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In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites.Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate.Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized.In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

SummaryThe term cirrhosis-associated immune dysfunction refers to the main syndromic abnormalities of immune function, immunodeficiency and systemic inflammation that are present in cirrhosis. The course of advanced cirrhosis, regardless of its aetiology, is complicated by cirrhosis-associated immune dysfunction and this constitutes the pathophysiological hallmark of an increased susceptibility to bacterial infection, distinctive of the disease. Cirrhosis impairs the homeostatic role of the liver in the systemic immune response. Damage to the reticulo-endothelial system compromises the immune surveillance function of the organ and the reduced hepatic synthesis of proteins, involved in innate immunity and pattern recognition, hinders the bactericidal ability of phagocytic cells. Systemic inflammation, in form of activated circulating immune cells and increased serum levels of pro-inflammatory cytokines, is the result of persistent episodic activation of circulating immune cells from damage-associated molecular patterns, released from necrotic liver cells and, as cirrhosis progresses, from pathogen-associated molecular patterns, released from the leaky gut. Cirrhosis-associated immune dysfunction phenotypes switch from predominantly “pro-inflammatory” to predominantly “immunodeficient” in patients with stable ascitic cirrhosis and in patients with severely decompensated cirrhosis and extra-hepatic organ failure (e.g. acute-on-chronic liver failure), respectively. These cirrhosis-associated immune dysfunction phenotypes represent the extremes of a spectrum of reversible dynamic events that take place during the course of cirrhosis. Systemic inflammation can affect the functions of tissue somatic cells and modify the clinical manifestation of cirrhosis. The best characterized example is the contribution of systemic inflammation to the haemodynamic derangement of cirrhosis, which correlates negatively with prognosis.

Depletion of circulating CD4(+) T-helper (Th) lymphocytes, especially naive Th cells, is common in cirrhosis. Little is known about the pathogenetic mechanisms involved in Th-cell depletion in cirrhosis. We investigated the mechanisms involved in circulating Th-cell lymphopenia in cirrhosis.Circulating naive and memory Th cells were analyzed by flow cytometry in 60 patients with cirrhosis and 40 sex- and age-matched healthy controls. Thymopoiesis, apoptosis, cell activation, and proliferation were assessed through CD31, annexin-V, HLA-DR and Ki-67 expression, respectively. Lipopolysaccharide (LPS)-binding protein (LBP) and spleen size were measured as indicators of bacterial translocation and splenic pooling, respectively.Compared to controls, patients showed reduced numbers of Th cells involving a greater depletion of the naive than memory Th-cell compartment (2.7- vs. 1.5-fold, respectively). Recent thymic emigrants were diminished (p0.01), and each patient had a lower number of CD31(+) naive Th cells than the matched-control. Spontaneous and induced apoptosis (Annexin-V(+)) of Th cells was increased in patients. Activated (HLA-DR(+)) and proliferating (Ki-67(+)) memory Th cells were increased in patients (p0.01), and they directly correlated with plasma LBP (p0.05) and negatively with naive Th cells (p0.01), respectively. Naive Th cells were inversely correlated (p0.01) with their frequencies of apoptosis and of activated memory Th cells, LBP, and spleen size. On multivariate analysis, defective thymic generation of naive Th cells, increased memory Th-cell activation, and splenomegaly were independently associated with Th-cell depletion.Th-cell immunodeficiency in cirrhosis is explained by a universal defect in thymopoiesis exacerbated by splenic pooling and activation-driven cell-death induced by bacterial translocation.

Cirrhosis with ascites is associated with a high rate of gut bacterial translocation (GBT) and spontaneous bacterial infections of enteric origin. We addressed the activation state and role of intestinal dendritic cells (DCs) in experimental ascitic cirrhosis and their relationship with GBT. Cirrhosis with ascites was CCl4 induced in rats. To examine their activation state and functions, DCs (CD103+RT1B+CD3−CD45RA−) were isolated from the intestinal lamina propria and mesenteric lymph nodes (MLNs), and the following parameters were determined by flow cytometry: surface antigen expression; spontaneous or lipopolysaccharide-stimulated tumor necrosis factor alpha (TNF-α) production; and in vitro capacity to phagocytose latex beads and to migrate toward the chemokine (C-C motif) ligand 21. GBT was defined as the growth of bacteria in MLNs culture. Bacterial DNA (Bact-DNA) in MLNs was identified by polymerase chain reaction. In rats with Bact-DNA in MLNs without GBT, intestinal and MLNs CD103+-DCs showed features of activation, expansion of the proinflammatory CD4+-DC subpopulation, augmented TNF-α production, and increased phagocytic and migratory capacities. In contrast, in rats with GBT, CD103+-DCs showed the absence of an activated phenotype, lowered TNF-α production, and relatively deficient phagocytosis and migration capacities. The CD103+-DC of rats without Bact-DNA in MLNs or GBT were similar to controls. In cirrhotic rats, bowel decontamination with antibiotics eliminated Bact-DNA in MLNs and GBT, normalized the activation state and functions of CD103+-DCs, and increased their TNF-α production. Conclusion: In experimental cirrhosis with ascites, continuous pressure of gut bacteria shapes the phenotypic and functional profile of intestinal DCs to produce effects that range from their activation and enhanced functions to their exhaustion and tolerance. (HEPATOLOGY 2012;56:1861–1869)

Systemic activation of the inflammatory immune system contributes to the progression of cirrhosis with ascites. Immune cells become activated after interacting at the mesenteric lymph nodes (MLNs) with bacteria translocated from the gut, and thereafter reach the bloodstream through recirculation. It is unknown whether systemic activation of the immune system is present in pre-ascitic cirrhosis, in which gut bacterial translocation has not been described. The purpose of this study was to determine whether systemic activation of the immune system initiates in rats with compensated carbon tetrachloride (CCl4)-induced cirrhosis, and if so to establish the activation site of immune cells. We studied the activation status of immune cells in peripheral blood, MLNs, and hepatic lymph nodes (HLNs). Systemic inflammation was present in rats with cirrhosis, as shown by expansion (P < 0.01) of circulating total and inflammatory monocytes and recently activated CD134+ T helper (Th) cells. The same populations of cells were increased (P < 0.01) in MLNs and HLNs. Bacterial translocation was absent in rats with cirrhosis or control rats, but bacterial DNA fragments were present in the MLNs of 54% of rats with cirrhosis. The liver was the source of activated immune cells present in the blood, as shown by the direct correlation between activated Th cells in the blood and HLNs, but not in MLNs, and the normalization by gut decontamination with antibiotics of activated cells in MLNs, but not in the blood or HLNs. Conclusion: In experimental cirrhosis, systemic activation of the immune system occurs before ascites development and is driven by recirculation of cells activated in HLNs. In addition, in compensated cirrhosis, bacterial DNA fragments reach the MLNs, where they elicit a local inflammatory response. (HEPATOLOGY 2010;52:2086-2095)

This paper presents results of numerical simulations made with a high-resolution multiscale four-dimensional data assimilation system applied over California during two episodes associated with high ozone concentrations in the San Joaquin Valley. The model used here is the nonhydrostatic Pennsylvania State University–National Center for Atmospheric Research Mesoscale Model (MM5). The focus of the paper is the objective validation of the regional (mesoalpha scale) meteorological results. The mulliscale data assimilation approach produces highly reliable simulations of the wind, temperature, mixed-layer depth, and moisture, each of which is vital to air quality modeling and a host of other mesoscale applications. The significance of this research is threefold. First, it is the first evaluation of this multiscale assimilation system in strongly heated summertime conditions and with comparatively fine grid resolution (4-km inner mesh). Second, the assimilation system has been extended so that tempera...

Visibility in Grand Canyon National Park has shown degradation during the last decade. The Penn State University/National Center for Atmospheric Research (PSU/NCAR) regional atmospheric model is ap...

Background An inflammatory immune system response ensues in the liver and in the systemic circulation in cirrhosis, where it contributes to hepatic fibrosis and peripheral vasodilation. Modulation of the inflammatory response without increasing susceptibility to infection is a therapeutic target in cirrhosis. AM3 is a low-toxicity biological response modifier with regulatory effects on innate and adaptative immunity, and the ability to normalise the production of tumour necrosis factor α (TNFα). Aims This was an experimental study to investigate the effects of oral AM3 on the systemic and hepatic inflammatory response, liver fibrosis and on the haemodynamic abnormalities of portal hypertension in rats with biliary cirrhosis. Design Bile-duct ligated rats received a 3-week oral course of AM3 or placebo. Results In cirrhotic rats, AM3 blunted the inflammatory switch of circulating and intrahepatic monocytes and T-cells to TNFα and interferon γ (IFNγ) production, respectively. AM3 modified the intrahepatic polarisation pattern of the regulatory cytokines, decreasing the mRNA expression of transforming growth factor β1 (TGFβ1), interleukin 4 (IL4), and IFNγ, and increasing that of IL10. Total and IFNγ-producing natural killer (NK) cells were lowered by AM3 in the peripheral blood and liver of cirrhotic rats. The immunomodulatory effects of AM3 led to reduced hepatic fibrogenesis in cirrhotic rats, as shown by decreased area of liver fibrosis, hydroxyproline content and mRNA expression of procollagen α1(I). Besides, AM3 lowered portal pressure and systemic hyperaemia. Conclusions The biological response modifier AM3 reverses the concurrent inflammatory immune system activation in peripheral blood and liver of experimental established cirrhosis, which results in reductions of hepatic fibrosis, portal pressure and peripheral vasodilation.

We read with interest the article by Wiest et al about spontaneous bacterial peritonitis guidelines.1 The authors point out the relevance of pathological bacterial translocation (BT) from the gut to mesenteric lymph nodes (MLN) as the pathophysiological hallmark of spontaneous bacterial peritonitis in cirrhosis, and the need of further research in the factors involved in BT. The study of BT, ascites and related immune system abnormalities in carbon tetrachloride (CCl4) cirrhosis is usually conducted in rats. This model has proved useful for research into the pathogenesis and role of the systemic pro-inflammatory response associated with cirrhosis and its link to BT.2–4 We recently showed that modulation of the immune system response in cirrhotic rats …

macrophages during conditions of excess iron by blocking the iron exporter ferroportin. In addition, IL-6 mediated upregulation of hepcidin during inflammation is the major mechanism of the socalled ‘anemia of chronic disease’. We here identify H2O2 which is co-released by inflammatory cells as potent transcriptional activator of hepcidin independent of upstream regulators IL-6 and BMP6. Material andmethods: H2O2 release (1–8mM) by immune cells was mimicked using purified enzymes glucose oxidase and catalase as recently described (GOX/CAT system). Huh7 hepatoma cells were treated with either IL-6 or BMP6 alone, or in combination with steady state H2O2 over 24h. Hepcidin regulation was assessed by quantitative real time PCR. Members of the intracellular signaling cascade such as STAT3 were assessed by Western blotting. Promotor studies were performed using hepcidin-promoter constructs with various deletions fused to luciferase as reporter gene with Renilla as control reporter gene. Results: Steady state non-toxic H2O2 concentrations comparable to those by inflammatory cells rapidly and drastically upregulate hepcidin in a dose-dependent way in Huh7 cells by a factor of 10. In addition, H2O2 further potentiates IL6 and BMP6 mediated upregulation of hepcidin 5-fold. Interestingly, induction was also not blunted by confounding hypoxia (2%). The H2O2mediated hepcidin response is an early response reaching the maximum mRNA-concentration already after 6h. Promoter studies identified the STAT3 element as major promoter region of H2O2 mediated hepcidin induction. Indeed, phosphorylation of STAT3 was confirmed under these conditions using Western blotting. Conclusions: Our studies establish H2O2 as as an important regulatory link between systemic iron regulation and oxidative stress. Impaired H2O2-hepcidin signaling in chronic liver diseases could contribute to hepatic accumulation of iron in cirrhotic patients.

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS L. Munoz, M. Ubeda, M.J. Borrero, M. Lario, R. Frances, D. Diaz, J. Monserrat, L. Lledo, J. Such, M. Alvarez-Mon, A. Albillos. University of Alcala, Ciberehd, Alcala de Henares, Madrid, Hospital General Universitario de Alicante, Ciberhed, Alicante, Hospital Universitario Pŕincipe de Asturias, University of Alcala, Ciberehd, Alcala de Henares, Hospital Universitario Ramon y Cajal, University of Alcala, Ciberehd, Madrid, Spain E-mail: leticia.munoz@uah.es

Peppler and Smith (1984) discussed truncation errors associated with second-order and fourth-order finite difference approximations used to calculate the geostrophic wind and relative vorticity. They found that these errors were, in general, smaller for longer wavelengths, finer-grid resolution, and fourth-order differencing. Second-order differencing produced fields that were numerically less than their corresponding analytic values and yielded errors which decreased with reduced grid interval. Fourth-order differencing decreased the errors when the grid interval was reduced, but only while the wavelength was ten limes or more greater than the grid interval. Results presented here indicate that the wind speed and Vorticity errors estimated by the fourth-order scheme decreased when the grid interval was decreased independent of wavelength. Comparison with Peppler and Smith's actual computations showed only one difference: in their finite-(difference equations the coefficients were truncated to th...

The effects of horizontal and vertical data resolution, data density, data location, different objective analysis algorithms, and measurement error on mesoscale-forecast accuracy are studied with observing-system simulation experiments. Domain-averaged errors are shown to generally decrease with time. It is found that the vertical distribution of error growth depends on the initial vertical distribution of the error itself. Larger gravity-inertia wave noise is produced in forecasts with coarser vertical data resolution. The use of a low vertical resolution observing system with three data levels leads to more forecast errors than moderate and high vertical resolution observing systems with 8 and 14 data levels. Also, with poor vertical resolution in soundings, the initial and forecast errors are not affected by the horizontal data resolution.