Stefan Lüth

Ectopic expression of neural autoantigen in mouse liver suppresses experimental autoimmune neuroinflammation by inducing antigen-specific Tregs

Tregs are important mediators of immune tolerance to self antigens, and it has been suggested that Treg inactivation may cause autoimmune disease. Therefore, immunotherapy approaches that aim to restore or expand autoantigen-specific Treg activity might be beneficial for the treatment of autoimmune disease. Here we report that Treg-mediated suppression of autoimmune disease can be achieved in vivo by taking advantage of the ability of the liver to promote immune tolerance. Expression of the neural autoantigen myelin basic protein (MBP) in the liver was accomplished stably in liver-specific MBP transgenic mice and transiently using gene transfer to liver cells in vivo. Such ectopic MBP expression induced protection from autoimmune neuroinflammation in a mouse model of multiple sclerosis. Protection from autoimmunity was mediated by MBP-specific CD4+CD25+Foxp3+ Tregs, as demonstrated by the ability of these cells to prevent disease when adoptively transferred into nontransgenic mice and to suppress conventional CD4+CD25- T cell proliferation after antigen-specific stimulation with MBP in vitro. The generation of MBP-specific CD4+CD25+Foxp3+ Tregs in vivo depended on expression of MBP in the liver, but not in skin, and occurred by TGF-beta-dependent peripheral conversion from conventional non-Tregs. Our findings indicate that autoantigen expression in the liver may generate autoantigen-specific Tregs. Thus, targeting of autoantigens to hepatocytes may be a novel approach to prevention or treatment of autoimmune diseases.

Serologic markers compared with liver biopsy for monitoring disease activity in autoimmune hepatitis.

Goals/Background Disease activity and response to treatment in autoimmune hepatitis is assessed best by liver biopsy, which does not suit for regular disease monitoring. It is frequent clinical practice to follow disease by assessment of serologic markers. Here, we assessed the diagnostic fidelity of this clinical practice. Study One hundred thirty-one biopsies from 82 patients with autoimmune hepatitis were analyzed for histologic activity. Serum samples, taken at the time of biopsy, were analyzed for aminotransferases [alanine aminotransferase (ALT), aspartate aminotransferase], IgG, and γ-globulin levels and compared with histology. Results All serum parameters were significantly associated with histologic activity (P<0.0075); ALT and IgG were most complementary. Presence of both elevated ALT and IgG were associated with high inflammatory activity (histologic activity scores ≥6) with 99% sensitivity. Elevation of either IgG or ALT was associated with residual inflammatory activity in almost all patients. Histologic remission is reliably indicated by normalization of both serum parameters, but about half of the patients with normal serum parameters still showed residual histologic activity of histologic activity index (HAI) 4 or 5. However, our patients with HAI scores 4 or 5 were at significantly lower risk of fibrosis progression than patients with scores ≥6 (P<0.02; odds ratio 14.2). Conclusions Histologic activity seems to be reliably indicated by elevated serum parameters. Normalization of serum parameters is not a reliable marker for complete histologic remission (HAI 1 to 3); however, normalized serum parameters identified patients at low risk of fibrosis progression. Thus, the common clinical practice of disease monitoring by serum markers seems to be suitable for regular follow-up.

Activin A Promotes the TGF-β-Induced Conversion of CD4+CD25-T Cells into Foxp3+ Induced Regulatory T Cells

TGF-β induces the conversion of CD4+CD25− T cells into CD4+CD25+Foxp3+ regulatory T cells (Treg). Activin A is a pleiotropic TGF-β family member and is expressed in response to inflammatory signals. In this study, we report on the effects of activin A on the conversion of CD4+CD25− T cells into Foxp3-expressing induced Treg (iTreg). Activin A was able to promote the conversion of CD4+CD25− T cells into iTreg in a dose-dependent manner in vitro. Activin A together with TGF-β1 had synergistic effects on the rate of iTreg conversion in vitro. Intact TGF-β1 signaling seemed to be essential for the effects of activin A on iTreg generation because cells overexpressing a dominant negative TGF-β type II receptor could not be converted by activin A in vitro. In vivo, the frequency of peripheral, but not central, Treg was increased in transgenic mice with elevated activin A serum levels and the in vivo conversion rate of CD4+CD25− T cells into Foxp3-expressing iTreg was increased as compared with wild type mice. These data suggest a role for activin A as a promoter of the TGF-β dependent conversion of CD4+CD25− T cells into iTreg in vitro and in vivo. Therefore, besides promoting inflammation, activin A may contribute to the regulation of inflammation via the expansion of peripheral Treg.

Delayed versus immediate treatment for patients with acute hepatitis C: a randomised controlled non-inferiority trial

BACKGROUND Early treatment of acute hepatitis C virus (HCV) infection with interferon alfa monotherapy is very effective, with cure rates of greater than 85%. However, spontaneous clearance of HCV occurs in 10-50% of cases. We aimed to assess an alternative treatment strategy of delayed antiviral therapy in patients who do not eliminate the virus spontaneously compared with immediate treatment. METHODS In our open-label phase 3 non-inferiority trial, we enrolled adults (≥18 years) with acute hepatitis C but no HIV or hepatitis B co-infection at 72 centres in Germany. We randomly allocated patients with symptomatic acute hepatitis C (1:1) to receive immediate pegylated interferon alfa-2b treatment for 24 weeks or delayed treatment with pegylated interferon alfa-2b plus ribavirin (for 24 weeks) starting 12 weeks after randomisation if HCV RNA remained positive. We used a computer-generated randomisation sequence and block sizes of eight, stratified by bilirubin concentration. We assigned all asymptomatic patients to immediate treatment with pegylated interferon alfa-2b for 24 weeks. The primary endpoint was sustained HCV RNA negativity in all randomly allocated participants who completed screening (intention-to-treat analysis), with a non-inferiority margin of 10%. For the primary analysis, we calculated the virological response of patients in the immediate and delayed treatment groups and an absolute risk difference stratified by bilirubin status. The trial was stopped early on advice from the study advisory committee because of slow recruitment of participants. This study is registered, number ISRCTN88729946. FINDINGS Between April, 2004, and February, 2010, we recruited 107 symptomatic and 25 asymptomatic patients. 37 (67%) of 55 symptomatic patients randomly allocated to receive immediate treatment and 28 (54%) of 52 symptomatic patients randomly allocated to receive delayed treatment had a sustained virological response (difference 13·7%, 95% CI -4·6 to 32·0; p=0·071). 18 (72%) of 25 asymptomatic patients had a sustained virological response. 22 (42%) of 52 symptomatic patients allocated to receive delayed treatment did not complete follow-up compared with 20 (25%) of 80 symptomatic or asymptomatic patients assigned immediate treatment (p=0·037). 11 symptomatic patients (21%) assigned delayed treatment had spontaneous HCV clearance. 14 patients who received delayed pegylated interferon alfa-2b plus ribavirin treatment and completed follow-up achieved sustained virological response. INTERPRETATION Delayed treatment is effective although not of equal efficacy to immediate treatment; coupled with the rate of spontaneous clearance it can reduce unnecessary treatment in closely monitored populations. Immediate treatment seems preferable in populations where loss to follow-up is great. FUNDING German Network of Competence on Viral Hepatitis (HepNet, funded by the German Federal Ministry of Education and Research, grants 01KI0102, 01KI0401, and 01KI0601), MSD, Schering-Plough.

Characteristics and long-term prognosis of the autoimmune hepatitis/primary sclerosing cholangitis overlap syndrome.

Goals/Background Diagnosis, treatment, and prognosis of the overlap syndrome of autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC) are controversial. Our aim was to assess the clinical characteristics and long-term prognosis of the AIH/PSC overlap syndrome. Study We reviewed the data of 16 patients seen in our center who fulfilled the diagnostic criteria of both diseases at some stage of their medical history. Results All patients had initially presented with laboratory markers of both, cholestasis and definite AIH. Histologic reexamination of initial biopsies, available from 11 of 16 patients, revealed features of both AIH and PSC in all biopsies. Cholangiography was performed at initial presentation in 9 of 16 patients and appeared normal in 6 of 9 patients. During follow-up cholangiography, an additional 11 patients developed pathologic characteristics of PSC. The age and sex distribution was typical for PSC. Immunosuppressive therapy improved biochemical markers; however, fibrosis was observed to progress in all patients during a median observation period of 12 years. Three patients initially presented with cirrhosis, 12 of 16 patients developed cirrhosis at the end of the observation period, and 3 developed complications of cirrhosis. Conclusions Overlap of AIH and PSC was detected most reliably on grounds of serologic markers and histology; early bile duct changes were often missed by endoscopic retrograde cholangiography. Immunosuppression combined with ursodeoxycholic acid seems to be beneficial, but cannot prevent long-term progression toward cirrhosis in the majority of patients.

Evaluation of F-Actin ELISA for the Diagnosis of Autoimmune Hepatitis

OBJECTIVE:Antibodies to F-actin have been proposed to increase specificity in the diagnosis of autoimmune hepatitis (AIH). We compared the diagnostic value of a new F-actin enzyme-linked immunosorbent assay (ELISA) with the current gold standard of detection of smooth muscle antibodies by indirect immunofluorescence (SMA-IFT).METHODS:Archived sera of 47 patients with SMA positive AIH were tested with the F-actin ELISA and SMA-IFT. Prospectively collected sera of 123 patients with various liver diseases, 35 of whom had AIH, were analyzed by both assays. Different cutoff limits were considered for the F-actin ELISA (increments between 20 and 60 ELISA units) and SMA-IFT (titers of 1:40, 1:80, and 1:160).RESULTS:The F-actin ELISA had a sensitivity of 100% to detect all of 47 SMA positive AIH sera and the value of the ELISA units correlated with that of SMA titers (p < 0.0001). In prospective analysis, F-actin ELISA showed a superior sensitivity (74% vs 34%) and a similar specificity (98% vs 99%) and positive predictive value (88% vs 92%), compared with SMA-IFT. Combining both assays improved neither sensitivity nor specificity.CONCLUSIONS:The new F-actin ELISA seems to be a useful diagnostic tool with similar specificity and superior sensitivity for the diagnosis of AIH, compared with standard SMA-IFT. Due to its simplicity and operator independency, the F-actin ELISA may become a preferred screening technique for detection of autoantibodies in patients with suspected AIH.

CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, provide technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off-target effects. Here, we identified cross-genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection.

P38 MAP Kinase Signaling Is Required for the Conversion of CD4+CD25− T Cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25− T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25− T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.

Non-invasive diagnosis and monitoring of liver fibrosis and cirrhosis

The accurate staging of liver fibrosis in chronic liver diseases, especially the early diagnosis of liver cirrhosis, is crucial for prognostic assessment of the course of the disease. The histological evaluation of a liver biopsy cylinder is still the gold standard in assessing the stage of liver fibrosis. However, liver biopsy is an invasive procedure and carries the risk of complications. This has to be balanced against the information benefit of liver histology. To overcome this, non-invasive tests were developed assessing liver fibrosis based on combinations of laboratory markers or techniques measuring liver elasticity. In this review the current impact of the non invasive methods is discussed and weighted against liver biopsy.

Control of organ transplant-associated graft-versus-host disease by activated host lymphocyte infusions.

Background. Prolonged persistence of donor-derived T cells after organ transplantation has been proposed to improve long-term allograft survival. However, surviving transplant-derived T cells are also able to mediate devastating graft-versus-host disease (GvHD). Currently, GvHD after organ transplantation is usually refractory to conventional therapy and the disease outcome fatal. Methods. Graft-reactive host T cells were generated ex vivo from a patient suffering from a severe and refractory liver–transplant-associated GvHD. To control GvHD, activated alloreactive host T cells were repetitively retransferred into the patient (activated host lymphocyte infusion [aHLI]). Results. Adoptive transfer of ex vivo activated alloreactive host T cells (aHLI) led to the control and complete resolution of severe GvHD without inducing allograft rejection. Conclusions. aHLI opens a novel therapeutic window to control solid-organ transplant-associated GvHD while preserving allograft integrity.