Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. METHODS: Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. RESULTS: Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. CONCLUSION: HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

The effect of levofloxacin on the lung microbiota of laboratory rats.

Aim: The aim of this study was to investigate the short-term effect of levofloxacin on the microbiota of healthy lungs. Material and methods: Male F344 rats received either no levofloxacin (n = 9), intravenous levofloxacin (n = 12), oral levofloxacin (n = 12), or subcutaneous levofloxacin (n = 14). Rats received a clinically applicable dose (5.56 mg/kg) of levofloxacin via the assigned delivery route once daily for three days. On day four, lung tissue was collected and the lung microbiota composition was investigated using 16S ribosomal RNA gene sequencing. Results: Untreated lungs showed a microbiota dominated by bacteria of the genera Serratia. After treatment with levofloxacin, bacteria of the genus Pantoea dominated the lung microbiota. This was observed for all routes of antibiotic administration, with a significant difference compared to no-antibiotic control group (PERMANOVA: P < 0.001; homogeneity of dispersions: P = 0.656). Conclusion: Our study is the first to demonstrate the effects of levofloxacin therapy on lung microbiota in laboratory rats. Levofloxacin treatment by any route of administration leads to profound changes in the rat lung microbiota, resulting in the predominance of bacteria belonging to the genus Pantoea. Further studies regarding the role of long-term application of broad spectrum antibiotics on induction of lung, allergic and autoimmune diseases are indicated.

A mole rat's gut microbiota suggests selective influence of diet on microbial niche space and evolution.

IMPACT STATEMENT: The composition of the microbiota is of critical importance for health and disease, and is receiving increased scientific and medical scrutiny. Of particular interest is the role of changing diets as a function of agriculture and, perhaps to an even greater extent, modern food processing. To probe the connection between diet and the gut's microbial community, the microbiota from a mole rat, a rodent with a relatively unusual diet, was analyzed in detail, and the microbes found were compared with previously identified organisms. The results show evidence of an adaptive radiation of some microbial clades, but relative stability in others. This suggests that the microbiota, like the genome, carries with it housekeeping components as well as other components which can evolve rapidly when the environment changes. This study provides a very broad view of the niche space in the gut and how factors such as diet might influence that niche space.

Early Immune Response to Acute Gastric Fluid Aspiration in a Rat Model of Lung Transplantation.

OBJECTIVES: Chronic aspiration of gastric fluid contents can decrease long-term survival of pulmonary transplants due to development of obliterative bronchiolitis. However, little is known about the early immune response and the cascade of events involved in the development of obliterative bronchiolitis. MATERIALS AND METHODS: We utilized a rat orthotopic pulmonary transplant model and a single aspiration of either gastric fluid or normal saline to investigate the histologic, cellular, and cytokine changes associated with an acute gastric fluid aspiration event compared with normal saline at 2 and 10 days after aspiration. RESULTS: Our observations included a decrease in pulmonary compliance and increased airway inflammation and acute rejection of the transplanted lung, as well as increases in macrophages, granulocytes, and proinflammatory cytokines such as interleukin 1ß, transforming growth factor ß1 and ß2, and tumor necrosis factor α in bronchoalveolar lavage fluid from the transplanted lung of gastric fluid-aspirated rats compared with normal saline-aspirated rats. CONCLUSIONS: The acute inflammatory response observed in the present study is consistent with changes found in chronic models of aspiration-associated injury and suggests a potentially important role for mast cells in the development of obliterative bronchiolitis.

Temporary autonomic modulation with botulinum toxin type A to reduce atrial fibrillation after cardiac surgery.

BACKGROUND: Postoperative atrial fibrillation (POAF) frequently complicates cardiac surgery and is associated with worse outcomes. The cardiac autonomic nervous system is implicated in the pathogenesis of POAF. OBJECTIVE: The purpose of this study was to determine the efficacy and safety of selective cardiac autonomic modulation in preventing POAF. METHODS: In this randomized, double-blind, placebo-controlled trial, adults undergoing cardiac surgery were randomized 1:1 to intraoperative injection of 250 units onabotulinumtoxinA (botulinum toxin type A [BoNTA]) or placebo into epicardial fat pads. The study was powered to detect a 40% reduction in relative risk of POAF. Time to first episode of in-hospital POAF was the primary outcome, evaluated in patients receiving injection. Additionally, incidence of POAF, length of stay (LOS), and adverse events were examined. RESULTS: The trial assigned 145 patients to injection, 15 of whom were dropped before treatment, leaving 130 patients for analysis. Overall, 36.5% (23/63) of BoNTA-treated patients developed POAF compared with 47.8% (32/67) of placebo-treated patients. The time-to-event analysis revealed a hazard ratio of 0.69 (95% confidence interval 0.41-1.19; P = .18) for the BoNTA vs placebo arm. There were no significant differences in postoperative hospital LOS (median [interquartile range] 6.0 [3.4] vs 6.2 [3.7] days; P = .51) or adverse events prolonging LOS (27/63 [42.9%] vs 30/67 [44.8%]; P = .83) in patients receiving BoNTA vs placebo. CONCLUSION: Epicardial injection of onabotulinumtoxinA was without discernible adverse effects, but we failed to detect a significant difference in risk of POAF. Future large-scale studies of epicardial onabotulinumtoxinA injection as a potential POAF prevention strategy should be designed to study smaller, but clinically meaningful, treatment effects.

Effect of gastric fluid aspiration on the lung microbiota of laboratory rats.

AIM OF THE STUDY: The pulmonary microbiota is important for both normal homeostasis and the progression of disease, and may be affected by aspiration of gastric fluid. The aim of this study was to investigate changes in the lung microbiota induced by aspiration of gastric fluid in a laboratory rat model. MATERIAL AND METHODS: Using the intratracheal application method, male rats received aspiration with 0.9% normal saline (n = 11); gastric fluid (n = 24) or sterilized (gamma-irradiated) gastric fluid (n = 12) once-weekly for four weeks. On the fifth week, the animals were sacrificed, and the microbiota of the lung was assessed by 16S ribosomal RNA gene sequencing. RESULTS: Lungs without aspiration and lungs after aspiration with normal saline had similar microbial compositions, dominated by bacteria of the genera Serratia, Ralstonia and Brucella. Evaluation of the microbiota following aspiration of gastric fluid revealed a much different profile that was dominated by bacteria from the genera Romboutsia and Turicibacter and largely independent of sterilization of the gastric fluid. CONCLUSION: In a laboratory rat model, aspiration with gastric fluid caused a substantial shift of the lung microbiota that could be characterized as a shift from Proteobacteria towards Firmicutes, possibly of enteric origin. Bacteria contained in the gastric fluid are not apparently responsible for this change.

Murine model of oropharyngeal gastric fluid aspiration-A new assessment method for intrapulmonary liquid distribution using digital pixel calculation.

AIM OF THE STUDY: The aim of this study was to investigate a new method for visualization and quantification of intrapulmonary liquid distribution after oropharyngeal gastric fluid aspiration in mice. MATERIAL AND METHODS: Eleven mice received oropharyngeal aspiration with a gastric fluid, India ink, and saline solution. Digital imaging and pixel calculation were used to analyze intrapulmonary fluid distribution selectively. RESULTS: Digital pixel analysis and orophanryngeal aspiration are both safe techniques in mice and deliver reproducible/valid results. Analysis revealed an average aspirate distribution of 86.8% of the total lung area. The proportional amount of the left lung was significantly greater than that of the right lung (P = 0.023). The lobe with the lowest mean distribution was the right lower lobe (79.2% ± 4.4%). CONCLUSION: Digital pixel calculation is a reliable method for quantitative, macroscopic evaluation of fluid distribution in the lung. This method is a useful tool for training purposes and it can be used to ensure interinvestigator reproducibility.

Production and Use of Hymenolepis diminuta Cysticercoids as Anti-Inflammatory Therapeutics.

Helminthic therapy has shown considerable promise as a means of alleviating some inflammatory diseases that have proven resistant to pharmaceutical intervention. However, research in the field has been limited by a lack of availability to clinician scientists of a helminth that is relatively benign, non-communicable, affordable, and effectively treats disease. Previous socio-medical studies have found that some individuals self-treating with helminths to alleviate various diseases are using the rat tapeworm (cysticercoid developmental stage of Hymenolepis diminuta; HDC). In this study, we describe the production and use of HDCs in a manner that is based on reports from individuals self-treating with helminths, individuals producing helminths for self-treatment, and physicians monitoring patients that are self-treating. The helminth may fit the criteria needed by clinical scientists for clinical trials, and the methodology is apparently feasible for any medical center to reproduce. It is hoped that future clinical trials using this organism may shed light on the potential for helminthic therapy to alleviate inflammatory diseases. Further, it is hoped that studies with HDCs may provide a stepping stone toward population-wide restoration of the biota of the human body, potentially reversing the inflammatory consequences of biota depletion that currently affect Western society.

The role of oxidative stress, inflammation and acetaminophen exposure from birth to early childhood in the induction of autism.

The wide range of factors associated with the induction of autism is invariably linked with either inflammation or oxidative stress, and sometimes both. The use of acetaminophen in babies and young children may be much more strongly associated with autism than its use during pregnancy, perhaps because of well-known deficiencies in the metabolic breakdown of pharmaceuticals during early development. Thus, one explanation for the increased prevalence of autism is that increased exposure to acetaminophen, exacerbated by inflammation and oxidative stress, is neurotoxic in babies and small children. This view mandates extreme urgency in probing the long-term effects of acetaminophen use in babies and the possibility that many cases of infantile autism may actually be induced by acetaminophen exposure shortly after birth.

Clearance of bile and trypsin in rat lungs following aspiration of human gastric fluid.

PURPOSE: In the clinical setting, there is no reliable tool for diagnosing gastric aspiration. A potential way of diagnosing gastric fluid aspiration entails bronchoalveolar lavage (BAL) with subsequent examination of the BAL fluid for gastric fluid components that are exogenous to the lungs. The objective of this study was to determine the longevity of the gastric fluid components bile and trypsin in the lung, in order to provide an estimate of the time frame in which assessment of these components in the BAL might effectively be used as a measure of aspiration. MATERIALS AND METHODS: Human gastric fluid (0.5 mg/kg) was infused in the right lung of intubated male Fischer 344 rats (n = 30). Animals were sacrificed at specified times following the experimentally induced aspiration, and bronchoalveolar lavage fluid (BALF) was collected. Bile concentrations were analyzed by an enzyme-linked chromatogenic method, and the concentration of trypsin was quantified using an ELISA. Data were analyzed using non-linear regression and a one-phase decay equation. RESULTS: In this experimental model, the half-life of bile was 9.3 hours (r(2) = 0.81), and the half-life of trypsin was 9.0 hours (r(2) = 0.68). CONCLUSIONS: The half-lives of bile and trypsin in the rodent aspiration model suggest that the ability to detect aspiration may be limited to a few days post-aspiration. If studies using rats are any indication, it may be most effective to collect BAL samples within the first 24 hours of suspected aspiration events in order to detect aspiration.

The role of soluble and insoluble gastric fluid components in the pathogenesis of obliterative bronchiolitis in rat lung allografts.

Repetitive gastric fluid aspirations have been shown to lead to obliterans bronchiolitis (OB), but the component or components of gastric fluid that are responsible are unknown. This study investigates the role of particulates and, separately, soluble material in gastric fluid during the development of OB. Whole gastric fluid (WGF) was collected from male Fischer 344 (F344) rats and separated by centrifugation into particle reduced gastric fluid (PRGF) and particulate components resuspended in normal saline (PNS). Orthotopic left lung transplants from male Wistar-Kyoto rats into F344 rats were performed using a modification of the nonsuture external cuff technique with prolonged cold ischemia. Rats were subjected to weekly aspiration of 0.5 ml/kg of WGF (n = 9), PRGF (n = 10), PNS (n = 9), or normal saline (control, NS; n = 9) for 8 weeks following transplantation. Lung allografts treated with WGF, PRGF, or PNS developed a significantly greater percentage of OB-like lesions compared with the control. No statistical difference was observed when comparing the fibrosis grades or the percentage of OB lesions of WGF, PRGF, and PNS groups, suggesting that both soluble and insoluble components of gastric fluid can promote the development of aspiration-induced OB and fibrosis in lung allografts.

Epigenetic profiling identifies novel genes for ascending aortic aneurysm formation with bicuspid aortic valves.

BACKGROUND:   Bicuspid aortic valves predispose to ascending aortic aneurysms, but the mechanisms underlying this aortopathy remain incompletely characterized.  We sought to identify epigenetic pathways predisposing to aneurysm formation in bicuspid patients. METHODS:   Ascending aortic aneurysm tissue samples were collected at the time of aortic replacement in subjects with bicuspid and trileaflet aortic valves.  Genome-wide DNA methylation status was determined on DNA from tissue using the Illumina 450K methylation chip, and gene expression was profiled on the same samples using Illumina Whole-Genome DASL arrays.  Gene methylation and expression were compared between bicuspid and trileaflet individuals using an unadjusted Wilcoxon rank sum test.   RESULTS:   Twenty-seven probes in 9 genes showed significant differential methylation and expression (P<5.5x10-4).  The top gene was protein tyrosine phosphatase, non-receptor type 22 (PTPN22), which was hypermethylated (delta beta range: +15.4 to +16.0%) and underexpressed (log 2 gene expression intensity: bicuspid 5.1 vs. trileaflet 7.9, P=2x10-5) in bicuspid patients, as compared to tricuspid patients.  Numerous genes involved in cardiovascular development were also differentially methylated, but not differentially expressed, including ACTA2 (4 probes, delta beta range:  -10.0 to -22.9%), which when mutated causes the syndrome of familial thoracic aortic aneurysms and dissections CONCLUSIONS:   Using an integrated, unbiased genomic approach, we have identified novel genes associated with ascending aortic aneurysms in patients with bicuspid aortic valves, modulated through epigenetic mechanisms.  The top gene was PTPN22, which is involved in T-cell receptor signaling and associated with various immune disorders.  These differences highlight novel potential mechanisms of aneurysm development in the bicuspid population.

Increased biodiversity in the environment improves the humoral response of rats.

Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of "natural" antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.

An assessment of human gastric fluid composition as a function of PPI usage.

The standard of care for chronic gastro-esophageal reflux disease (GERD), which affects up to 40% of the population, is the use of drugs such as proton pump inhibitors (PPIs) that block the production of stomach acid. Despite widespread use, the effects of PPIs on gastric fluid remain poorly characterized. In this study, gastric fluid was collected from patients undergoing cardiac surgery who were not (n = 40) or were (n = 25) actively taking PPIs. Various enzymatic and immunoassays as well as mass spectrometry were utilized to analyze the concentrations of bile, gastricsin, trypsin, and pepsin in the gastric fluid. Proteomic analyses by mass spectrometry suggested that degradation of trypsin at low pH might account, at least in part, for the observation that patients taking PPIs have a greater likelihood of having high concentrations of trypsin in their gastric fluid. In general, the concentrations of all analytes evaluated varied over several orders of magnitude, covering a minimum of a 2000-fold range (gastricsin) and a maximum of a 1 × 10(6) -fold range (trypsin). Furthermore, the concentrations of various analytes were poorly correlated with one another in the samples. For example, trypsin and bile concentrations showed a significant (P < 0.0001) but not strong correlation (r = 0.54). Finally, direct assessment of bacterial concentrations by flow cytometry revealed that PPIs did not cause a profound increase in microbial load in the gastric fluid. These results further delineate the profound effects that PPI usage has on the physiology of the stomach.