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Abgesehen von histopathologischen, allerdings eingeschrankt reproduzierbaren Merkmalen sind keine Kriterien bekannt, die die Entwicklung einer Myelofibrose (MF) als typische Komplikation myeloproliferativer Neoplasien (MPN; chronische myeloproliferative Erkrankungen nach alter Nomenklatur) in fruhen noch unverfaserten Stadien vorhersagen. Altersassoziierte klonale Hamatopoese („age-related clonal hematopoiesis“, ARCH, oder „clonal hematopoiesis of indetermined potential“, CHIP) wird ab dem 65. Lebensjahr in bis zu 10 % dieser Alterskohorte angetroffen. ARCH wurde kombiniert mit typischen Treibermutationen (JAK2, MPL und CALR) bei MPN beschrieben. Um die Auswirkung von ARCH auf das Fibroserisiko von primarer Myelofibrose (PMF) im unverfaserten Fruhstadium (MF 0) zu ermitteln, wurden in dieser Studie 77 Falle von fruher PMF mit spaterer Entwicklung einer Myelofibrose (MF 2 und 3) im Verlauf (Median 6,2 Jahre) hinsichtlich ihres Mutationsstatus untersucht. Als Vergleichskollektiv dienten PMF-Falle (n = 27) ohne Entwicklung einer Myelofibrose mit einer medianen Beobachtungszeit von 7,2 Jahren. Die haufigsten ARCH-Mutationen, die Gene TET2, ASXL1 und DNMT3A betreffend, zeigten dabei keine Korrelation mit dem fibrotischen Progress, wenn sie bereits in der initialen Biopsie bestanden. Andere, eher beim myelodysplastischen Syndrom (MDS) und nicht bei ARCH anzutreffende Mutationen der Hamatopoese wie SRSF2, U2AF1, SF3B1, IDH1/2 und EZH2 fanden sich bei 24,7 % der prafibrotischen PMF-Falle mit spaterem fibrotischen Progress, aber in keinem der prafibrotischen PMF-Falle ohne Fibroseentwicklung (p = 0,0028). Aus diesen Befunden ergibt sich, dass die haufigen ARCH/CHIP-Mutationen TET2, ASXL1 und DNMT3A kein gesteigertes Fibroserisiko bei PMF im unverfaserten Fruhstadium bewirken, wahrend die MDS- und Nicht-ARCH-assoziierten Mutationen SRSF2, U2AF1, SF3B1, IDH1/2 und EZH2 einen signifikanten Risikofaktor fur den schnellen Progress in eine MF (Median 2 Jahre) darstellen.

Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed, paraffin-embedded trephines are frequently used in hematologic diagnostic tests and provide relevant access to leukemic cells for further studies, for example, phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. Sixty-three formalin-fixed, paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia, and myeloproliferative neoplasms were analyzed for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next-generation sequencing approach. Fusion transcripts relevant for diagnosis and therapy could be detected and validated (by RT-PCR) in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose, and only one sample failed in the RNA analysis (1.6%). This study concludes that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p

Nonalcoholic fatty liver disease (NAFLD) is a growing concern in the aging population with human immunodeficiency virus (HIV). Screening for NAFLD is recommended in patients with metabolic risk factors or unexplained transaminitis. This study aimed to prospectively assess the prevalence and associated factors of liver steatosis and advanced fibrosis (AF) in HIV-monoinfected patients at risk of NAFLD.We conducted a multicenter study in HIV-monoinfected patients, nonexcessive drinkers with metabolic syndrome, and/or persistently elevated liver enzymes, and/or clinical lipodystrophy. All participants had magnetic resonance imaging proton density fat fraction (MRI-PDFF), Fibroscan/controlled attenuation parameter (CAP), and cytokine and genetic analysis.From March 2014 to November 2015, we enrolled 442 participants and analyzed 402: male (85%); median age, 55 years (interquartile range [IQR], 50-61 years); body mass index, 27.0 kg/mIn a large cohort of HIV-moninfected patients at risk of NAFLD, steatosis is present in two-thirds of cases, and around 10% have AF. The CAP technique is accurate for screening steatosis in this population.

The priming of T cells in the liver is widely accepted. Nonetheless, it is controversial whether immune activation in autoimmune hepatitis (AIH) occurs in the liver or in the spleen. To address this issue, we splenectomized mice and induced experimental murine AIH (emAIH) with an adenovirus (Ad)-expressing formiminotransferase cyclodeaminase (FTCD). Post-splenectomy, the experimental mice developed emAIH to a higher extent than the control mice. In addition, splenectomized mice harboured more intrahepatic B cells and a disproportionately small number of regulatory T cells (Tregs) within a reduced T cell population at the site of inflammation. These results imply that the spleen is not the site of AIH induction. In contrast, the spleen seems to have a protective function since the pathological score was more severe in splenectomized animals. These findings have important implications for the aetiology of AIH.

Background and Aims Sporadic hepatitis E is an emerging indigenous disease in Europe induced by genotype 3 of the virus. While the disease takes an acute self‐limited course in immunocompetent individuals, under immunocompromised conditions chronic hepatitis E might develop. The histology of chronic hepatitis E has not been described in detail systematically. Methods Liver biopsies from 19 immunosuppressed patients with chronic hepatitis E were collected: 17 were organ transplant recipients, one had a CD4‐deficiency and one had received steroid therapy because of ulcerative colitis. Biopsies were processed with standard stains. Evaluation of histologic activity and fibrosis was performed according to Ishak. Additionally, immunohistochemistry with antibodies directed against open reading frame 2 and 3 of the virus was performed and liver biopsies were tested for hepatitis E virus RNA. Results Biochemical data showed an increase in alanine transaminase, aspartate transaminase, gamma‐glutamyl transferase and total bilirubin. Histopathology displayed typical features of chronic hepatitis with mild to moderate activity. The number of polymorphonuclear leucocytes was considerably increased and all patients had a florid cholangitis that presented as a destructive form in five of them. Hepatocytes and bile duct epithelia stained positive for hepatitis E virus by immunohistochemistry. Conclusions Chronic hepatitis E in immunocompromised individuals runs a similar course as hepatitis B and C and shows similar histopathology. However, the presence of destructive cholangitis in some cases accompanied by an increased number of polymorphonuclear leucocytes is markedly different. Immunohistochemically the virus is present in bile duct epithelia, seemingly the cause for cholangitis.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High-fat high-carbohydrate (HF-HC) diet feeding of NASH-resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)-deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF-HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen-driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1−/− mice was substantially worsened and accompanied by a sharp increase of M1-like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti-cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it. OA Förderung 2022

Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires life-long immunosuppression. Frequent relapses after discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current therapies. As steroid therapy preferentially depletes intrahepatic regulatory T cell (Tregs), immune regulation might be re-established by increasing and functionally strengthening intrahepatic Tregs. In recent clinical trials with low dose IL-2, the Treg compartment was strengthened in autoimmune diseases. Therefore, we tested complexed IL-2/anti-IL-2 to increase the selectivity for Tregs. We used our model of experimental murine AIH (emAIH) and treated the mice with complexed IL-2/anti-Il-2 in the late course of the disease. The mice showed increased intrahepatic and systemic Treg numbers after treatment and a reduction in activated, intrahepatic effector T cells (Teffs). This resulted in a reduction in liver-specific ALT levels and a molecular pattern similar to that of healthy individuals. In conclusion, complexed IL-2/anti-IL-2 restored the balance between Tregs and Teffs within the liver, thereby improving the course of emAIH. Treg-specific IL-2 augmentation offers new hope for reestablishing immune tolerance in patients with AIH.

Objectives Myeloproliferative neoplasms (MPN) comprising Poycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) follow a bi-phasic course of disease with fibrotic and/or blastic progression. At presentation in the chronic phase, currently there are only insufficient tools to predict the risk of progression in individual cases. Methods In this study chronic phase MPN (16 PMF, 11 PV, and 11 MPN unclassified) with blastic transformation during course of disease (n = 38, median follow-up 5.3 years) were analyzed by high-throughput sequencing. MPN cases with a comparable follow-up period and without evidence of blast increase served as control (n = 63, median follow-up 5.8 years). Results Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) found at presentation were not significantly associated with blastic transformation. By contrast, mutations of SRSF2, U2AF1, and IDH1/2 at first presentation were exclusively observed in the progression cohort (13/38, 34.2%) and were completely missing in the control group without blast transformation during follow-up (P = 0.0007 for SRSF2; P = 0.0063 for U2AF1 and IDH1/2). Conclusion Unlike frequent ARCH/CHIP alterations (TET2, ASXL1, DNMT3A) mutations in SRSF2, IDH1/2, and U2AF1 when manifest already at first presentation provide an independent risk factor for blast transformation of MPN.

Aim Acute liver failure (ALF) is a syndrome of severe liver injury that may need urgent liver transplantation and is associated with significant risk of death. Early outcome prediction and further possibilities to increase accuracy of prognosis scores are important. Methods We examined 30 patients with ALF, according to the novel criteria of the Intractable Hepato-Biliary Diseases Study Group, who underwent transjugular liver biopsy (TJLB) and investigated the relevance of histology for correct diagnosis and etiology. We assessed the suitability of necrosis (%), hepatic venous pressure gradients (HVPG), and hepatocentral venous gradients of serum biomarkers for outcome prediction. For this purpose, we calculated the difference of biomarker levels between hepatic vein (HV) and superior vena cava (SVC) blood samples. Results Histology of TJLB specimens contributed to finding the etiology in 83%. Necrosis (%) and HVPGs were not significantly different between outcome groups. In gradient measurements, caspase 3/7 activity and total cytokeratin 18 (CK-18) (M65) had significant and relevant levels different from zero. Although they were not accurate for outcome prediction, differences between outcome groups were detected in caspase activation: levels of caspase 3/7 activity in the HV and caspase-cleaved CK-18 (M30) in the SVC were significantly higher in spontaneously recovered patients. Conclusions Our results underline the role of caspase activation in spontaneous recovery from ALF. Furthermore, the calculation of hepatocentral venous biomarker gradients could represent a new diagnostic tool whose clinical potential needs to be further investigated.

Background Chronic viral hepatitis is linked to fibrotic liver injury that can progress to liver cirrhosis with its associated complications. Recent evidence suggests a role of senescence in liver fibrosis, although the senescence regulators contributing to fibrosis progression remain unclear. Aim To investigate the role of senescence and different senescence markers for fibrosis progression in patients with chronic hepatitis C virus (HCV) infection. Methods The expression of the cell cycle inhibitors p21, p27 and p16 as well as the senescence markers p-HP1γ and γ-H2AX was analysed in liver tissue with different fibrosis stages. Senescence-associated chitotriosidase activity was measured in sera of HCV patients (n = 61) and age-matched healthy individuals (n = 22). Results We found a remarkable up-regulation of the cell cycle inhibitors and senescence markers in chronic HCV infection compared to healthy liver tissue. Liver tissue with relevant fibrosis stages (F2-3) or cirrhosis (F4) revealed a significant increase in senescent cells compared to livers with no or minimal fibrosis (F0-1). In cirrhotic livers, a significantly higher number of p-HP1γ, p21 and p27 positive cells was detected compared to liver tissue with F2-3 fibrosis. Importantly, we identified T-cells as the dominant cell type contributing to increased senescence during fibrosis progression. Compared to healthy individuals, serum chitotriosidase was significantly elevated and correlated with histological fibrosis stages and liver stiffness as assessed by transient elastography. Conclusions Senescence of hepatic T-cells is enhanced in chronic viral hepatitis and increases with fibrosis progression. Serological detection of senescence-associated chitotriosidase might allow for the non-invasive detection of relevant fibrosis stages.

BACKGROUND Percutaneous liver biopsy is an established diagnostic procedure for the assessment of liver pathologies. Limited data are available on the clinical impact of liver biopsies in liver transplant recipients. MATERIAL AND METHODS Liver transplant recipients undergoing liver biopsy between 2000 and 2013 were analyzed. Demographic characteristics and transplantation data were extracted from the transplantation database. RESULTS A total of 496 liver biopsies were performed in 312 patients. The main biopsy indications were suspected rejection (66%, 327/496), protocol biopsy (22%, 108/496), and suspected recurrence of the primary disease (7%, 34/496). Histological findings showed acute cellular rejection in 36% (179/496), idiopathic chronic hepatitis in 28% (141/496), and normal histology in 11% (54/496). Liver biopsies in patients with clinically suspected rejection showed histological findings compatible with acute or chronic rejection in 46% (151/327). In 41% (205/496) of the patients, the immunosuppressive therapy was adjusted due to the biopsy result. For alanine-aminotransferase and bilirubin, significant differences were detected between baseline and week 4 and 12 after treatment modification (p

Besides histopathological findings there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPN). Age-related clonal hematopoiesis (ARCH/CHIP) is a frequent finding in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared with prefibrotic PMF samples without development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, and DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations which are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of the tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when manifest already at first presentation.

An association of human leukocyte antigen (HLA) class II alleles with autoimmune conditions is increasingly being used for diagnostic purposes. The aim of our study was to examine whether HLA class II alleles in pediatric-onset autoimmune liver disease (pAILD) may serve as diagnostic markers and if they correlate with clinical outcome parameters. HLA-DRB1 alleles of 76 children with pAILD (autoimmune hepatitis [AIH], autoimmune sclerosing cholangitis [AISC], primary sclerosing cholangitis [PSC]) and of 50 healthy blood donors as control group were analyzed retrospectively. Diagnosis of these patients was confirmed by the autoimmune hepatitis score including liver histology, which has been re-evaluated by a blinded liver pathologist, and by bile duct imaging, as appropriate. Our results showed significant association of HLA-DRB1*03 with AIH1 and AISC with 82 % specificity for AIH. For pAILD (excluding AIH2), HLA-DRB1*03 homozygosity had specificity of 98 %, whereas sensitivity is low. Remission in HLA-DRB1*03-positive patients appears to be less likely. HLA-DRB1*13 is significantly associated with PSC and also with AIH1.HLA-DRB1 alleles provide supportive information for diagnostic workup in patients with liver disease, but they were not suitable for differentiation within pAILD. Their prognostic value could be helpful but needs to be evaluated further.• HLA-DRB1*03 is NOT associated with pediatric AIH in a previous national study. • In other studies, HLA-DRB1*03 is associated with AIH1. • HLA-DRB1*13 is associated with PSC. • HLA-DRB1*04 is described as protective for AILD. What is New: • HLA-DRB1 four-digit typing for all alleles and for all subgroups of pAILD combined with re-assessment of liver histology • HLA-DRB1*03:01 is associated with pediatric AIH1 and AISC. • HLA-DRB1*03:01 appears to be a prognostic marker. • HLA-DRB1*13:01 is associated with pediatric AIH1 in mixed ethnicity cohort. • HLA-DRB1*04 does not show any protective effect for pAILD.

Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations of the autoimmune regulator (AIRE) gene. Mouse studies have shown that this results in defective negative selection of T cells and defective early seeding of peripheral organs with regulatory T cells (Tregs). Aire deficiency in humans and mice manifests as spontaneous autoimmunity against multiple organs, and 20% of patients develop an autoimmune hepatitis (AIH). To study AIH in APS-1, we generated a murine model of human AIH on a BALB/c mouse background, in which Aire is truncated at exon 2. A subgroup of 24% of mice is affected by AIH, characterized by lymphoplasmacytic and periportal hepatic infiltrates, autoantibodies, elevated aminotransferases, and a chronic and progressive course of disease. Disease manifestation was dependent on specific Aire mutations and the genetic background of the mice. Though intrahepatic Treg numbers were increased and hyperproliferative, the intrahepatic CD4/CD8 ratio was decreased. The targets of the adaptive autoimmune response were polyspecific and not focussed on essential autoantigens, as described for other APS-1-related autoimmune diseases. The AIH could be treated with prednisolone or adoptive transfer of polyspecific Tregs. Conclusion: Development of AIH in APS-1 is dependent on specific Aire mutations and genetic background genes. Autoimmune response is polyspecific and can be controlled by steroids or transfer with Tregs. This might enable new treatment options for patients with AIH. (Hepatology 2015;61:1295–1305)

Although autoimmune hepatitis (AIH) can be treated with corticosteroid-based first-line therapy, incomplete remission is associated with progressive liver fibrosis. So far accepted predictors of the subsequent treatment response of AIH patients are lacking. Therefore, we analysed baseline parameters, including iron homeostasis and cytokine levels, in 60 children with paediatric AIH (pAIH). In contrast to adults, elevated serum markers indicating iron overload were not commonly found in children. Therefore, ferritin was not predictive of the treatment response in pAIH. Although baseline immunoglobulins were lower in pAIH children with subsequent complete biochemical remission (BR) upon standard first-line therapy, only lower AIH scores (≤16 points) could predict BR upon standard therapy in our training and validation cohorts. Additionally, higher baseline IL-2 and MCP-1/CCL2 levels were associated with BR in a sub-cohort. A combined score of IL-2 level and a simplified AIH score predicted treatment response more precisely than both parameter alone in this sub-cohort. In conclusion, the baseline AIH score could be validated as a predictor of treatment response in pAIH. Additionally, low baseline IL-2 may help identify children who need salvage therapy. This could be important because the use of low-dose IL-2 therapies is being tested in various autoimmune diseases.

Regulatory T cells (Tregs) play an important role in controlling alloreactivity after solid organ transplantation, but they may also impair antiviral immunity. We hypothesized that the Treg frequency and the Treg phenotype are altered in hepatitis C virus (HCV)–infected recipients of liver transplantation (LT) with possible prognostic implications. Tregs from 141 individuals, including healthy individuals, LT recipients with or without persistent HCV infections, and nontransplant patients with chronic HCV, were studied. A comprehensive phenotypic analysis was performed with multicolor flow cytometry, which included standard Treg markers [CD4+, CD25hi, CD127–, and FoxP3+ in addition to HLA DR, CCR7, CD45RA, CD62L, CD49d, CD39, ICOS and LAP‐TGFβ stainings. Healthy individuals and LT patients displayed similar Treg frequencies and largely comparable Treg phenotypes, which were stable over time after transplantation. In contrast, Tregs with a CD45RA–CCR7– effector phenotype were enriched in LT recipients with chronic HCV versus HCV‐negative transplant patients. HCV infection, rather than LT, altered the expression of functional markers on Tregs. A principal component analysis revealed distinct Treg phenotypes in HCV‐infected LT recipients with rejection and patients with recurrent graft HCV. In conclusion, Treg phenotypes are altered in HCV‐infected LT patients. An investigation of Tregs may possibly help to distinguish recurrent HCV from graft rejection. Further functional studies are needed to define the role of Tregs in determining the balance between antiviral and allogenic immunity. Liver Transpl 21:381–395, 2015. © 2015 AASLD.

Fibrotic progression in Polycythemia vera is associated with early concomitant driver-mutations besides JAK2

Fine needle aspiration (FNA) is a sensitive and specific method (95%), often helpful in characterizing suspected liver lesions. It is appropriate to distinguish between primary and secondary liver neoplasia. Moreover, in most cases, the use of cell block preparations of small specimens allows immunocytochemical evaluation to determine the nature of the primary tumour. In a retrospective study at Hannover Medical School (MHH) from 1998 to 2012 (14 years), 4,136 sonographically guided FNAs were performed. The patients provided consent and the study protocol was approved by the local ethics committee. There were 39.6% malignant and 57.5% benign lesions in the liver, while 2.8% of the cases were undetermined. FNA was non-representative in 1.1% of the cases. The diagnostic utility of highly differentiated hepatocellular carcinoma (HCC; G1) remains difficult; cell bridges with cell atypia are pathognomonic for diagnosis. Ancillary techniques and immunocytochemical investigations will increase the sensitivity and specificity, particularly by using the cell block technique.

Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%-90% of patients can be treated with a life-long immunosuppression. Unfortunately, there are strong drug-related side effects and steroid-refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self-limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver-specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T-cell tolerance against hepatic self-antigens was also broken and CD4+ T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. Conclusion: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression. (Hepatology 2013;58:718–728)

Background & aims The autoimmune hepatitis (AIH) is a chronic hepatitis driven by the adaptive immunity that affects all age groups. A functional and numerical regulatory T cell (Treg) defect has been reported in pediatric AIH (pAIH), while an intrahepatic increase in adult AIH (aAIH) patients has been detected in current research findings. Methods Therefore, we quantified the intrahepatic numbers of Treg, T and B cells, as well as serum cytokine levels before and during therapy in pAIH. Results We found a disproportional intrahepatic enrichment of Tregs in untreated pAIH compared to pediatric non-alcoholic fatty liver disease. The increase of Treg/total T cells was even more pronounced than in aAIH due to fewer infiltrating T and B cells. Portal densities of Treg, as well as total T and B cells, declined significantly during therapy. However, portal Treg densities decreased disproportionately, leading to even decreasing ratios of Treg to T and B cells during therapy. Out of 28 serum cytokines IL-2 showed the strongest (10fold) decrease under therapy. This decline of IL-2 was associated with decreasing intrahepatic Treg numbers under therapy. None of the baseline T and B cell infiltration parameters were associated with the subsequent treatment response in pAIH. Conclusions Intrahepatic Tregs are rather enriched in untreated pAIH. The disproportional decrease of Tregs during therapy may be caused by a decrease of IL-2 levels. New therapies should, therefore, aim in strengthening intrahepatic immune regulation.

For the development of autoimmune hepatitis (AIH), genetic predisposition and environmental triggers are of major importance. Although experimental AIH can be induced in genetically susceptible mice, the low precursor frequency of autoreactive T cells hampers a deeper analysis of liver-specific T cells. Here, we established a system where the model antigen hemagglutinin (HA) is expressed exclusively in hepatocytes of Rosa26-HA mice following administration of a replication deficient adenovirus expressing Cre recombinase (Ad-Cre). Under these conditions, hepatocytes mimic the generation of altered-self neoantigens. To follow autoreactive T cells during AIH, we adoptively transferred HA-­specific Cl4-TCR and 6.5-TCR T cells into Ad-Cre infected ­Rosa26-HA mice. Alternatively, Rosa26-HA mice have been crossed with TCR transgenic mice that were infected with Ad-Cre to break hepatic tolerance and induce the expression of the HA antigen as a hepatic self-antigen. Surprisingly, neither adoptive transfer nor a very high precursor frequency of autoreactive T cells was able to break tolerance in the context of adenoviral infection. The low proliferation of the antigen experienced autoreactive T cells despite the presence of the autoantigen and inflammation points to anergy as a potential tolerance mechanism. This model underscores the crucial importance of genetic susceptibility to break tolerance against hepatic autoantigens.

Background: Iron overload is a common problem in patients with primary myelofibrosis and anemia due to transfusion dependency. This results in organ damage and toxic effects on hematopoietic cells in the bone marrow. At present, iron chelation therapy is not recommended in patients with myeloproliferative syndromes. Case Report: We describe a very interesting development in a patient with primary myelofibrosis receiving iron chelation. Transfusion independency and a nearly complete histological remission of the underlying disease occurred within a few weeks of therapy. In addition, a change in molecular genetic findings was observed. Initially a JAK2 and a U2AF1 mutation were detected in the core biopsy. During and after therapy the U2AF1 mutation progressed, whereas the JAK2 mutation could no longer be verified. Conclusion: The improvement in hematopoiesis might results from reduction of oxidative stress on hematopoietic progenitor cells or other unclear deferasirox-mediated effects, whereas the reason for the change in molecular genetic findings is unclear. It appears that deferasirox might have a modulating effect on JAK2-kinase mutations. However, further investigation of selective molecular suppression properties of deferasirox are warranted.

Zu den myeloproliferativen Neoplasien (chronische myeloproliferative Erkrankungen nach fruherer Nomenklatur) zahlen die chronische myeloische Leukamie, die Polycythamia vera, die essenzielle Thrombozythamie, die primare Myelofibrose, die chronische Eosinophilenleukamie, die chronische Neutrophilenleukamie und die systemische Mastozytose. Allen ist gemeinsam, dass eine oder mehrere hamatopoetische Differenzierungslinien exzessiv vermehrt sind und dass sie mit unterschiedlichen Wahrscheinlichkeiten in eine Blastenkrise oder Knochenmarkfibrose ubergehen konnen. Ferner sind bei allen Tyrosinkinasen und assoziierte Signalwege von Mutationen betroffen (BCR-ABL, JAK2V617F, MPLW515L/K, KITD816V und FIP1L1-PDGFRA), welche die gesteigerte Proliferation auslosen. Diese Mutationen sind diagnostisch wie therapeutisch von groser Bedeutung, da sie reaktive Myeloproliferationen ausschliesen, mit unterschiedlicher Spezifitat zur Subtypisierung beitragen und zumindest z. T. medikamentos gezielt zu hemmen sind. Die molekularen Mechanismen der blastaren und fibrotischen Progression sind noch unbekannt.

Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme-linked immunosorbent assay (ELISA), which detects a caspase-cleaved cytokeratin-18 (CK-18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase-cleaved and uncleaved CK-18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls. Conclusion: Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis. (HEPATOLOGY 2012)

Outcome of patients with hepatocellular carcinoma is influenced by their histological grade. Invasive biopsy of the lesions is the gold standard in this regard.We therefore analysed the diagnostic accuracy of contrast enhanced ultrasound for non-invasive grading of hypervascular hepatocellular carcinoma in liver cirrhosis.According to the tumour perfusion kinetics on contrast enhanced ultrasound two grading groups were prospectively defined: well-differentiated hepatocellular carcinoma (US-G1) and higher grade hepatocellular carcinoma (US-G2/G3). Immediately after contrast enhanced ultrasound-grading, biopsies of hepatocellular carcinoma-lesions (n=95, 1.2-12.5 cm) were obtained and analysed for tumour grading (G). Descriptive statistics, sensitivity, specificity positive and negative predictive values, diagnostic likelihood ratios and interoperator reproducibility were calculated (κ).Histologically 77 (81.1%) patients had G2-G3 and 18 (18.9%) had G1 tumours. Higher grade hepatocellular carcinoma showed more often a washout in the portal or late phase (p0.0001). The sensitivity, specificity, positive predictive values and negative predictive values of contrast enhanced ultrasound for grading of hepatocellular carcinoma for all patients were 94% (CI: 72-99%), 95% (CI: 88-99%), 81% and 99% and for patients with tumours5 cm 100%(95% CI: 79-100), 96% (95% CI: 80-99), 92% and 100%. Positive and negative diagnostic likelihood ratios' were 18 and 26 and 0.06 and 0, respectively. κ=0.941 (p0.001).Contrast enhanced ultrasound has a high diagnostic value and reproducibility for non-invasive grading of hypervascular hepatocellular carcinoma1cm in patients with liver cirrhosis.

SRSF2 and U2AF1 mutations in primary myelofibrosis are associated with JAK2 and MPL but not calreticulin mutation and may independently reoccur after allogeneic stem cell transplantation

Hepatitis E is an infectious disease caused by the hepatitis E virus (HEV). Hepatitis E is mainly a self-limiting travel-associated disease without chronic evolution. In recent years an increasing number of authochtonous HEV-infections has been described in industrialized countries. HEV-infections frequently take a mild clinical silent course of disease in immunocompetent individuals and thus HEV infection is largely underdiagnosed. Therefore, the anti-HEV-prevalence is much higher than anticipated with 2 - 20% in Western Europe. HEV genotype 3 infections must be considered as zoonotic infections with several animals including swines serving as reservoirs. Cases of HEV-transmissions by blood transfusion were described not only in Asia but also in France and the UK. HEV-infections may take severe courses in pregnant women and patients with chronic liver diseases, sometimes leading to acute liver failure. In addition several centres described cases of progressive chronic HEV-infection in organ transplant recipients and HIV-positive patients during the last two years. In this review we summarize the current state-of-the-art on the knowledge of HEV-infections in industrialised countries.

Background & Aims The quantifiable level of HBsAg has been suggested as a predictor of treatment response in chronic hepatitis B. However, there is limited information on HBsAg levels considering the dynamic natural course of HBV-infection. This study aimed to determine HBsAg levels in the different phases of HBV-infection in European HBsAg-positive patients. Methods 226 HBV-monoinfected patients, not undergoing antiviral therapy, were analyzed in a cross-sectional study. Patients were categorized according to the phase of HBV-infection: HBeAg(+) immune tolerance phase (IT, n =30), immune clearance phase (IC, n =48), HBeAg(−) low-replicative phase (LR, n =68), HBeAg(−) hepatitis (ENH, n =68), and acute hepatitis B ( n =12). HBsAg was quantified and correlated with HBV-DNA, HBV-genotypes and clinical parameters. In addition, 30 LR-patients were followed longitudinally. Results HBsAg levels were higher in IT-patients and IC-patients compared to LR-patients and ENH-patients (4.96/4.37/3.09/3.87-log 10 IU/ml, p 0.001). HBsAg showed a strong correlation with HBV-DNA during acute hepatitis B ( R =0.79, p 0.01). Correlation of HBsAg and HBV-DNA was weak or missing when analyzing different phases of persistent HBV-infection separately. However, associations between HBsAg and HBV-DNA were observed in patients infected with HBV-genotype D but not with HBV-genotype A. LR-patients with HBV-reactivation during follow-up (increase of HBV-DNA >2000IU/ml) showed >3-fold higher baseline HBsAg levels with a NPV of 95% for an HBsAg cut-off of 3500IU/ml. Conclusions HBsAg levels show significant differences during the natural course of HBV-infection and between HBV-genotypes. These findings may have important implications for understanding the natural history of HBV-infection and for using quantitative HBsAg as a diagnostic tool, i.e. as a marker for predicting HBV-reactivation.

Hepatitis E virus (HEV) infection induces self-limiting liver disease in immunocompetent individuals. Cases of chronic hepatitis E have recently been identified in organ transplant recipients. We questioned if chronic hepatitis E plays a role in graft hepatitis after liver transplantation in a low endemic area. Two hundred twenty-six liver transplant recipients, 129 nontransplanted patients with chronic liver disease, and 108 healthy controls were tested for HEV antibodies. HEV RNA was investigated in all sera from transplanted patients. HEV antibodies were detected in 1 healthy control (1%), 4 patients with chronic liver disease (3%), and 10 liver transplant recipients (4%). Three liver transplant patients also tested positive for HEV RNA. Two of them developed persistent viremia with HEV genotype 3. The patients were anti-HEV immunoglobulin G-negative and HEV RNA-negative before transplantation and had an episode of acute hepatitis 5 or 7 months after transplantation, which led to advanced liver fibrosis after 22 months in 1 patient. Seroconversion to anti-HEV occurred not before 4 months after the first detection of HEV RNA. The possibility of reverse zoonotic transmission was experimentally confirmed by the infection of 5 pigs with a patient's serum. The pigs showed histological inflammation in the liver, and HEV RNA was detectable in different organs, including muscle. In conclusion, the prevalence of HEV infection in Central European liver transplant recipients is low; however, chronic hepatitis E may occur and needs to be considered in the differential diagnosis of graft hepatitis. The diagnosis of HEV infection should be based on HEV RNA determination in immunosuppressed patients. We suggest that immunocompromised individuals should avoid eating uncooked meat and contact with possibly HEV-infected animals.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor beta-1 (TGFbeta-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (Por = 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFbeta-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFbeta-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFbeta-1.

The suppressor of cytokine signalling-1 (SOCS-1) is a negative regulator of signal transduction mediated by cytoplasmic tyrosine kinases such as the Janus kinases (JAKs). We investigated SOCS-1 expression in bone marrow cells from Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD) and normal haematopoiesis (n=121), and additionally in peripheral blood samples (n=18). Except for chronic idiopathic myelofibrosis harbouring wild-type JAK2, other Ph(-) CMPD expressed significantly higher SOCS-1 levels of up to 14-fold compared to the control group (p0.001) independent of the JAK2 status. The mononuclear cell fraction but not granulocytes in patients with Polycythaemia vera also significantly overexpressed SOCS-1. We conclude that up-regulation of the SOCS-1 gene might reflect a compensatory feedback mechanism with different emphasis among Ph(-) CMPD subtypes independent of an underlying JAK2 (V617F) mutation.

Homing of allogeneic donor T cells to recipient tissue is imperative for the development of acute graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this study we show that alteration of T cell homing due to integrin-β7 deficiency on T cells or its ligand MAdCAM-1 in BMT recipients contributes to the pathophysiology of experimental GVHD. In contrast, lack of CC chemokine receptor 9 on donor T cells alters tissue homing but does not impact GVHD survival. We further demonstrate that MAdCAM-1 is aberrantly expressed in hepatic murine GVHD as well as in patients with active liver GVHD. However, infiltration of donor T cells in gut but not liver was dependent of MAdCAM-1 expression, indicating, that homing and/or retention of donor T cells rests on divergent molecular pathways depending on the GVHD target tissue.

Objectives: Friend leukemia integration- 1 (Fli-1), a member of the Ets gene family of transcription factors, has been demonstrated to be a target of a leukaemia inducing virus in mice, and is known to be part of a fusion gene in Ewings' sarcoma in humans. Wild-type Fli-1 is involved in lineage commitment of megakaryocytes and myeloid progenitors through induction of Janus kinases (JAKs) following ligand binding to cytokine and growth factor receptors. Proliferation of atypical megakaryocytes is a predominant histopathological feature in Philadelphia chromosome negative chronic myeloproliferative disorders (Ph - CMPD) and a potential aberrant expression of Fli-1 has not been investigated so far. Methods: Fli-1 expression was investigated by real-time RT-PCR and immunohistochemistry in bone marrow cells derived from Ph - CMPD (n = 80) and non-neoplastic haematopoiesis (n = 21) following determination of the JAK2 status. Results: Fli-1 mRNA expression was significantly higher in Essential thrombocythaemia (ET) with JAK2 (V617F) compared with other Ph - CMPD and control (P < 0.001). By immunohistochemistry, Fli-1 protein could be detected in nuclei of atypical megakaryocytes in Ph - CMPD and, less accentuated, in non-neoplastic megakaryocytes. Fli-1 protein expression by myeloid progenitors was considerably heterogenous in Ph - CMPD independent of an underlying JAK2 (V617F) mutation and without notable differences to non-neoplastic haematopoiesis. Conclusion: Fli-1 is rather constitutively expressed by bone marrow cells in Ph - CMPD independent of the underlying JAK2 status. The overall stronger labelling for Fli-1 in megakaryocytes in Ph - CMPD most likely reflects the degree of polyploidisation but aberrant activation of nuclear target genes can not be excluded.

Transforming growth factor β-1 (TGFβ-1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph− CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGFβ-1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGFβ-1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGFβ-1 expression in IMF and other Ph− CMPD, total bone marrow cells as well as laser-microdissected megakaryocytes were quantitatively analysed by real-time RT-PCR. OPG mRNA expression in fibrotic IMF was correlated with TGFβ-1 mRNA expression in a case-specific manner. Both OPG and TGFβ-1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGFβ-1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non-neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGFβ-1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGFβ-1 protein was predominantly expressed by the myeloid lineage in Ph− CMPD and non-neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGFβ-1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGFβ-1 expression occurs in megakaryocytes as well as myeloid cells in Ph− CMPD. TGFβ-1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non-fibrotic Ph− CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGFβ-1 expression. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.