finding consistent with the known role of NS1 as a viral mRNA translational enhancer. NS1 mutant analysis confirms NS1-RNA binding is not required for the translational enhancement and reveals the NS1-CPSF30 interaction surface is essential. CONCLUSIONS: del20NLS-NP is a nuclear localized NP mutant able to bind nucleic acids but inefficient for assembly of functional vRNPs inside the host cell. Our results add to growing evidence the N-terminus of NP plays important roles aside from vRNP nuclear localization. We demonstrate the utility of this partially functional NP mutant to characterize the influence of additional proteins on viral gene expression. Our studies reveal the NS1-CPSF30 interaction surface is required for the ability of NS1 to enhance viral protein translation, supporting a function for this NS1 domain in the cytoplasm.

[Severe acute pancreatitis associated with gallbladder gangrene]. / Pancreatitis aguda grave asociada a gangrena vesicular.

We present a diabetic patient who developed severe acute pancreatitis associated to gallbladder gangrene, in this case we assessed the applicability of classification criteria and management of the pathways for acute pancreatitis and also we suggest some topics that could be investigated in the future.

An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.

UNLABELLED: Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. CONCLUSION: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.

Methylene blue for the treatment of refractory septic shock secondary to listeriosis in a paediatric patient.

Current therapies frequently used for refractory septic shock include hydrocortisone, vasopressin, extracorporeal membrane oxygenation (ECMO) support, inodilators, levosimendan and methylene blue. The evidence for these treatments is very limited. We present a case of a 5-year-old patient with refractory septic shock, secondary to Listeria monocytogenes meningitis. She presented with status epilepticus and developed septic shock. Shock persisted despite multiple high-dose vasoactive medications. ECMO support was not available. The medical team decided to use methylene blue to revert the vasoplegia, with excellent results. Shortly after the administration, vasopressors were weaned off and the high lactate cleared. She developed severe neurological sequelae due to brain haemorrhage secondary to the Listeria meningitis. The evidence supporting methylene blue for refractory septic shock in paediatric patients is limited. This case represents the effectiveness of this therapy without secondary effects.

The establishment of public health policies and the burden of non-alcoholic fatty liver disease in the Americas.

Non-alcoholic fatty liver disease (NAFLD) affects 20-25% of the general population and is associated with morbidity, increased mortality, and elevated health-care costs. Most NAFLD risk factors are modifiable and, therefore, potentially amenable to being reduced by public health policies. To date, there is no information about NAFLD-related public health policies in the Americas. In this study, we analysed data from 17 American countries and found that none have established national public health policies to decrease NAFLD-related burden. There is notable heterogeneity in the existence of public health policies to prevent NAFLD-related conditions. The most common public health policies were related to diabetes (15 [88%] countries), hypertension (14 [82%] countries), cardiovascular diseases (14 [82%] countries), obesity (nine [53%] countries), and dyslipidaemia (six [35%] of countries). Only seven (41%) countries had a registry of the burden of NAFLD, and efforts to raise awareness in the Americas were scarce. The implementation of public health policies are urgently needed in the Americas to decrease the burden of NAFLD.

Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation.

Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. We now demonstrate that these hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Moreover, stable transposition could also be achieved in iPS, which retained their ability to differentiate along neuronal, cardiac, and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon-mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD(+) myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic "molecular switch" in iPS. Hence, this hyperactive transposon system represents an attractive nonviral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy.

IL-10 dampens TNF/inducible nitric oxide synthase-producing dendritic cell-mediated pathogenicity during parasitic infection.

Antiparasite responses are associated with the recruitment of monocytes that differentiate to macrophages and dendritic cells at the site of infection. Although classically activated monocytic cells are assumed to be the major source of TNF and NO during Trypanosoma brucei brucei infection, their cellular origin remains unclear. In this study, we show that bone marrow-derived monocytes accumulate and differentiate to TNF/inducible NO synthase-producing dendritic cells (TIP-DCs) in the spleen, liver, and lymph nodes of T. brucei brucei-infected mice. Although TIP-DCs have been shown to play a beneficial role in the elimination of several intracellular pathogens, we report that TIP-DCs, as a major source of TNF and NO in inflamed organs, could contribute actively to tissue damage during the chronic stage of T. brucei brucei infection. In addition, the absence of IL-10 leads to enhanced differentiation of monocytes to TIP-DCs, resulting in exacerbated pathogenicity and early death of the host. Finally, we demonstrate that sustained production of IL-10 following IL-10 gene delivery treatment with an adeno-associated viral vector to chronically infected mice limits the differentiation of monocytes to TIP-DCs and protects the host from tissue damage.

GASTRIC AND ORAL FEEDING IN SEVERE ACUTE PANCREATITIS.

BACKGROUND: There is controversy about the initiation of gastric or oral feeding in patients with severe acute pancreatitis (SAP) because they could increase pancreatic stimulation and exacerbate symptoms and complications. OBJECTIVE: To describe the clinical characteristics and results of patients with SAP who underwent gastric tube or oral feeding versus parenteral or jejunal feeding. METHODS: A retrospective study was carried out on patients over 18 years old with SAP diagnostic, who had been treated in critical care units. We excluded patients coming from other hospitals and those with incomplete medical records. RESULTS: Thirty patients with SAP were included, fifty three percent of them tolerated the gastric tube or oral feeding, and most of them were females and older than patients who received parenteral or jejunal feeding. Other clinical characteristics and outcomes were similar in both groups. Conclusion: Gastric tube or oral feeding is no absolute contraindication for SAP.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting.

Factores de riesgo para destete fallido de la ventilación mecánica en adultos

Introducción: El destete o liberación de la ventilación mecánica (VM) es un proceso complejo y cuando es fallido aumentará los riesgos de complicaciones y gastos. Objetivo: Identificar factores de riesgo modificables para destete fallido en adultos con VM. Materiales y Métodos: Estudio de casos y controles realizado en pacientes ≥ 18 años ingresados en la unidad de cuidados intensivos de un hospital de tercer nivel. Los casos se identificaron como destete fallido (DF) del VM y los controles como destete simple. Se excluyeron los pacientes procedentes de otro hospital con VM. Los factores estudiados fueron el alto riesgo nutricional por el Nutric score modificado, la nutrición enteral tardía, el balance hídrico (BH) positivo y la ausencia de fisioterapia previos al destete. Se calculo el OR con una significancia < 0,05 para el análisis bivariado, multivariado y ajustado. Resultados: Se incluyeron 105 pacientes, 35 casos y 70 controles. El análisis bivariado encontró que el alto riesgo nutricional (OR = 2.5; IC 95% = 1,1 5,9; p=0,027) fue factor de riesgo, pero el análisis multivariado no lo confirmó. La nutrición enteral tardía (OR = 1,2; IC 95% = 0,4 3,4), el BH positivo (OR = 0,7; IC 95% = 0,3 1,7) y la ausencia de fisioterapia respiratoria (OR = 0,2; IC 95% = 0,004 0,011) no fueron factores de riesgo para DF. Conclusiones: El alto riesgo nutricional, la nutrición enteral tardía, el BH positivo y la ausencia de fisioterapia respiratoria antes del destete no fueron factores de riesgo para DF.

Background: Weaning of the mechanical ventilation (MV) is a complex process and when it fails, it can increase the risks of complications and expenses in health systems. Objective: To identify risk factors for failed weaning in adults with MV. Materials and Methods: Case-control study carried out in patients older than 18 years admitted to the intensive care unit of a tertiary care hospital. Cases were identified as failed weaning (FW) of MV, and controls were simple weaning. Patients from another hospital with MV were excluded. Risk factors studied were high nutritional risk by the modified Nutric score, late enteral nutrition, positive water balance (WB) and the absence of physical therapy prior to weaning. OR was calculated with a significance < 0.05 for bivariate, multivariate, and adjusted analysis. Results: 105 patients were included, 35 cases and 70 controls. The bivariate analysis found that high nutritional risk (OR = 2.5; 95% CI = 1.1 5.9; p = 0.027) was a risk factor, but the multivariate analysis did not confirm it. Late enteral nutrition (OR = 1.2; 95% CI = 0.4 3.4), positive WB (OR = 0.7; 95% CI = 0.3 1.7) and the absence of respiratory physiotherapy (OR = 0.2; 95% CI = 0.004 0.011) were not risk factors for FW. Conclusions: High nutritional risk, late enteral nutrition, positive BH and the absence of respiratory physiotherapy before weaning were not risk factors for FW.

Germination fitness of two temperate epiphytic ferns shifts under increasing temperatures and forest fragmentation.

Ferns are an important component of ecosystems around the world. Studies of the impacts that global changes may have on ferns are scarce, yet emerging studies indicate that some species may be particularly sensitive to climate change. The lack of research in this subject is much more aggravated in the case of epiphytes, and especially those that live under temperate climates. A mathematical model was developed for two temperate epiphytic ferns in order to predict potential impacts on spore germination kinetics, in response to different scenarios of global change, coming from increasing temperature and forest fragmentation. Our results show that an increasing temperature will have a negative impact over the populations of these temperate epiphytic ferns. Under unfragmented forests the germination percentage was comparatively less influenced than in fragmented patches. This study highlight that, in the long term, populations of the studied epiphytic temperate ferns may decline due to climate change. Overall, epiphytic fern communities will suffer changes in diversity, richness and dominance. Our study draws attention to the role of ferns in epiphytic communities of temperate forests, emphasizing the importance of considering these plants in any conservation strategy, specifically forest conservation. From a methodological point of view, the model we propose could be easily used to dynamically monitor the status of ecosystems, allowing the quick prediction of possible future scenarios, which is a crucial issue in biodiversity conservation decision-making.