Liver Antigen Research Articles

Analysis of avian Usutu virus infections in Germany from 2011 to 2018 with focus on dsRNA detection to demonstrate viral infections

Usutu virus (USUV) is a zoonotic arbovirus causing avian mass mortalities. The first outbreak in North-Western Germany occurred in 2018. This retrospective analysis focused on combining virological and pathological findings in birds and immunohistochemistry. 25 common blackbirds, one great grey owl, and one kingfisher collected from 2011 to 2018 and positive for USUV by qRT-PCR were investigated. Macroscopically, most USUV infected birds showed splenomegaly and hepatomegaly. Histopathological lesions included necrosis and lymphohistiocytic inflammation within spleen, Bursa fabricii, liver, heart, brain, lung and intestine. Immunohistochemistry revealed USUV antigen positive cells in heart, spleen, pancreas, lung, brain, proventriculus/gizzard, Bursa fabricii, kidney, intestine, skeletal muscle, and liver. Analysis of viral genome allocated the virus to Europe 3 or Africa 2 lineage. This study investigated whether immunohistochemical detection of double-stranded ribonucleic acid (dsRNA) serves as an alternative tool to detect viral intermediates. Tissue samples of six animals with confirmed USUV infection by qRT-PCR but lacking viral antigen in liver and spleen, were further examined immunohistochemically. Two animals exhibited a positive signal for dsRNA. This could indicate either an early state of infection without sufficient formation of virus translation products, occurrence of another concurrent virus infection or endogenous dsRNA not related to infectious pathogens and should be investigated in more detail in future studies.

The Spectrum of Autoimmune Liver Disorders, Clinical Presentation, and Autoantibodies in Patients From a Tertiary Care Center in Pakistan.

BackgroundThe autoimmune illnesses that affect the liver include autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and overlap syndrome. In our patients, we aimed to address the complete spectrum of autoimmune liver disorders, clinical presentation, and autoantibodies.MethodsThe study included all the patients diagnosed with autoimmune liver disorder irrespective of age, gender, and ethnic background presented at the liver clinic of the hospital in the last two years. The diagnosis was based on characteristic clinical and laboratory findings, the presence of one or more characteristic autoantibodies, and/or histological abnormalities. The diagnosis of AIH was further validated by revised International AIH Group criteria using a scoring calculator. The diagnostic criteria for PBC required the presence of chronic elevation of alkaline phosphatase (ALP) with positive antimitochondrial antibody (AMA) or positive PBC-specific anti-nuclear antibodies (ANA) (sp-100, gp-210) tests and/or compatible histology. The patients of AIH-PBC overlap syndrome fulfilled the criteria for AIH in the setting of PBC. Patients having liver involvement in other autoimmune disorders were included in the study.ResultsThe total number of patients was 124; 83 (67%) were females; mean age ± standard error of mean (SEM) was 44.97 ± 1.47 years with a range of 09-84 years. Type-1 AIH was seen in 68 (54.8%) patients, type-2 AIH in 10 (8.1%) patients, PBC in 22 (17.7%) patients, overlap of PBC with AIH in 10 (8.1%) patients, IgG4 disease in four (3.2%) patients, psoriasis-specific immune hepatitis in four (3.2%) patients, celiac disease-related hepatitis in three (2.4%) patients, sarcoidosis in two (1.6%) patients, and ichthyosis-associated hepatitis in one (0.8%) patient. There was a high prevalence of cirrhosis (50%) at the time of presentation; 19% of patients had decompensated liver disease. ANA was positive in 52/68 cases of AIH type-1, but anti-smooth muscle antibody (ASMA) was reactive only in nine cases and anti-soluble liver antigen (SLA) in five cases. There was no female preponderance in type-2 AIH (M:F = 6:4). AMA was reactive in 25 (78%) cases of PBC and overlap syndrome. Antibodies prevalent in PBC (AMA-M2, AMA-M2-3E, sp-100, gp-210, anti-Ro52) were also seen in some cases of AIH, though they did not fulfill the criteria of the overlap syndrome.ConclusionThere is an unmet need for the early diagnosis of autoimmune liver diseases and the initiation of appropriate management to prevent complications.

Nimbolide attenuated the inflammation in the liver of autoimmune hepatitis's mice through regulation of HDAC3

A chronic liver disease named autoimmune hepatitis (AIH) will carry elevated levels of inflammatory cytokines, but there is currently no effective treatment to cure it. Histone deacetylase 3 (HDAC3) takes an important position in regulating the expression of inflammatory genes. Nimbolide (NIB) is a limonoid extracted from the neem tree (Azadirachta indica) that has been found to be effective against many diseases, including cancer, scleroderma, and acute respiratory distress syndrome. Here, we investigated the protective effect of nimbolide on AIH liver. Mice and AML12 cells were employed to establish AIH model with liver antigen S100 and cell injury model of LPS, and then treated with different concentrations of nimbolide. After the successful establishment of the animal model and cell model, inflammatory cytokines of IL-1β, IL-6 and TNF-α as well as cellular signaling related to inflammation such as STAT3, IκB-α and NF-κB were examined. We observed for the first time about nimbolide can effectively inhibit inflammation in AIH mice's liver and AML12 cells by inhibiting HDAC3 expression. HDAC3 knocked down by siRNA in cells can also effectively alleviate the inflammation in AML12 cells, further confirming that HDAC3 plays an important role in the inflammation of liver cells. These results suggest nimbolide could be a potential new treatment for autoimmune hepatitis, and HDAC3 may become a new target for autoimmune hepatitis.

Maintenance immunosuppressive therapy in autoimmune hepatitis: To stop or not to stop, that is the question.

Autoimmune hepatitis (AIH) is a rare and challenging disease. Its presentation ranges from mildly elevated transaminases to jaundice with decompensated liver disease. Blood tests often reveal elevated immunoglobulin G levels and several antibody tests like anti-smooth muscle antibodies, liver-kidney microsomal antibodies or soluble liver antigen antibodies can be positive. Viral hepatitis (including hepatitis E viral infection), drug-induced liver injury and (especially in young patients) Wilson disease should be excluded.

Expression of Cancer Testis Antigens in Tumor-Adjacent Normal Liver Is Associated with Post-Resection Recurrence of Hepatocellular Carcinoma.

Simple SummaryHigh recurrence rates after resection of liver cancer (hepatocellular carcinoma) with curative intent impair clinical outcomes of patients diagnosed with liver cancer. Cancer/testis antigens (CTAs) are expressed in cancer and can serve as therapeutic targets. We identified 12 CTAs expressed in 80% of liver cancer patients, and each one individually in at least 10%. Furthermore, we found that patients with expression of CTAs in macroscopically tumor-free liver tissue, experience more tumor recurrence and poor survival after surgical tumor removal. The increased risk of tumor recurrence in patients with CTA expression in tumor-free liver suggests that these patients already have micro-metastasis at the time of operation. These CTA-expressing (pre-)malignant cells may thus be a source of liver cancer recurrence, reflecting the relevance of targeting these to prevent liver cancer recurrence.High recurrence rates after resection of hepatocellular carcinoma (HCC) with curative intent impair clinical outcomes of HCC. Cancer/testis antigens (CTAs) are suitable targets for cancer immunotherapy if selectively expressed in tumor cells. The aims were to identify CTAs that are frequently and selectively expressed in HCC-tumors, and to investigate whether CTAs could serve as biomarkers for occult metastasis. Tumor and paired tumor-free liver (TFL) tissues of HCC-patients and healthy tissues were assessed for mRNA expression of 49 CTAs by RT-qPCR and protein expression of five CTAs by immunohistochemistry. Twelve CTA-mRNAs were expressed in ≥10% of HCC-tumors and not in healthy tissues except testis. In tumors, mRNA and protein of ≥ 1 CTA was expressed in 78% and 71% of HCC-patients, respectively. In TFL, CTA mRNA and protein was found in 45% and 30% of HCC-patients, respectively. Interestingly, CTA-expression in TFL was an independent negative prognostic factor for post-resection HCC-recurrence and survival. We established a panel of 12 testis-restricted CTAs expressed in tumors of most HCC-patients. The increased risk of HCC-recurrence in patients with CTA expression in TFL, suggests that CTA-expressing (pre-)malignant cells may be a source of HCC-recurrence, reflecting the relevance of targeting these to prevent HCC-recurrence.

Manipulating antigen presentation for antigen-specific immunotherapy of autoimmune diseases.

Current treatments for autoimmune diseases do not address the immune pathology underlying their initiation and progression and too often rely on non-specific immunosuppressive drugs for control of symptoms. Antigen-specific immunotherapy aims to induce tolerance selectively among the cells causing the disease while leaving the rest of the adaptive immune system capable of protecting against infectious diseases and cancers. Here we describe how novel approaches for antigen-specific immunotherapy are designed to manipulate antigen presentation and promote tolerance to specific self-antigens. This analysis points to liver antigen presenting cells, targeted by carrier particles, and steady-state dendritic cells, to which antigen-processing independent T-cell epitopes (apitopes) bind directly, as the principal targets for antigen-specific immunotherapy. Delivery of antigens to these cells holds great promise for effective control of this rapidly expanding group of diseases.

Differential Yellow Fever Susceptibility in New World Nonhuman Primates, Comparison with Humans, and Implications for Surveillance.

A major outbreak of yellow fever (YF) occurred in Brazil during 2016–2018. Epizootics in New World nonhuman primates are sentinel events for YF virus circulation. However, genus-specific susceptibilities and suitability for YF surveillance remain poorly understood. We obtained and compared epidemiologic, histopathologic, immunohistochemical, and molecular results from 93 human and 1,752 primate cases submitted during the recent YF outbreak in Brazil (2017), with the support of the Brazilian National YF Surveillance Program. We detected heterogeneous YF-associated profiles among the various genera of primates we analyzed. Alouatta primates were the most reliable sentinel; Sapajus and Callicebus primates had higher viral loads but lower proportional mortality rates. Callithrix primates were the least sensitive, showing lower viral loads, lower proportional mortality rates, and no demonstrable YF virus antigen or extensive lesions in liver, despite detectable viral RNA. These differences in susceptibility, viral load, and mortality rates should be considered in strategic surveillance of epizootics and control measures for YF.

From the Editor's Desk…

From the Editor's Desk…

S2629 A Case of Autoimmune Hepatitis Induced After Treatment of Syphilitic Hepatitis

INTRODUCTION: Liver involvement manifested by increased liver enzymes has been reported in approximately 10% of cases of syphilis. Syphilitic hepatitis has been reported to cause cholestatic hepatitis, however has not been associated with hepatocellular injury. We present a case of autoimmune hepatitis flare after completion of treatment for biopsy proven syphilitic hepatitis. CASE DESCRIPTION/METHODS: Fifty-one-year-old Hispanic man with history of HTN presented for new onset jaundice and RUQ pain. Initial labs revealed Tbili 19.9, Dbili 15.5, ALP 147, ALT 1652 and INR 1.6. No new history of medications/herbs use, new sexual contact, body piercing, drug use or family history of liver disease. Work-up was negative for acute viral hepatitis, CMV and EBV. ANA positive (1:160), ASMA negative, anti-LKM 1.8, Anti-SLA 1.3 and IGG 1301. RPR reactive confirmed with FTA antibodies. Liver biopsy showed acute hepatitis with mixed infiltrates, confluent necrosis and cholestasis. Immunostaining for spirochetes was positive on liver biopsy. The patient received IM 2.4 million IU Benz Pen Q weekly x 3 doses with substantial improvement in liver enzymes (AST 481, ALT 422, Tbili 4.6) and INR 1.2. On 3-week follow-up, however liver enzymes were, AST 1007, ALT 1000, TBili 10.3, INR 1.6, requiring readmission. Further work-up showed: IgG 2275, ANA positive 1:160 and repeat liver biopsy revealed interface hepatitis with negative stains for spirochetes. The patient was treated with IV solumedrol 40 mg with significant improvement of liver enzymes and was discharged on prednisone 40 mg daily. Follow-up labs revealed: AST 90, ALT 139, Tbili 2.5 and INR 1.1. The prednisone dose was tapered with continued biochemical improvement. DISCUSSION: Treponema pallidum causes Syphilis and transmission is sexual and vertical. In cases of syphilitic hepatitis liver biopsy reveals focal hepatocyte necrosis and portal tracts with inflammatory infiltrates. Immunohistochemical stain for spirochetes is confirmatory but only identifies in about 50% of cases. Autoimmune hepatitis is T cell medicated immune attack on liver antigens with progressive necroinflammatory and fibrotic process in the liver. The diagnosis of AIH needs biochemical, immunologic and histologic confirmation. We report a unique case of biopsy proven syphilitic hepatitis with significant elevation of liver enzymes which improved with antibiotic therapy but subsequently triggered immunogenic response identical to AIH with recovery after steroid therapy.

S2627 A Case of K2-Induced Hyperbilirubinemia

INTRODUCTION: Over the last decade, synthetic cannabinoids (SCB) have been marketed as a natural and safer version of marijuana, leading to an increase in its recreational use. We present a case of isolated hyperbilirubinemia caused by K2 and, to our knowledge, this is the first reported case of K2-induced indirect hyperbilirubinemia. CASE DESCRIPTION/METHODS: An 18-year-old male with no significant medical history presented with behavioral changes. He admitted to using K2 daily. His sibling and paternal uncle had Gilbert's syndrome. On physical exam he was withdrawn and agitated. He had Scleral icterus and jaundice. Slit Lamp exam was equivocal. On labs: Total Bilirubin 11.2 mg/dL, Indirect bilirubin 10.8 mg/dL, Urine toxicology positive for marijuana.Serum ceruloplasmin, serum copper, 24h urinary copper excretion, anti-mitochondrial Ab, anti-Nuclear Ab, anti-LKM, Soluble Liver antigen, ferritin, iron, TIBC, A1AT, A1AT phenotype, IgG, IgM, IgA, hepatitis A, B, C serologies, LDH, haptoglobin, uric acid, Coombs test, and G6PD levels were all within normal limits. His peripheral blood smear was normal. Imaging: MRI brain: unremarkable, Liver biopsy:normal hepatic architecture without severe pathological process (Figures 2a and 2b). He was treated with supportive care and indirect hyperbilirubinemia improved. DISCUSSION: • There is no current literature that correlates indirect hyperbilirubinemia and SCB use. In our otherwise healthy patient, this hyperbilirubinemia was likely a consequence of his K2 ingestion. We hypothesize that K2 may impair hepatic glucuronidation which is the process by which indirect bilirubin is converted to direct bilirubin. • During the time of exacerbation of Gilbert's disease, most often the indirect bilirubin levels remain < 5mg/dL. In the case of our patient, he presented with an indirect bilirubin level of 10.8 mg/dL. His work-up excluded other causes of indirect hyperbilirubinemia. • This leads us to hypothesize that K2 may induce significant isolated indirect hyperbilirubinemia in patients with a predisposition to impaired hepatic glucuronidation. • We believe that removal of the insulting agent is required for resolution of the hyperbilirubinemia. • With the use of SCB increasing recreationally, it is crucial we understand the effects these drugs can have. SCB do not appear on standard hospital drug screens. Therefore, a focused history and exam should be obtained from the patient. Our case serves to increase awareness of SCB use as a cause of indirect hyperbilirubinemia.Figure 1Figure (2a).: Liver biopsy stained with Hematoxylin & Eosin, showing normal hepatic architecture with no periductal or ductular inflammatory infiltration.Figure (2b).: Hepatic tissue stained with Periodid acid-Schiff Diastase and for copper; normal pathology & negative copper staining.

Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH. We used brief exvivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis. Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5-CCR6-CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA81-100 and SLA177-204 contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1+CXCR5-CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH. We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells. One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.

Molecular detection, histopathological analysis, and immunohistochemical characterization of equine infectious anemia virus in naturally infected equids.

Equine infectious anemia (EIA), a disease caused by equine infectious anemia virus (EIAV), is considered an obstacle to the development of the horse industry. There is no treatment or vaccine available for EIA, and its pathogenesis, as well as the immune response against the virus, is not fully understood. Therefore, an immunohistochemistry assay was developed for the detection of viral antigens in tissues of equids naturally infected with EIAV. Sections of organs of six equids from Apodi-RN, Brazil, that tested positive for EIA by serological tests (ELISA and AGID) were fixed in 10% formalin solution and embedded in paraffin. Immunohistochemistry was performed using a polyclonal anti-EIAV antibody. EIAV antigens were observed in red spleen pulp cells and hepatic sinusoids, as well as bronchiolar and alveolar epithelial cells of the lungs and proximal and distal tubules of the kidneys. The presence of EIAV in the spleen and liver was expected due to viral tropism by macrophages, which are abundantly present in these organs. However, EIAV was also found in lung and kidney epithelial cells, indicating that the virus infects cell types other than macrophages. In conclusion, the immunohistochemical assay standardized in this study was able to detect EIAV antigens in spleen, liver, kidney and lung cells from naturally infected EIAV equids. Immunostaining observed in the spleen confirms viral tropism by mononuclear phagocytes; however, the presence of EIAV in lung and kidney epithelial cells indicates that virus may be eliminated in urine and/or oronasal secretions, suggesting new routes for viral excretion.

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice

Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Higher seroprevalence of hepatitis E virus in autoimmune hepatitis: Role of false-positive antibodies.

Recent studies have found an increase in the seroprevalence of hepatitis E virus (HEV) infection in patients with autoimmune hepatitis (AIH). We aimed to assess the prevalence of positive anti-HEV IgM and IgG, and HEV-RNA in a cohort of patients with AIH, to determine the impact of positive HEV serology on patient outcome, and to evaluate the role of hypergammaglobulinemia and positive autoantibodies in the presence of positive anti-HEV serology. One hundred and five patients tested for HEV infection between 2014 and 2018 were included in the study: 50 with chronic AIH (more than 1year on treatment), and 55 with an acute hepatitis (30 patients with acute AIH and 25 with non-AIH). Seroprevalence of HEV was higher in patients with acute AIH (17% vs 10% in patients with chronic AIH and 8% in patients with non-AIH). Patients with acute AIH and positive anti-HEV IgG were older (58 vs 40; P=.006), had higher IgG levels (27g/dL vs 13g/dL; P=.03) and antismooth muscle antibodies (ASMA) titres (1:160 vs 1:80; P=.045), and were more likely to have another autoimmune disease (60% vs 16%; P=.03). At the time of HEV testing, anti-HEV IgG positive patients had significantly higher serum IgG levels (17g/L vs 11g/L; P=.009), ANA (1:160 vs 1:60; P=.026) and ASMA titres (1:80 vs 1:40; P=.021). Seroprevalence of HEV in patients with AIH in Catalonia does not differ from that of the general population. The higher HEV seroprevalence in patients with acute AIH with higher levels of gammaglobulins and high antibody titres suggest the presence of cross-reactivity between HEV and liver antigens.

Seroprevalence of AIH-related autoantibodies in patients with acute hepatitis E viral infection: a prospective case–control study in China

ABSTRACT The seroprevalenc of autoimmune hepatitis (AIH)-related antibodies in patients, particularly Asians, with acute hepatitis E (AHE) is unclear. In this study, we investigated whether acute hepatitis E virus (HEV) infection is associated with the seroprevalence of AIH-related autoantibodies and assessed their impact on the disease characteristics. AIH-related autoantibodies were detected by indirect immunofluorescence in 198 AHE patients and 50 type 1 AIH patients. The positivity rates of against nuclear antigen (ANA) and smooth muscles antibody (SMA) in AHE patients were 37.4% and 22.7%, and the total positivity rate was 50%. Compared to those in AIH patients, the positivity rates of ANA-H and SMA-AA were significantly lower (35.1% vs. 82.1% and 4.4% vs. 88.4%). Female gender and the ALT level, but not immunosuppressive or antiviral drugs, were independently predictive of the presence of AIH-related autoantibodies in AHE patients. Fifty-two patients positive for AIH-related autoantibodies were followed up for 12 months. During this period, 33 of them became negative and 19 remained positive, albeit with significantly decreased titres. In conclusions, the seroprevalence of AIH-related autoantibodies in AHE patients was elevated, particularly in females, but their subspecificities and titres differed from those of type 1 AIH. Acute HEV infection may be related to AIH. Abbreviations: AIH: autoimmune hepatitis; AHE: acute hepatitis E; ANA: against nuclear antigen; SMA: smooth muscles antibody; ANA-H: ANA with homogeneous pattern; SMA-AA: SMA with anti-actin pattern; Anti-LKM1: anti- liver-kidney microsomes-1 antibody; ANCA: anti-neutrophil cytoplasmic antibody; AMA: anti-mitochondrial antibody; Anti-SLA: anti-soluble liver antigen; Anti-LC1: anti-liver cytoplasmic type 1 antibody; pANCA: perinuclear antineutrophil cytoplasmic antibody

Analysis of the autoantibodies characteristics of 77 anti-soluble liver antigen positive patients with liver diseases

Objective To understand the characteristics and clinical significance of anti-soluble liver antigen antibody (anti-SLA) in patients with liver diseases. Methods Serum samples from seventy-seven patients with anti-SLA were collected from Beijing You'An Hospital during the period between January 2010 and December 2018. Anti-SLA, anti-liver cytosol type 1 antibody (anti-LC1), anti-glycoprotein 210 antibody(anti-gp210) and anti-nuclear body protein sp100 antibody(anti-sp100) were detected by immunoblotting; indirect immunofluorescence assay used for detecting anti-nuclear antibody (ANA), anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (SMA), and anti-liver kidney microsome antibody (anti-LKM). One-way analysis of variance was used to compare the ages of different anti-SLA groups. The non-parametric rank sum test was used to compare the liver function indexes and immunoglobulins in different intensity groups of anti-SLA. P<0.05 was considered statistically significant. Further comparisons were made between the two groups, the correction level α′=0.008 3, P<0.008 3 was considered statistically significant. Results The average age of 77 anti-SLA positive patients was (52.50±1.25) years old, 70 females (90.9%) and 7 males (9.1%). 80.5% of anti-SLA-positive patients (62/77 cases) were strongly positive at the time of initial diagnosis (+++ to ++++).The Alanine aminotransferase (ALT) level in the SLA++ group was higher than that in the SLA++++ group (232.7 U/L vs 65.6 U/L,χ2=7.751,P=0.005) and the immunoglobulin M(IgM) level in the SLA+++ group was lower than that in the SLA++++ group (1 270 mg/L vs 2 270 mg/L,χ2=8.337,P=0.004).There was no significant difference in age, other liver function and immunological indicators among the different groups.Seventy cases (90.9%) were both anti-SLA and ANA positive, 13 cases (16.9%) were positive with SMA, and none positive with anti-LKM and anti-LC1. Among anti-SLA positive patients, 58 cases were diagnosed with autoimmune hepatitis (AIH), 12 were AIH/primary biliary cholangitis (PBC) overlap syndrome (OS), 2 were drug-induced liver injury, 2 were chronic hepatitis B, and 3 were hepatitis A, hepatitis E and acquired immune deficiency syndrome (AIDS) with liver injury, respectively.Cases of AIH and AIH/PBC OS accounted for 90.9% (70/77 cases) of anti-SLA-positive patients, and 5 of 7 patients diagnosed with non-AIH (and OS) had elevated IgG, showing AIH feature.92.3% (12/13 cases) of anti-SLA with high titers of AMA were diagnosed as AIH/PBC overlap syndrome. Of the 77 anti-SLA-positive patients, 28 (36.4%) had advanced or end-stage liver disease, including decompensated cirrhosis (22 cases), chronic acute liver failure (4 cases), and liver transplantation (1 case) and death from liver failure (1 case). Conclusions Anti-SLA has high diagnostic specificity for AIH;anti-SLA positive in patients with PBC should be an important biomarker for the diagnosis of AIH/PBC overlap syndrome. Key words: Liver diseases; Hepatitis, autoimmune; Autoantibodies

Osthole delays hepatocarcinogenesis in mice by suppressing AKT/FASN axis and ERK phosphorylation

Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies worldwide. Inhibition of the lipogenic enzymes involved in hepatic de novo lipogenesis can both effectively restrain proliferation of HCC cells in vitro and reduce the risk of hepatocarcinogenesis in vivo. Although a natural coumarin derivative osthole shows efficacy in suppressing cell proliferation and inducing apoptosis in cultured hepatoma cells and HCC xenograft tumors, the molecular mechanism by which osthole delays hepatocellular malignant transformation during lipogenesis-driven hepatocarcinogenesis remains unknown. Here, we evaluate the efficacy of osthole in a rapid HCC mouse model featuring excessive levels of hepatic steatosis established via hydrodynamic transfection of activated forms of AKT and c-Met proto-oncogenes. Moreover, human hepatoma cell lines were employed for in vitro assessment. Hematoxylin and eosin staining, immunoblotting and immunohistochemistry were applied for mechanistic investigations. The results revealed that if osthole was administered in the early stage of AKT/c-Met-driven HCC, it led to disease stabilization. Moreover, osthole alleviated hepatic steatosis in the AKT/c-Met mice. Further evidence at the molecular level suggested that osthole reduced the expression of phosphor-extracellular signal-regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA) and Ki67 in livers of the AKT/c-Met mice. Mechanically, osthole efficiently repressed the phospho-AKT (Thr308) / ribosomal protein S6 (RPS6) / fatty acid synthase (FASN) signaling both in mice and in vitro. Altogether, this study suggests that osthole exerts its antilipogenic and antiproliferative efficacy by suppressing the AKT/FASN axis and ERK phosphorylation, which contributes to its capacity to delay hepatocarcinogenesis.

2479 Autoimmune Hepatitis Triggered by Hepatitis A: An Interesting Case and Review of Pathophysiology

INTRODUCTION: Autoimmune hepatitis (AIH) is an immune-mediated disorder of the liver, whose pathophysiology involves triggering of T-cell mediated events by environmental agents in the liver in a host genetically predisposed to this disease. One trigger described in literature has been the hepatitis A virus (HAV). Hence, occasionally cases which clinically appear to be that of relapsing HAV are in fact concealing underlying autoimmune hepatitis that has been provoked from the initial episode of HAV. We describe one such case. CASE DESCRIPTION/METHODS: A 45 year old Caucasian male with past medical history of chronic hepatitis C and acute hepatitis two months prior to admission was admitted for multifocal pneumonia. During his initial evaluation, the patient was found to have generalized jaundice, and laboratory evaluation revealed elevated transaminases with a hepatocellular pattern. He was initially thought to have reactivation of the hepatitis A, however non-improvement with conservative measures led to further serological testing, which revealed positive anti-smooth muscle antibody. Autoimmune hepatitis was confirmed by biopsy, and patient was started or oral steroids. The patient's clinical status and liver function improved significantly, and he was discharged with outpatient follow-up. DISCUSSION: It is hypothesized that environmental agents trigger T-cell–mediated events directed at liver antigens in a host genetically predisposed to AIH, leading to a progressive necro-inflammatory and fibrotic process in the liver. Studies have shown hepatotropic viruses including HAV to be associated with AIH. Defects in suppressor-inducer T lymphocytes specifically controlling immune responses to the asialoglycoprotein receptor may be the cause of susceptibility. It has also been shown that humoral immune responses to liver cell surface antigens are frequently provoked by hepatitis A and B viruses. Newer research is also exploring molecular mimicry between the infectious particle and the liver constituent as the mechanism, although specific data has not been discovered on HAV. In the setting of the recent outbreaks of HAV, such pathology may become more commonplace. It is important for a clinician to distinguish between relapses of HAV (documented to be between 3-20% of patients in the first 6 months) and onset of AIH, as treatment modalities and prognosis are vastly different. A high level of awareness of this pathology allows for its prompt recognition and the potential for successful treatment.

Steroid receptor coactivator 3 inhibits hepatitis B virus gene expression through activating Akt signaling to prevent HNF4\u03b1 nuclear translocation

BackgroundChronic hepatitis B virus (HBV) infection is one of the most serious global public health problems. The role of steroid receptor coactivator 3 (SRC-3) in HBV biosynthesis is unknown. The aim of this study is to investigate the function of SRC-3 in regulating HBV biosynthesis both in vitro and in vivo and to identify the underlying mechanism.ResultsIn this study, we found that knockdown of SRC-3 could increase the levels of HBV mRNA and HBV proteins HBsAg and HBeAg in human liver cancer cell line HepG2 transfected with pHBV1.3 plasmids. In contrast, enforced expression of SRC-3 in SRC-3-knockdown HepG2 cells reduced the levels of HBV mRNA and HBV proteins HBsAg and HBeAg. Knockdown of SRC-3 dampened the Akt signaling, which has been shown to play a negative role in HBV transcription. Ectopic expression of constitutively activated Akt impaired the enhancement of HBV transcription by SRC-3 knockdown, indicating that SRC-3 inhibits HBV transcription by enhancing Akt signaling. Both SRC-3 and constitutively activated Akt could inhibit hepatocyte nuclear factor 4α (HNF4α)-mediated upregulation of HBV core promoter activity by preventing HNF4α nuclear translocation. Consistent with the in vitro results, in an in vivo chronic HBV replication mouse model developed by hydrodynamic injection of pHBV1.3 plasmids into mouse tail vein, enforced expression of SRC-3 in mouse liver reduced the levels of HBV mRNA in the liver and HBV antigens in serum, whereas knockout of SRC-3 in mouse increased the levels of HBV mRNA in the liver and HBV antigens in the serum.ConclusionOur study suggests that SRC-3 inhibits HBV gene expression by activating Akt signaling to prevent HNF4α nuclear translocation.

Clinical, Histopathologic, and Immunohistochemical Characterization of Experimental Marburg Virus Infection in A Natural Reservoir Host, the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (Rousettus aegyptiacus) are natural reservoir hosts of Marburg virus (MARV), and Ravn virus (RAVV; collectively called marburgviruses) and have been linked to human cases of Marburg virus disease (MVD). We investigated the clinical and pathologic effects of experimental MARV infection in Egyptian rousettes through a serial euthanasia study and found clear evidence of mild but transient disease. Three groups of nine, captive-born, juvenile male bats were inoculated subcutaneously with 10,000 TCID50 of Marburg virus strain Uganda 371Bat2007, a minimally passaged virus originally isolated from a wild Egyptian rousette. Control bats (n = 3) were mock-inoculated. Three animals per day were euthanized at 3, 5–10, 12 and 28 days post-inoculation (DPI); controls were euthanized at 28 DPI. Blood chemistry analyses showed a mild, statistically significant elevation in alanine aminotransferase (ALT) at 3, 6 and 7 DPI. Lymphocyte and monocyte counts were mildly elevated in inoculated bats after 9 DPI. Liver histology revealed small foci of inflammatory infiltrate in infected bats, similar to lesions previously described in wild, naturally-infected bats. Liver lesion severity scores peaked at 7 DPI, and were correlated with both ALT and hepatic viral RNA levels. Immunohistochemical staining detected infrequent viral antigen in liver (3–8 DPI, n = 8), spleen (3–7 DPI, n = 8), skin (inoculation site; 3–12 DPI, n = 20), lymph nodes (3–10 DPI, n = 6), and oral submucosa (8–9 DPI, n = 2). Viral antigen was present in histiocytes, hepatocytes and mesenchymal cells, and in the liver, antigen staining co-localized with inflammatory foci. These results show the first clear evidence of very mild disease caused by a filovirus in a reservoir bat host and provide support for our experimental model of this virus-reservoir host system.