Contrast-Enhanced Ultrasound Algorithms (CEUS-LIRADS/ESCULAP) for the Noninvasive Diagnosis of Hepatocellular Carcinoma - A Prospective Multicenter DEGUM Study.

BACKGROUND: This prospective multicenter study funded by the DEGUM assesses the diagnostic accuracy of standardized contrast-enhanced ultrasound (CEUS) for the noninvasive diagnosis of hepatocellular carcinoma (HCC) in high-risk patients. METHODS: Patients at high risk for HCC with a histologically proven focal liver lesion on B-mode ultrasound were recruited prospectively in a multicenter approach. Clinical and imaging data were entered via online entry forms. The diagnostic accuracies for the noninvasive diagnosis of HCC were compared for the conventional interpretation of standardized CEUS at the time of the examination (= CEUS on-site) and the two CEUS algorithms ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) and CEUS LI-RADS (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System). RESULTS: 321 patients were recruited in 43 centers; 299 (93.1 %) had liver cirrhosis. The diagnosis according to histology was HCC in 256 cases, and intrahepatic cholangiocarcinoma (iCCA) in 23 cases. In the subgroup of cirrhotic patients (n = 299), the highest sensitivity for the diagnosis of HCC was achieved with the CEUS algorithm ESCULAP (94.2 %) and CEUS on-site (90.9 %). The lowest sensitivity was reached with the CEUS LI-RADS algorithm (64 %; p < 0.001). However, the specificity of CEUS LI-RADS (78.9 %) was superior to that of ESCULAP (50.9 %) and CEUS on-site (64.9 %; p < 0.001). At the same time, the negative predictive value (NPV) of CEUS LI-RADS was significantly inferior to that of ESCULAP (34.1 % vs. 67.4 %; p < 0.001) and CEUS on-site (62.7 %; p < 0.001). The positive predictive values of all modalities were high (around 90 %), with the best results seen for CEUS LI-RADS and CEUS on-site. CONCLUSION: This is the first multicenter, prospective comparison of standardized CEUS and the recently developed CEUS-based algorithms in histologically proven liver lesions in cirrhotic patients. Our results reaffirm the excellent diagnostic accuracy of CEUS for the noninvasive diagnosis of HCC in high-risk patients. However, on-site diagnosis by an experienced examiner achieves an almost equal diagnostic accuracy compared to CEUS-based diagnostic algorithms.

Long-term follow-up of patients with difficult to treat type 1 autoimmune hepatitis on Tacrolimus therapy.

INTRODUCTION: Autoimmune hepatitis (AIH) is an immune-mediated liver disease, which requires long-term immunosuppression. Ten to fifteen percent of patients experience insufficient/intolerance response to standard therapy. Although alternate immunosuppression has been applied, there is little long-term data reported on safety, efficacy, steroid-dose reduction and disease evolution in patients with difficult AIH who were on Tacrolimus therapy. MATERIALS AND METHODS: Clinical, biochemical, immunological profiles, treatment response and side effects of 17 AIH patients treated with Tacrolimus between 2003 and 2014 were analyzed from two tertiary referral liver centers. RESULTS: Tacrolimus was started on 16/17 (94%) patients due to insufficient response to standard therapy. The median duration of treatment was 24 months and patients were followed up for median of 60 months. Tacrolimus dosage was 2 mg/day (median). During first year of therapy, there was a significant improvement in immunoglobulin G and Aspartate transaminase level. 9/17 (52%) compliant and definite AIH patients remained on Tacrolimus at end of follow-up and prednisolone dose reduction was achieved from 10 to 5 mg. All patients are alive and one patient underwent liver transplantation. 4/17 (24%) patients developed overlap with primary sclerosing cholangitis over follow-up period. No significant side effects were observed with Tacrolimus therapy. CONCLUSION: Tacrolimus could be used in compliant patients with difficult to treat AIH in experienced centers. Its use is safe and can improve liver biochemistry, IgG and reduce steroid requirement. However, due to the lack of immunomodulatory effect, unmet need for effective immune-regulatory therapies still remain for AIH patients.

Health-related quality of life, depression, and anxiety in patients with autoimmune hepatitis.

BACKGROUND & AIMS: Improving health related quality of life (HrQoL) in patients with chronic diseases such as autoimmune hepatitis (AIH) should be a major treatment goal. However, little is known on the HrQoL in patients with AIH, and the topic is not given attention in current practice guidelines. We therefore conducted a single center study evaluating HrQoL in 103 consecutive outpatients with AIH. METHODS: Patient-reported HrQoL data were analysed in relation to clinical disease parameters and compared to representative data of the German population as well as control patients. RESULTS: Based on patient-reported data, a major depressive syndrome (10.8%) was found to be five times more frequent in AIH patients compared to the general population (p<0.001). The rate of severe symptoms of anxiety was also found to be significantly increased compared to the general population (p=0.006). In seven of the eleven patients who scored for a major depressive syndrome a psychiatric comorbidity had not been diagnosed before. Major factors associated with depression and anxiety were concerns with regard to the progression of the liver disease. CONCLUSIONS: This study identified--for the first time--a high rate of previously unrecognized severe symptoms of depression and anxiety in patients with AIH. Of importance for daily clinical practice, the factors associated with these symptoms may in part be amenable to targeted counselling and adequate treatment of the disease, thereby offering the chance to improve the care and HrQoL of AIH-patients.

Reduced FOXP3(+) regulatory T cells in patients with primary sclerosing cholangitis are associated with IL2RA gene polymorphisms.

BACKGROUND & AIMS: Recently, genome wide association studies in primary sclerosing cholangitis (PSC) revealed associations with gene polymorphisms that potentially could affect the function of regulatory T cells (Treg). The aim of this study was to investigate Treg in patients with PSC and to associate their numbers with relevant gene polymorphisms. METHODS: Treg frequency in blood was assessed by staining for CD4(+)CD25(high)FOXP3(+)CD127(low) lymphocytes and determination of Treg-specific FOXP3 gene locus demethylation. Single nucleotide polymorphisms (SNP) in the interleukin-2 receptor alpha (IL2RA), the interleukin-2 (IL2) and interleukin-21 (IL21) gene locus were analysed. Liver biopsies taken at the time of diagnosis were stained for FOXP3 and CD3. Treg function was assessed in a CFSE-based suppression assay. RESULTS: The frequency of Treg in peripheral blood of PSC patients was significantly decreased. We confirmed this finding by demonstrating a reduction of non-methylated DNA in the Treg-specific demethylated FOXP3 gene region of peripheral blood cells in PSC patients. Reduced peripheral Treg numbers were significantly associated with homozygosity for the major allele of the SNP "rs10905718" in the IL2RA gene. Intrahepatic FOXP3(+) cell numbers at the time of initial diagnosis were decreased in PSC as compared to PBC. In addition to reduced numbers, the suppressive capacity of Treg isolated from PSC patients seemed to be impaired as compared to healthy controls. CONCLUSIONS: Our findings indicate that Treg impairment may play a role in the immune dysregulation observed in PSC. Reduced Treg numbers in patients with PSC are associated with polymorphisms in the IL2RA gene.

Infliximab as a rescue treatment in difficult-to-treat autoimmune hepatitis.

BACKGROUND & AIMS: Autoimmune hepatitis is a chronic inflammatory liver disease that leads to liver cirrhosis and corresponding complications, if left untreated. Current standard treatment with azathioprine and prednisolone induces remission in the vast majority of patients. However, for those patients not responding to standard treatment or not tolerating these drugs, few alternatives can be used and their effectiveness might be limited. We sought to analyze the safety and efficacy of off-label treatment with infliximab in a cohort of eleven patients with difficult-to-treat autoimmune hepatitis. METHODS: Patients with difficult-to-treat autoimmune hepatitis who could not be brought into remission with standard treatment, either due to drug intolerance or to insufficient drug impact, were treated off-label with infliximab for a minimum of six months. Patient files were reviewed retrospectively. RESULTS: Treatment with infliximab led to reduction of inflammation, evidenced by a decrease in transaminases (mean AST prior treatment 475 U/L ± 466, mean AST during treatment 43 U/L ± 32) as well as in immunoglobulins (pretreatment mean IgG 24.8 mg/dl ± 10.1, mean IgG during treatment 17.38 mg/dl ± 6). Infectious complications occurred in seven out of eleven patients and close monitoring was necessary. CONCLUSIONS: Infliximab may be considered as rescue therapy in patients with difficult-to-treat autoimmune hepatitis, albeit treatment may be associated with infectious complications.

FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency.

BACKGROUND & AIMS: The pathogenesis of autoimmune hepatitis (AIH) is not understood, but it was suggested that AIH may be related to a numerical or functional impairment of CD4+CD25+FOXP3+ regulatory T cells (Treg), which are important mediators of immune tolerance to self-antigens. However, the role of Treg in AIH is not clear, since earlier studies reporting Treg impairment had used only CD25 as marker that cannot unambiguously distinguish Treg from activated effector T cells. METHODS: We assessed the frequency and suppressor function of Treg using current staining protocols that can distinguish Treg from activated effector T cells. RESULTS: The frequency of CD4+CD25(high)CD127(low)FOXP3+ Treg cells in blood of AIH patients was not reduced compared to healthy subjects, as shown by flow cytometry and confirmed by quantifying Treg-specific demethylation of the FOXP3 gene. Moreover, the suppressor function of Treg isolated from AIH patients was similar to that of Treg isolated from healthy subjects, indicating that Treg function was not impaired in AIH patients. However, we observed that the Treg frequency was significantly higher in those AIH patients with active disease than in those who were in a state of remission, suggesting that the Treg frequency may increase with the degree of inflammation. Indeed, analysis of FOXP3+ Treg in liver histology revealed that the intrahepatic Treg frequency was higher in AIH patients than in NASH patients and correlated with the inflammatory activity of the liver. CONCLUSIONS: The frequency and function of circulating Treg cells is not impaired in AIH.

Transabdominal Ultrasound and Magnetic Resonance Enterography in Inflammatory Bowel Disease: Results of an Observational Retrospective Single-Center Study.

Purpose Transabdominal ultrasound (US) and magnetic resonance enterography (MRE) are used to assess disease activity and extent in IBD, but their impact on therapeutic decisions is unclear. Therefore, our study has two goals: to compare the usefulness of US and MRE in assessing disease extent and activity in the small and large bowel, and to determine the relevance for clinical decisions in IBD. Materials and Methods We included 54 IBD patients who had undergone both MRE and US within three months. We used the construct reference standard model to compare MRE and US for detecting inflammation and examined the impact on clinical decisions in IBD patients. Results In 54 IBD patients (44 patients Crohn's disease (CD), 5 ulcerative colitis (UC), 5 indeterminate colitis (IC)), 42 patients (77.8%) showed inflammation either in the small or large bowel. Small bowel disease was present in 34 patients (77.3%). Complications were found in 19 patients (35.2%). MRE and US both showed high sensitivity (90.5 and 88.1%) and moderate specificity (50% in MRE and US) for detecting inflammation. MRE revealed higher sensitivity than US for detecting conglomerate tumors without statistical significance (85.7 vs. 71.4%, p=1.0) and equal specificity (97.9 vs 97.7, p=1.0). Therapeutic decisions included steroids in 20 patients (47.6%) and surgery/percutaneous drainage in six patients (14.3%), these decisions were triggered by results of US or MRE in equal distribution. Conclusion US and MRE have comparable sensitivity and specificity for detecting intestinal inflammation and complications in IBD patients. Therefore, both methods are sufficient for making clinical decisions.

Scoring systems for the diagnosis of autoimmune hepatitis: past, present, and future.

The diagnosis of autoimmune hepatitis (AIH) can be challenging due to the variable clinical and laboratory findings. The original diagnostic criteria published in 1993 by the International Autoimmune Hepatitis Group (IAIHG) were revised in 1999 in an attempt to standardize the diagnosis. However, these criteria are complex and can be cumbersome in clinical practice. In 2008, simplified diagnostic criteria were reported to facilitate the bedside diagnosis of AIH. The scoring systems have been evaluated in several retrospective case series and tested for their ability to reliably diagnose and exclude AIH. However, the scoring systems did not fare as well in patients with concomitant cholestatic disease, fatty liver disease, fulminant hepatitis, and pediatric patients. Both positive and negative predictive values are low in these patients. Prospective studies are needed to compare the two scoring systems to determine which (if not all) patients require liver biopsy and which patients would benefit from immunotherapy.

Defective T helper response of hepatocyte-stimulated CD4 T cells impairs antiviral CD8 response and viral clearance.

BACKGROUND & AIMS: In hepatitis, hepatocytes gain the ability to express major histocompatibility complex (MHC) class II molecules and to present antigen to CD4 T cells. Here, we investigated whether MHC class II-expressing hepatocytes influence in vitro the differentiation of CD4 T cells and in vivo the T-cell response to and control of viral infection. METHODS: Class II transactivator-transgenic hepatocytes that constitutively express MHC class II molecules were used to stimulate CD4 T cells in vitro, and the effector response type of the stimulated CD4 T cells was determined. The in vivo relevance of the obtained findings was confirmed by infecting nontransgenic or class II transactivator-transgenic mice with lymphocytic choriomeningitis virus. RESULTS: MHC II-expressing hepatocytes induced T helper cell (Th) 2 differentiation of uncommitted CD4 T cells and abrogated the ability of previously differentiated Th1 to secrete interferon-gamma, even in the presence of proinflammatory microbial signals. The suppression of Th1 responses by hepatocytes was associated with poor expression levels of Th1-promoting Delta-like Notch ligands. In vivo, MHC II expression by hepatocytes impaired the interferon-gamma production by lymphocytic choriomeningitis virus-specific CD4 and CD8 T cells and prolonged viral persistence. CONCLUSIONS: By instructing infiltrating CD4 T cells to differentiate into a less inflammatory phenotype, MHC II-expressing hepatocytes seem to impair antiviral CD8 T-cell responses and viral clearance. Thus, hepatocytes may contribute to the chronicity of hepatitis virus infection.

Mycophenolate mofetil as second line therapy in autoimmune hepatitis?

INTRODUCTION: In patients with autoimmune hepatitis, efficient immunosuppressive therapy is essential to avoid progression to cirrhosis. There is no established second line therapy for patients failing standard therapy with steroids and azathioprine. The aim of this study was to examine the possible role of mycophenolate mofetil (MMF) as second line treatment of autoimmune hepatitis (AIH). PATIENTS AND METHODS: We were able to identify 37 patients (29 women, 8 men) with AIH proven according to International AIH Group criteria who failed standard therapy. One patient on MMF was excluded due to non-compliance. A total of 28 of 36 patients had experienced side effects necessitating stop of treatment. One patient stopped azathioprine due to pregnancy. A total of nine patients did not respond sufficiently to azathioprine. A total of four patients with a treatment duration of 3 months or less because of severe side effects were considered as intolerant to MMF. Remission was defined as aspartate transaminase (ASP) < twice upper normal limit (UNL). RESULTS: Of 36 patients on MMF included in the analysis, 14 patients (39%) experienced remission. A total of 22 patients (61%) did not respond sufficiently to MMF. The response rate to MMF was dependent on the cause of treatment cessation of azathioprine. Of eight patients with prior nonresponse to azathioprine, six (75%) did not respond to MMF and only two (25%) reached biochemical remission. Of 28 patients with azathioprine intolerance in 16 (57%) patients, the response to MMF was insufficient and in 12 patients (43%) remission was reached. The difference did not reach statistical significance due to the relatively small numbers included. CONCLUSION: In the light of its good tolerability, MMF seems to be an alternative for patients who could not tolerate azathioprine previously. However, our data suggest that a majority of patients fail MMF particularly if they are switched because of an insufficient response to azathioprine.

Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils.

TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.

CD4+ T-cell help is required for effective CD8+ T cell-mediated resolution of acute viral hepatitis in mice.

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA-/- mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA-/- mice. The impaired CD8+ T-cell function in CIITA-/- mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.

Diagnostic criteria for autoimmune hepatitis.

The clinical spectrum of autoimmune hepatitis is very wide. In addition, autoimmune hepatitis can present in any age group. Diagnosis is usually made by a combination of clinical, laboratory and histological features. Diagnostic scores can help both in the daily diagnostic work-up of patients, and in allowing comparability of clinical scientific studies. However, all diagnostic scores have limitations in individual cases, which are discussed in this review.

Murine liver antigen presenting cells control suppressor activity of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T cells (Treg) are important mediators of peripheral immune tolerance; however, whether Treg participate also in hepatic immune tolerance is not clear. Therefore, we tested the potential of Treg to suppress stimulation of CD4(+) T cells by liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), or hepatocytes. In the absence of Treg, all 3 types of liver cells could stimulate CD4(+) T cell proliferation; in the presence of Treg, however, CD4(+) T cell proliferation was suppressed. Interaction with KC even stimulated the expansion of the Treg population; LSEC or hepatocytes, in contrast, could not induce proliferation of Treg. Because liver inflammation can be induced by infection, we tested the potential of liver cells to modify Treg suppressor activity in the presence of microbial signals. In the presence of immune-stimulatory CpG-oligonucleotides, LSEC, KC, and hepatocytes could indeed overcome Treg-mediated suppression; in the presence of lipopolysaccharide (LPS), however, only KC and hepatocytes, but not LSEC, could overcome Treg suppressor activity. Hepatocytes from mice with deficient toll-like receptor-4 signaling failed to abrogate Treg suppression in response to LPS, indicating that overcoming Treg suppressor activity was indeed a response of the liver cell and not of the Treg. In conclusion, Treg can suppress CD4(+) T cell stimulation by liver cells. However, in response to microbial signals, the liver cells can overcome the suppressive activity of Treg. Thus, liver cells may facilitate the transition from hepatic immune tolerance to hepatic inflammation by controlling Treg suppressor activity.

MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocyutes.

The ability to activate CD4 T cells is restricted to antigen-presenting cells that express major histocompatibility complex (MHC) class II molecules. Parenchymal cells normally do not express MHC class II molecules; however, in clinical hepatitis, viral or autoimmune, hepatocytes often exhibit aberrant MHC class II expression. It is not known whether MHC class II-expressing hepatocytes can function as antigen-presenting cells, but it has been suggested that aberrant MHC class II expression by parenchymal cells may cause autoimmune disease. Therefore, we generated transgenic mice that specifically overexpress class II transactivator molecules in hepatocytes. Hepatocytes from these mice exhibited stable MHC class II expression and were used to stimulate CD4 T cells from T-cell receptor transgenic mice and CD4 T-cell lines. MHC II-expressing hepatocytes featured costimulatory CD80 molecules and could serve as antigen-presenting cells that were able to process protein antigen and to activate specific CD4 T cells. Nevertheless, the transgenic mice with aberrant hepatocellular MHC class II expression did not exhibit any symptoms of autoimmune disease. In conclusion, MHC II-expressing hepatocytes, as found in clinical hepatitis, can present antigen and activate CD4 T cells. The ability of hepatocytes to present antigen on MHC II molecules does not seem to be a sufficient cause for inflammatory autoimmunity and hepatitis. However, we still need to explore whether such antigen presentation is occurring in vivo. The transgenic mice described in this study may serve as a model to study the immune interaction of hepatocytes and CD4 T cells in both in vitro and in vivo.