Mouse Models of Nonalcoholic Steatohepatitis: Head-to-Head Comparison of Dietary Models and Impact on Inflammation and Animal Welfare.

A variety of dietary nonalcoholic steatohepatitis (NASH) mouse models are available, and choosing the appropriate mouse model is one of the most important steps in the design of NASH studies. In addition to the histopathological and metabolic findings of NASH, a sufficient mouse model should guarantee a robust clinical status and good animal welfare. Three different NASH diets, a high-fat diet (HFD60), a western diet (WD), and a cafeteria diet (CAFD), were fed for 12 or 16 weeks. Metabolic assessment was conducted at baseline and before scheduled sacrifice, and liver inflammation was analyzed via fluorescence-associated cell sorting and histopathological examination. Clinical health conditions were scored weekly to assess the impact on animal welfare. The HFD60 and WD were identified as suitable NASH mouse models without a significant strain on animal welfare. Furthermore, the progression of inflammation and liver fibrosis was associated with a decreased proportion of CD3+ NK1.1+ cells. The WD represents a model of advanced-stage NASH, and the HFD60 is a strong model of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. However, the CAFD should not be considered a NASH model.

A Newly Established Murine Cell Line as a Model for Hepatocellular Cancer in Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has become a major risk factor for hepatocellular cancer (HCC) due to the worldwide increasing prevalence of obesity. However, the pathophysiology of NASH and its progression to HCC is incompletely understood. Thus, the aim of this study was to generate a model specific NASH-derived HCC cell line. A murine NASH-HCC model was conducted and the obtained cancer cells (N-HCC25) were investigated towards chromosomal aberrations, the expression of cell type-specific markers, dependency on nutrients, and functional importance of mTOR. N-HCC25 exhibited several chromosomal aberrations as compared to healthy hepatocytes. Hepatocytic (HNF4), EMT (Twist, Snail), and cancer stem cell markers (CD44, EpCAM, CK19, Sox9) were simultaneously expressed in these cells. Proliferation highly depended on the supply of glucose and FBS, but not glutamine. Treatment with a second generation mTOR inhibitor (KU-0063794) resulted in a strong decrease of cell growth in a dose-dependent manner. In contrast, a first generation mTOR inhibitor (Everolimus) only slightly reduced cell proliferation. Cell cycle analyses revealed that the observed growth reduction was most likely due to G1/G0 cell cycle arrest. These results indicate that N-HCC25 is a highly proliferative HCC cell line from a NASH background, which might serve as a suitable in vitro model for future investigations of NASH-derived HCC.

Biomechanical analyses of prosthetic mesh repair in a hiatal hernia model.

Recurrence rate of hiatal hernia can be reduced with prosthetic mesh repair; however, type and shape of the mesh are still a matter of controversy. The purpose of this study was to investigate the biomechanical properties of four conventional meshes: pure polypropylene mesh (PP-P), polypropylene/poliglecaprone mesh (PP-U), polyvinylidenefluoride/polypropylene mesh (PVDF-I), and pure polyvinylidenefluoride mesh (PVDF-S). Meshes were tested either in warp direction (parallel to production direction) or perpendicular to the warp direction. A Zwick testing machine was used to measure elasticity and effective porosity of the textile probes. Stretching of the meshes in warp direction required forces that were up to 85-fold higher than the same elongation in perpendicular direction. Stretch stress led to loss of effective porosity in most meshes, except for PVDF-S. Biomechanical impact of the mesh was additionally evaluated in a hiatal hernia model. The different meshes were used either as rectangular patches or as circular meshes. Circular meshes led to a significant reinforcement of the hiatus, largely unaffected by the orientation of the warp fibers. In contrast, rectangular meshes provided a significant reinforcement only when warp fibers ran perpendicular to the crura. Anisotropic elasticity of prosthetic meshes should therefore be considered in hiatal closure with rectangular patches.

Cytoprotective Actions of FTY720 Modulate Severe Preservation Reperfusion Injury in Rat Renal Transplants.

BACKGROUND: Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia. METHODS: Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-beta, and tumor necrosis factor-alpha expression. Rat proximal tubular cells were incubated with 0.1 to 30 micromol/L of phosphorylated FTY720 to test for in vitro cytopathic effects. RESULTS: FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3 and ED1 infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-alpha and transforming growth factor-beta expression. In vitro, pharmacologic concentrations up to 1 micromol/L of phosphorylated FTY720 did not affect tubular cell viability. CONCLUSION: FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.

Pharmacodynamic evaluation of the first dose of mycophenolate mofetil before kidney transplantation.

BACKGROUND AND OBJECTIVES: The effect of mycophenolate mofetil (MMF) on T cell function has not been evaluated in patients undergoing kidney transplantation. The aim of this study was to assess the effect of 1g of MMF on T cell function, that is, intralymphocyte cytokine expression, T cell activation (CD25 and CD71), and T cell proliferation, as well as inosine monophosphate dehydrogenase (IMPDH) activity, to better understand the relationship between pharmacokinetic and pharmacodynamic markers in patients receiving the first dose of MMF before kidney transplantation. PATIENTS: Twenty-four patients undergoing a kidney transplantation from a living donor were enrolled in this study. RESULTS: Compared with baseline (before MMF intake), T cell proliferation (93%), IMPDH activity (74%), CD25 (46%), and CD71 (50%) expression significantly decreased during the first hour after MMF intake, in parallel to the rise in MPA concentration. Thereafter, all pharmacodynamic markers, except IMPDH activity, returned back to baseline level. There was a complex inverse relationship between pharmacokinetic and pharmacodynamic markers. The inhibition of T cell proliferation was highly correlated to IMPDH activity, but also to T cell activation markers. CONCLUSION: The administration of MMF to patients is associated not only with a dramatic decrease in both T cell proliferation and IMPDH activity, but also with in a decrease in CD25 and CD71 expression.

Tabebuia avellanedae extracts inhibit IL-2-independent T-lymphocyte activation and proliferation.

In order to identify new, immune modulating compounds, aqueous extracts of plants pre-selected on ethno-pharmacological knowledge were screened for inhibitory effects in an anti-CD3 driven lymphocyte proliferation assay (MTT-assay). We found for the extract of the inner bark of Tabebuia avellanedae (Tabebuia) dose dependent and reproducible inhibitory effects on lymphocyte proliferation. We further analyzed Tabebuia in flow cytometry based whole blood T-cell function assays. We found that Tabebuia inhibited dose dependent ConA stimulated T-cell proliferation. Decreased T-lymphocyte proliferation was associated with dose dependent reduction of CD25 and CD71 expression on T-lymphocytes. In contrast Tabebuia exerted no effects on cytokine expression (Il-2 and TNF-alpha) by PMA/Ionomycin stimulated T-lymphocytes. Concentrations of Tabebuia used were not toxic for lymphocytes as verified by trypan blue exclusion assay. Further experiments showed that the immune inhibitory effects by Tabebuia were not mediated by its pharmacological lead compound beta-lapachone and only observed in aqueous but not in ethanol plant extracts.

Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans.

Pharmacodynamic monitoring (PD) can evaluate the efficacy of immunosuppressive drug therapies. In this study, the expressions of PD biomarkers [lymphocyte proliferation, CD25 and CD71 expression, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha) synthesis] were determined in whole-blood assays and were validated for their application in PD of immune modulators in future clinical trials. Initially, the assay conditions were re-evaluated. The measurement of T-lymphocyte proliferation and activation marker expression in whole-blood cultures resulted in optimized stimulation for 72 hours with 7.5 microg/mL concavalin A. Intracellular cytokine expression of CD3+ T-cells received optimized stimulation for 4 hours with 15 ng/mL phorbol 12-myristate 13-acetate and 0.75 microg/mL ionomycin. Statistical assay parameters (intra-assay, intra-individual, and interindividual variabilities) were determined. It was found that blood storage for up to 24 hours is possible without any change in biomarker expression. Dosage effects of immunosuppressive drugs (tacrolimus, cyclosporin A, sirolimus, mycophenolic acid, and methylprednisolone) were evaluated in vitro and the assay was applied successfully to dialysis, renal transplant, and liver transplant patients. We conclude that these biomarkers used in whole-blood assays are suitable for PD of immune modulators in clinical trials.