Screening HIV Patients at Risk for NAFLD Using MRI-PDFF and Transient Elastography: A European Multicenter Prospective Study.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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/m2 (IQR, 23.6-28.7 kg/m2); metabolic syndrome (67%); and CD4 cell count, 630/mm3 (IQR, 510-832/mm3). Overall 257 of 402 (64%) had NAFLD (MRI-PDFF ≥5%). Among them, 11.3% had a liver stiffness ≥9.6 kPa, suggestive of AF. Multivariable analysis identified 7 factors of steatosis: high CD4-cell count (odds ratio [OR], 4.04; 95% confidence interval [CI], 1.92-8.51), high leptin level (OR, 2.12; 95% CI, 1.14-3.93), non-CC PNPLA3s738409 genetic polymorphism (OR, 1.92; 95% CI, 1.11-3.33), low high-density lipoprotein (OR, 1.83; 95% CI, 1.03-3.27), high triglycerides (OR, 1.48; 95% CI, 1.18-1.84), elevated alanine transaminase (OR, 1.23; 95% CI, 1.16-1.31), and hyper ferritinemia (OR, 1.05; 95% CI, 1.03-1.07). Two factors were associated with AF: high body mass index (OR, 1.23 ; 95% CI, 1.07-1.42 ; P = .005, and elevated aspartate aminotransferase (OR, 1.03; 95% CI, 1.01-1.05; P = .001). Using MRI-PDFF as a reference, CAP (best cutoff, 280 dB/m) had good accuracy (area under the receiver operating characteristic curve = 0.86; 95% CI, 0.82-0.90) for the diagnosis of moderate to severe steatosis. CONCLUSIONS: In 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.

Splenectomy induces biochemical remission and regeneration in experimental murine autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. It is known that AIH originates not from the spleen but from the liver itself. Nonetheless, most details of the etiology and pathophysiology are unknown. We induced experimental murine AIH (emAIH) in NOD/Ltj mice by single administration of a replication-deficient adenovirus and performed splenectomy during late-stage disease. Biochemical disease remission occurred, which was characterized by improvement in transaminase levels. The causes of this remission included a shift in the transcriptomic signature of serum proteins toward regeneration. At the cellular level, there was a marked decrease in activated CD8+ T cells and an increase in intrahepatic regulatory T cells (Tregs). Here, intrahepatic Treg numbers correlated with biochemical remission. Notably, an imbalance in the T-cell/B-cell ratio was observed, with a disproportionate increase in total B cells. In summary, intrahepatic increases in Tregs, biochemical remission, and regeneration could be induced by splenectomy in the late stage of emAIH.

The Detrimental Role of Regulatory T Cells in Nonalcoholic Steatohepatitis.

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.

Inter-observer agreement for the histological diagnosis of invasive lobular breast carcinoma.

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 < 0.001). Agreement with the reference diagnosis was substantial in set A (median κ = 0.67, IQR: 0.57-0.75) and almost perfect in set B (median κ = 0.86, IQR: 0.73-0.93, p < 0.001). The median frequency of CDH1/E-cadherin mutations in specimens classified as ILC was 65% in set A (IQR: 56-72%) and 73% in set B (IQR: 65-75%, p < 0.001). Cases with variable subtype calls included E-cadherin-positive ILCs harboring CDH1 missense mutations, and E-cadherin-negative ILCs with tubular elements and focal P-cadherin expression. ILCs with trabecular growth pattern were often misclassified as non-lobular BC in set A but not in set B. In conclusion, subtyping of BC as ILC achieves almost perfect agreement with a pre-defined reference standard, if assessment is supported by E-cadherin IHC. CDH1 missense mutations associated with preserved E-cadherin protein expression, E- to P-cadherin switching in ILC with tubular elements, and trabecular ILC were identified as potential sources of discordant classification.

Anti-CD20 Therapy Alters the Protein Signature in Experimental Murine AIH, but Not Exclusively towards Regeneration.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires lifelong immunosuppression. Frequent recurrences after the discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current treatments. Studies of other autoimmune diseases suggest that temporary depletion of B cells can improve disease progression in the long term. METHODS: We tested a single administration of anti-CD20 antibodies to reduce B cells and the amount of IgG to induce intrahepatic immune tolerance. We used our experimental murine AIH (emAIH) model and treated the mice with anti-CD20 during the late stage of the disease. RESULTS: After treatment, the mice showed the expected reductions in B cells and serum IgGs, but no improvements in pathology. However, all treated animals showed a highly altered serum protein expression pattern, which was a balance between inflammation and regeneration. CONCLUSIONS: In conclusion, anti-CD20 therapy did not produce clinically measurable results because it triggered inflammation, as well as regeneration, at the proteomic level. This finding suggests that anti-CD20 is ineffective as a sole treatment for AIH or emAIH.

Genome-wide DNA methylation profiling is able to identify prefibrotic PMF cases at risk for progression to myelofibrosis.

BACKGROUND: Patients suffering from the BCR-ABL1-negative myeloproliferative disease prefibrotic primary myelofibrosis (pre-PMF) have a certain risk for progression to myelofibrosis. Accurate risk estimation for this fibrotic progression is of prognostic importance and clinically relevant. Commonly applied risk scores are based on clinical, cytogenetic, and genetic data but do not include epigenetic modifications. Therefore, we evaluated the assessment of genome-wide DNA methylation patterns for their ability to predict fibrotic progression in PMF patients. RESULTS: For this purpose, the DNA methylation profile was analyzed genome-wide in a training set of 22 bone marrow trephines from patients with either fibrotic progression (n = 12) or stable disease over several years (n = 10) using the 850 k EPIC array from Illumina. The DNA methylation classifier constructed from this data set was validated in an independently measured test set of additional 11 bone marrow trephines (7 with stable disease, 4 with fibrotic progress). Hierarchical clustering of methylation ß-values and linear discriminant classification yielded very good discrimination between both patient groups. By gene ontology analysis, the most differentially methylated CpG sites are primarily associated with genes involved in cell-cell and cell-matrix interactions. CONCLUSIONS: In conclusion, we could show that genome-wide DNA methylation profiling of bone marrow trephines is feasible under routine diagnostic conditions and, more importantly, is able to predict fibrotic progression in pre-fibrotic primary myelofibrosis with high accuracy.

Splenectomy Prior to Experimental Induction of Autoimmune Hepatitis Promotes More Severe Hepatic Inflammation, Production of IL-17 and Apoptosis.

Autoimmune hepatitis (AIH) is detected at a late stage in the course of the disease. Therefore, induction and etiology are largely unclear. It is controversial if the induction of autoimmunity occurs in the liver or in the spleen. In our experimental murine AIH model, the induction of autoimmunity did not occur in the spleen. Instead, a protective role of the spleen could be more likely. Therefore, we splenectomized mice followed by induction of experimental murine AIH. Splenectomized mice presented more severe portal inflammation. Furthermore, these mice had more IL-17, IL-23 receptor (IL-23R) and caspase 3 (casp3) and a decreased amount of erythropoietin in serum, while intrahepatic T cell compartments were unaffected. These results indicate that the spleen is not necessary for induction of AIH, and splenectomy disrupts the ability to immune regulate the intensity of hepatic inflammation, production of IL-17 and apoptosis.

Treg-specific IL-2 therapy can reestablish intrahepatic immune regulation in autoimmune hepatitis.

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.

Combination of myeloproliferative neoplasm driver gene activation with mutations of splice factor or epigenetic modifier genes increases risk of rapid blastic progression.

OBJECTIVES: Myeloproliferative neoplasms (MPN) comprising polycythemia 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 frequently observed in the progression cohort (13/38, 34.2%) and were completely missing in the control group without blast transformation during follow-up (P = .0007 for SRSF2; P = .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 rapid blast transformation of MPN.

Feasibility of Combined Detection of Gene Mutations and Fusion Transcripts in Bone Marrow Trephines from Leukemic Neoplasms.

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.

Mutations associated with age-related clonal hematopoiesis in PMF patients with rapid progression to myelofibrosis.

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.

Autoimmune hepatitis induction can occur in the liver.

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.

Chronic Hepatitis E is associated with cholangitis.

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.

Comprehensive mutation profiling and mRNA expression analysis in atypical chronic myeloid leukemia in comparison with chronic myelomonocytic leukemia.

Atypical chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML) represent two histologically and clinically overlapping myelodysplastic/myeloproliferative neoplasms. Also the mutational landscapes of both entities show congruencies. We analyzed and compared an aCML cohort (n = 26) and a CMML cohort (n = 59) by next-generation sequencing of 25 genes and by an nCounter approach for differential expression in 107 genes. Significant differences were found with regard to the mutation frequency of TET2, SETBP1, and CSF3R. Blast content of the bone marrow revealed an inverse correlation with the mutation status of SETBP1 in aCML and TET2 in CMML, respectively. By linear discriminant analysis, a mutation-based machine learning algorithm was generated which placed 19/26 aCML cases (73%) and 54/59 (92%) CMML cases into the correct category. After multiple correction, differential mRNA expression could be detected between both cohorts in a subset of genes (FLT3, CSF3R, and SETBP1 showed the strongest correlation). However, due to high variances in the mRNA expression, the potential utility for the clinic is limited. We conclude that a medium-sized NGS panel provides a valuable assistance for the correct classification of aCML and CMML.

Diagnostic Accuracy of Noninvasive Markers of Steatosis, NASH, and Liver Fibrosis in HIV-Monoinfected Individuals at Risk of Nonalcoholic Fatty Liver Disease (NAFLD): Results From the ECHAM Study.

BACKGROUND: HIV-monoinfected individuals are at high risk of nonalcoholic fatty liver disease. Noninvasive tests of steatosis, nonalcoholic steatohepatitis (NASH), and fibrosis have been poorly assessed in this population. Using liver biopsy (LB) as a reference, we assessed the accuracy of noninvasive methods for their respective diagnosis: magnetic resonance imaging proton-density-fat-fraction (MRI-PDFF), FibroScan/controlled attenuation parameter (CAP), and biochemical tests. METHODS: We enrolled antiretroviral therapy-controlled participants with persistently elevated transaminases and/or metabolic syndrome, and/or lipodystrophy. All had hepatic MRI-PDFF, FibroScan/CAP, FibroTest/NashTest/SteatoTest, APRI, FIB-4, and nonalcoholic fatty liver disease-fibrosis score. A LB was indicated if suspected significant fibrosis (FibroScan ≥7.1 kPa and/or FibroTest ≥0.49). Performance was considered as good if area under a receiver operating characteristic curves (AUROCs) was >0.80. RESULTS: Among the 140 patients with suspected significant fibrosis out of the 402 eligible patients, 49 had had a LB: median age of 54 years (53-65), body mass index: 26 kg/m (24-30), steatosis in 37 (76%), NASH in 23 (47%), and fibrosis in 31 (63%) patients [F2: 7 (14%); F3: 6 (12%); and F4: 2 (4%)]. Regarding steatosis, MRI-PDFF had excellent and CAP good performances with AUROCs at 0.98 (95% confidence interval: 0.96 to 1.00) and 0.88 (0.76 to 0.99), respectively, whereas the AUROCs of SteatoTest was 0.68 (0.51 to 0.85). Regarding fibrosis (≥F2), APRI and FIB-4 had good performance with AUROCs at 0.86 (0.74 to 0.98) and 0.81 (0.67 to 0.95). By contrast, FibroScan and FibroTest had poor AUROCs [0.61 (0.43 to 0.79) and 0.61 (0.44 to 0.78)], with very low specificity. Regarding NASH, alanine aminotransferase ≥36 IU/L had good performance with AUROCs of 0.83 (0.71 to 0.94), whereas the NashTest had an AUROC of 0.60 (0.44 to 0.76). CONCLUSIONS: In HIV-monoinfected patients, MRI-PDFF and FibroScan/CAP are highly accurate for the diagnosis of steatosis. The alanine aminotransferase level and APRI should be considered for the detection of NASH and fibrosis.

Transjugular diagnostics in acute liver failure including measurements of hepatocentral venous biomarker gradients.

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.

Baseline IL-2 and the AIH score can predict the response to standard therapy in paediatric autoimmune hepatitis.

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.

Hepatic T Cell Tolerance Induction in An Inflammatory Environment.

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.

Pediatric autoimmune hepatitis shows a disproportionate decline of regulatory T cells in the liver and of IL-2 in the blood of patients undergoing therapy.

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.