Jf Schlaak

Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo.

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1–5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130–Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6–sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6–sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.

Non‐alcoholic fatty liver disease progresses to hepatocellular carcinoma in the absence of apparent cirrhosis

Non‐alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome (MS). Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. We recruited 162 adults with HCC between February 2007 and March 2008, investigated the underlying etiologies and determined the prevalence of the MS and related features within each group. Patients with NAFLD/NASH‐associated HCC exhibited a higher prevalence of metabolic features (Type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) compared to non‐NAFLD/NASH‐HCC. Intriguingly, a significant number (41.7%; p < 0.005) of individuals with NAFLD/NASH‐HCC had no evidence of cirrhosis. Patients with alcohol‐induced liver disease also displayed many features (14/19, 73.7%) of the MS, although, in contrast to NAFLD/NASH‐HCC, alcohol‐associated HCC was highly associated with cirrhosis (95.0%; p = 0.064). NAFLD/NASH as the hepatic entity of the MS may itself pose a risk factor for HCC, even in the absence of cirrhosis. The MS may also promote development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in the MS may instigate early interventions against developing HCC.

Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge

BACKGROUND/AIMS Kupffer cells are involved in local immunoregulation in the liver by secretion of cytokines and direct cellular contact. They are able to influence the function of other liver cells, i.e. sinusoidal endothelial cells, Ito cells and hepatocytes. The three known major functions of Kupffer cells are clearance of endotoxin from the portal circulation, release of soluble mediators and presentation of antigen. METHODS Human Kupffer cells were isolated by collagenase perfusion followed by centrifugal elutriation and analyzed for cytokine secretion after 3 days in culture. RESULTS We found that freshly isolated human Kupffer cells secreted the anti-inflammatory cytokine interleukin-10 in response to stimulation with lipopolysaccharide. The release of interleukin-10 was maximal 12-24 h after lipopolysaccharide challenge. Furthermore, we could show that exogenous interleukin-10 downregulated the release of the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha by Kupffer cells after lipopolysaccharide stimulation. The release of interleukin-6 was maximal 24 h after stimulation and interleukin-10 inhibited interleukin-6 release after 6 h. Tumor necrosis factor alpha showed maximal secretion 6 h after stimulation and exogenous interleukin-10 also downregulated the tumor necrosis factor alpha release after 6 h. CONCLUSIONS Kupffer cells are exposed physiologically to lipopolysaccharide present in portal venous blood. Given the known anti-inflammatory effect of interleukin-10, our findings of secretion of interleukin-10 by Kupffer cells in response to lipopolysaccharide and suppression of interleukin-6 and tumor necrosis factor alpha release by Kupffer cells in vitro through exogenous interleukin-10 suggest an important role for interleukin-10 in the regulation of the local immune response in the liver sinusoid.

Randomised trial of mycophenolate mofetil versus azathioprine for treatment of chronic active Crohn's disease.

BACKGROUND Crohns disease is a chronic inflammatory disease of the alimentary tract. Azathioprine is an effective agent in the management of chronic active Crohns disease leading to long term remission of disease activity. Such treatment leads to limited efficacy or side effects in a small subset of patients. AIMS To compare efficacy and side effects of treatment with azathioprine plus corticosteroids versus mycophenolate mofetil (MMF) plus corticosteroids in patients with chronic active Crohns disease. METHODS Seventy patients with chronic active Crohns disease (Crohns disease activity index (CDAI) greater than 150) were randomised for treatment with azathioprine/cortisone or MMF/cortisone. Corticosteroid dosage was tapered according to a standard protocol. Disease activity was monitored by clinical scores after one, two, three, and six months. RESULTS Treatment of patients with moderately active (CDAI 150-300) Crohns disease with MMF/cortisone led to a significant reduction in clinical activity scores comparable to treatment with azathioprine/cortisone. Treatment of patients with highly active Crohns disease (CDAI greater than 300) with MMF/cortisone caused significant suppression of clinical activity earlier than azathioprine/cortisone treatment. Treatment with MMF/cortisone was associated with few adverse effects. CONCLUSION Treatment of chronic active Crohns disease with MMF plus cortisone appears to be effective and well tolerated and should be considered in patients allergic to azathioprine or in whom azathioprine has failed.

Mycophenolate Mofetil Versus Azathioprine in Patients With Chronic Active Ulcerative Colitis: A 12-Month Pilot Study

OBJECTIVE:Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology frequently requiring long-term therapy for control of symptoms and prevention of relapse. Azathioprine (AZA) has been shown to be effective and safe in the treatment of chronic active UC. However, the alternatives to treatment with AZA are limited. Our aim was to compare the efficacy and safety of treatment with mycophenolate mofetil (MMF)/prednisolone versus standard immunosuppressive treatment with azathioprine (AZA)/prednisolone in patients with chronic active UC.METHODS:The study was designed as an open comparison of MMF versus AZA. Twenty-four patients with active UC (Rachmilewitz score ≥6 points) were randomly assigned to the MMF (20 mg/kg)/prednisolone or AZA (2 mg/kg)/prednisolone group. The initial prednisolone dosage was 50 mg and was tapered according to a standard protocol. Treatment was scheduled for 1 yr.RESULTS:The rates of remission were higher in the AZA/prednisolone group (n = 12) than in the MMF/prednisolone group (n = 12) throughout the study. Remission rates were 92% versus 67% after 4 wk, 92% versus 67% after 3 months, 92% versus 67% after 6 months, 83% versus 78% after 9 months, and 100% versus 88% after 1 yr. The number of patients not requiring steroids was higher in the AZA/prednisolone group than in the MMF/prednisolone group. Moreover, in the AZA/prednisolone group no severe adverse events were recorded, whereas in the MMF/prednisolone group two severe adverse events were observed: one patient discontinued MMF after 6 months because of recurrent upper airway infections, and one patient exhibited a bacterial meningitis after 9 months.CONCLUSIONS:Treatment with AZA/prednisolone appears to be more effective and safe compared to MMF/prednisolone in patients with chronic active UC. MMF might be an alternative treatment for patients with contraindications to AZA. To further evaluate the effects of MMF in active UC, a placebo-controlled double-blinded study appears warranted.

Analysis of the in vitro cytokine production by liver-infiltrating T cells of patients with autoimmune hepatitis.

The pathogenic mechanisms underlying the development of autoimmune hepatitis (AIH) are still unclear. Since AIH is associated with the presence of various autoantibodics and certain HLA subtypes, it is likely that T and B cells play a major role in this disease. In this study we have determined the functional capacities of in vivo preactivated liver‐infiltrating T cells (LTC) from patients with AIH. As controls we used LTC from patients with non‐autoimmune hepatitis (non‐AIH). Our results show that preactivated LTC from patients with AIH predominantly (190/255 clones) reside in the CD4+ population, whereas LTC in non‐AIH are dominated by the CD8+ phenotype (148/254 clones). In view of this finding we have investigated the cytokine secretion patterns of 102 randomly chosen CD4+ T cell clones from six patients with AIH. As controls we have used 58 CD4+ LTC from 11 patients with non‐AIH. All clones were stimulated by lectin and irradiated accessory cells and subsequent cytokine production was evaluated. LTC from patients with AIH have a lower interfcron‐gamma (IFN‐γ)/IL‐4 ratio compared with LTC from non‐AIH. Although clones from some patients with AIH produced very high amounts of IL‐4 in vitro, this was not a constant finding. These results show that in two preactivated LTC from patients with AIH are mostly CD4+ T cells that produce more IL‐4 than IFN‐γ. In contrast. LTC from patients with non‐AIH are dominated by CD8+ and CD4+ T cells that produce significantly less IL‐4 than IFN‐γ. Thus, liver‐infiltrating T cells from patients with AIH and non‐AIH belong to different functional T cell subsets. This may have implications for the regulation of humoral and cellular immune responses in inflammatory liver disease.

Lipopolysaccharide‐induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon‐independent pathways

Our previous studies have shown that Toll‐like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non‐parenchymal liver cells and trigger the production of interferon (IFN) to inhibit hepatitis B virus replication in vivo and in vitro. However, little is known about TLR‐mediated cellular responses in primary hepatocytes. By the model of woodchuck hepatitis virus (WHV) infected primary woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI‐3k/Akt pathways in PWHs. Further, inhibitors of MAPK‐ERK and PI‐3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN‐ independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.

A practical data processing workflow for multi-OMICS projects☆

Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner. Application of the software is presented for the detection of novel biomarkers, their ranking and annotation with existing knowledge using the example of corresponding Transcriptomics and Proteomics data sets obtained from patients suffering from hepatocellular carcinoma. Additionally, a linear regression analysis of Transcriptomics vs. Proteomics data is presented and its performance assessed. It was shown, that for capturing profound relations between Transcriptomics and Proteomics data, a simple linear regression analysis is not sufficient and implementation and evaluation of alternative statistical approaches are needed. Additionally, the integration of multivariate variable selection and classification approaches is intended for further development of the software. Although this paper focuses only on the combination of data obtained from quantitative Proteomics and Transcriptomics experiments, several approaches and data integration steps are also applicable for other OMICS technologies. Keeping specific restrictions in mind the suggested workflow (or at least parts of it) may be used as a template for similar projects that make use of different high throughput techniques. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

The presence of high amounts of HBV-DNA in serum is associated with suppressed costimulatory effects of interleukin 12 on HBV-induced immune response

BACKGROUND/AIMS The aim of this study was to examine the influence of the viral load on costimulatory effects of rhIL-12 on the hepatitis B virus (HBV)-induced immune response. METHODS Peripheral blood mononuclear cells of HBsAg positive patients without cirrhosis were stimulated with HBsAg, HBcAg, preS1Ag and tetanus toxoid in the absence or presence of IL-12 (0.01, 0.1 and 1 ng/ml). Stimulation by alpha-CD3+alpha-CD28, pokeweed mitogen (PWM) and lipopolysaccharide (LPS) were used as controls. Then, proliferation and cytokine production were determined by 3H-thymidine uptake and ELISA after 72 h. The patients were divided into group 1 (n=21): HBV-DNA: not detectable, group 2 (n=13): HBV-DNA: <300 pg/ml, and group 3 (n= 10): HBV-DNA: >300 pg/ml. RESULTS After stimulation with only HBV antigens, the highest amounts of IL-10 were found in group 3, while interferon (IFN)-gamma was rarely detectable. After stimulation with IL-12 and HBV antigens, strong costimulatory effects on IFN-gamma production, as well as proliferation, were observed in all patients except individuals from group 3. With regard to antigen-unrelated stimulation, significantly lower amounts of LPS-induced IFN-gamma production and alpha-CD3+28 induced proliferative responses, but higher amounts of LPS-induced IL-10 were observed in group 3. CONCLUSIONS These data suggest that the presence of high amounts of HBV-DNA in serum is associated with suppressed co-stimulatory and regulatory effects of IL-12 on the immune response to HBV antigens. This may be one explanation for the poor response to immunostimulating therapy in patients with a high viral load.

Interleukin 12 enhances deficient HCV‐antigen‐induced Th1‐type immune response of peripheral blood mononuclear cells

The aim of this study was to examine the possible immunomodulating effects of rhIL‐12 on the immune response induced by different hepatitis C virus (HCV) antigens. Freshly isolated peripheral blood mononuclear cells (PBMC) of 33 patients with chronic HCV infection were stimulated with optimal concentrations of antigens from the NS3, NS4, NS5, and core region of HCV in the absence or presence of interleukin12 (IL‐12). Stimulation by α‐CD3 + α‐CD28, lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were used as controls. Proliferation and cytokine production were determined by 3H‐thymidine uptake and enzyme‐linked immunosorbent assay (ELISA) after 72 hr. After stimulation with antigen or antigen + IL‐12, increased proliferation and production of interferon‐γ (IFNγ) and tumor necrosis factor‐α (TNFα) were observed in 23 of the 33 patients. Thus, a separation of the patients into HCV‐antigen/;IL‐12 responders (group 1, n = 23) and HCV‐antigen/;IL‐12 nonresponders (group 2, n = 10) was possible. Lower baseline IL‐12‐ and LPS‐induced IFNγ, TNFα, and IL‐12 production was observed in group 2 due to a possible dysfunction of accessory cells. Significant antigen‐induced Th2‐type cytokine (IL‐4, IL‐10, IL‐13) production was not found. According to clinical and serological parameters, group 2 comprised mostly patients with advanced liver disease. These data suggest an HCV‐related cellular immune defect in patients with hepatitis C that can be restored in most patients by IL‐12. J. Med. Virol. 56:112–117, 1998.