Anti-TNFα treatment in Crohn's disease: Impact on hepatic steatosis, gut-derived hormones and metabolic status.

BACKGROUND AND AIMS: An association between Crohn's disease (CD) and hepatic steatosis has been reported. However, the underlying mechanisms of steatosis progression in CD are not clear. Among the most effective CD treatments are agents that inhibit Tumor-Necrosis-Factor (TNF) activity, yet it is unclear why anti-TNFα agents would affect steatosis in CD. Recent studies suggest that microbiome can affect both, CD and steatosis pathogenesis. Therefore, we here analysed a potential relationship between anti-TNF treatment and hepatic steatosis in CD, focusing on the gut-liver axis. METHODS: This cross-sectional study evaluated patients with established CD, with and without anti-TNFα treatment, analysing serum markers of liver injury, measurement of transient elastography, controlled attenuation parameter (CAP) and MRI for fat detection. Changes in lipid and metabolic profiles were assessed by serum and stool lipidomics and metabolimics. Additionally, we analysed gut microbiota composition and mediators of bile acid (BA) signalling via stool and serum analysis. RESULTS: Patients on anti-TNFα treatment had less hepatic steatosis as assessed by CAP and MRI. Serum FGF19 levels were significantly higher in patients on anti-TNFα therapy and associate with reduced steatosis and increased bowel motility. Neutral lipids including triglycerides were reduced in the serum of patients on anti-TNF treatment. Bacteria involved in BA metabolism and FGF19 regulation, including Firmicutes, showed group-specific alterations with low levels in patients without anti-TNFα treatment. Low abundance of Firmicutes was associated with higher triglyceride levels. CONCLUSIONS: Anti-TNFα treatment is associated with reduced steatosis, lower triglyceride levels, alterations in FXR-signalling (eg FGF19) and microbiota composition in CD.

[Histopathological diagnosis and differential diagnosis of nonalcoholic fatty liver disease]. / Histopathologische Diagnostik und Differenzialdiagnostik der nichtalkoholischen Fettlebererkrankung.

Fatty liver disease is a rising problem worldwide, particularly due to metabolic syndrome. The current prevalence is 20-30%, but a further increase is expected whereby children will also be increasingly affected. The presence of fat in hepatocytes is known as steatosis or, in the case of nonalcoholic origin, nonalcoholic fatty liver (NAFL). It is basically reversible, but can progress to steatohepatitis (NASH) as an active and progressive form of fatty liver disease due to continuous cell damage. This leads to progressive liver fibrosis up to end-stage liver cirrhosis. The gold standard of diagnosis is liver biopsy, in which obesity, inflammation, and hepatocellular damage (hepatocellular ballooning) are assessed for the distinction between NAFL and NASH. The extent of fibrosis indicates the progress of the disease. Childhood and adult fatty liver diseases differ morphologically, particularly in the location and amount of fat, inflammation, and fibrosis. Alcoholic and nonalcoholic fatty liver disease/steatohepatitis cannot be reliably differentiated by histology. Clinical parameters must also be taken into consideration for the differential diagnosis of other diseases associated with fatty liver. The main therapeutic goal is to reduce insulin resistance, which can be achieved through weight loss and lifestyle changes. Recently, however, drug therapies have also become available as a promising therapeutic option.

[Liver transplantation. Current aspects of pretransplantation diagnosis and rejection]. / Lebertransplantation. Aktuelle Aspekte der Prätransplantationsdiagnostik und Abstoßung.

Liver transplantation is an established treatment option for patients with end-stage liver disease. The therapy management of these patients is interdisciplinary and requires pathologists to have both clinical and immunological knowledge. Continuous advances in treatment and increasing clinical experience are accompanied by the further development of pathological transplant diagnostics. This article presents and discusses the latest classification of T­cell-mediated rejection (TCMR), antibody-mediated rejection (AMR), and aspects of pretransplant diagnostics.

Spleen Stiffness Differentiates Between Acute and Chronic Liver Damage and Predicts Hepatic Decompensation.

OBJECTIVES: Spleen stiffness (SS) correlates with liver stiffness (LS) and hepatic venous pressure gradient. The latter is currently the most accurate predictor of hepatic decompensation. Our study aims to check whether SS has a similar predictive capability, while being an easy-to-perform noninvasive test in a real-life patient cohort. METHODS: Concomitantly, 210 successive patients were examined and received liver and SS measurements and a standard laboratory. Patients were observed for 1 year in terms of clinical signs of decompensation. RESULTS: One hundred fifty-nine of the initial 210 patients had a valid LS and SS measurement and were evaluable for clinical follow-up. Twelve patients developed a hepatic decompensation; with a SS >39 kPa (P=0.0005). Especially in a group with elevated LS, patients with a high risk of decompensation could be identified using SS. Patients with comparable LS who suffered from acute liver damage had significantly lower SS than respective patients with chronic liver damage (30.97 vs. 46.03 kPa; P=0.04). Acute liver failure was associated with elevated LS (16.47 kPa) but not with elevated SS (30.97 kPa). CONCLUSIONS: The risk of a hepatic decompensation can easily be assessed using SS measurement. Therefore SS measurement might be a powerful screening tool identifying patients who need closer monitoring. Moreover, SS is able to differentiate between acute and chronic or acute on chronic liver damage.

Immunohistochemical Markers Distinguishing Cholangiocellular Carcinoma (CCC) from Pancreatic Ductal Adenocarcinoma (PDAC) Discovered by Proteomic Analysis of Microdissected Cells.

Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumors deriving from PDAC.

Low transferrin and high ferritin concentrations are associated with worse outcome in acute liver failure.

BACKGROUND & AIMS: Serum ferritin and transferrin have been identified as prognostic markers in patients with chronic diseases. In this study, we investigated if these parameters can predict outcome in patients with acute liver failure. METHODS: A total of 102 consecutive patients with acute liver failure were retrospectively analysed. The patients were grouped by outcome: spontaneous recovery vs liver transplantation and/or death or survival vs death. Routine laboratory parameters, transferrin and ferritin concentrations in serum, and anthropomorphic data collected on admission were analysed. RESULTS: Non-spontaneously recovering patients had higher ferritin (12 252±25 791 vs 4434.4±9027.2 µg/L; P<.05) and lower transferrin levels (140.4±66.7 vs 206.9±65.8 mg/dL; P<.05) than spontaneously recovering patients. Similarly non-survivors exhibited higher serum ferritin and lower transferrin than non-transplanted survivors. Patients with severe hepatic inflammation (A3) had higher ferritin levels compared to patients with mild-moderate inflammation (A1-2) (5280±5094 vs 2361±2737 µg/L; P=.025). ROC analysis of single parameters was performed in non-transplanted patients, resulting in an area under the curve, sensitivity and specificity of 0.812%, 83.3%, and 77.1% for age, 0.871%, 84.1% and 75% for transferrin and 0.802%, 91.7% and 62.9% for ferritin. A model incorporating age, MELD and transferrin had the best predictive value with an area under the curve of 0.947, a sensitivity of 100% and corresponding specificity of 77.8%. CONCLUSIONS: High ferritin and low transferrin levels are associated with worse outcome in patients with acute liver failure. A model incorporating age, MELD score and transferrin outperformed MELD score for 90-day overall survival of non-transplanted patients.

Bathing in carbon dioxide-enriched water alters protein expression in keratinocytes of skin tissue in rats.

Beneficial effects of balneotherapy using naturally occurring carbonated water (CO2 enriched) have been known since the Middle Ages. Although this therapy is clinically applied for peripheral artery disease and skin disorder, the underlying mechanisms are not fully elucidated.Under controlled conditions, rats were bathed in either CO2-enriched water (CO2 content 1200 mg/L) or tap water, both at 37 °C, for 10 min daily over 4 weeks. Proliferation activity was assessed by Ki67 immunohistochemistry of the epidermis of the abdomen. The capillary density was assessed by immunodetection of isolectin-positive cells. Using cryo-fixed abdominal skin epidermis, follicle cells and stroma tissue containing capillaries were separately isolated by means of laser microdissection and subjected to proteomic analysis using label-free technique. Differentially expressed proteins were validated by immunohistochemistry.Proliferation activity of keratinocytes was not significantly different in the epidermis after bathing in CO2-enriched water, and also, capillary density did not change. Proteomic analysis revealed up to 36 significantly regulated proteins in the analyzed tissue. Based on the best expression profiles, ten proteins were selected for immunohistochemical validation. Only one protein, far upstream element binding protein 2 (FUBP2), was similarly downregulated in the epidermis after bathing in CO2-enriched water with both techniques. Low FUBP2 expression was associated with low c-Myc immune-expression in keratinocytes.Long-term bathing in CO2-enriched water showed a cellular protein response of epithelial cells in the epidermis which was detectable by two different methods. However, differences in proliferation activity or capillary density were not detected in the normal skin.

Serum sodium based modification of the MELD does not improve prediction of outcome in acute liver failure.

BACKGROUND: Acute liver failure (ALF) is a devastating clinical syndrome with a high mortality rate. The MELD score has been implied as a prognostic tool in ALF. Hyponatremia is associated with lethal outcome in ALF. Inclusion of serum sodium (Na) into the MELD score was found to improve its predictive value in cirrhotic patients. Therefore the aim of this study was to determine whether inclusion of serum Na improves the predictive value of MELD in ALF compared to established criteria. METHODS: In a prospective single center study (11/2006-12/2010), we recruited 108 consecutive ALF patients (64% females / 36% males), who met the criteria defined by the "Acute Liver Failure Study Group Germany". Upon admission, clinical and laboratory data were collected, King's College Criteria (KCC), Model of End Stage Liver Disease score (MELD), and serum sodium based modifications like the MELD-Na score and the United Kingdom Model of End Stage Liver Disease score (UKELD) were calculated and area under the receiver operating characteristic curve analyses were performed regarding the prediction of spontaneous recovery (SR) or non-spontaneous recovery (NSR; death or transplantation). RESULTS: Serum bilirubin was of no prognostic value in ALF, and Na also failed to predict NSR in ALF. The classical MELD score was superior to sodium-based modifications and KCC. CONCLUSIONS: We validated the prognostic value of MELD-Na and UKELD in ALF. Classic MELD score calculations performed superior to KCC in the prediction of NSR. Serum Na and Na-based modifications of MELD did not further improve its prognostic value.

Higher pNRF2, SOCS3, IRF3, and RIG1 Tissue Protein Expression in NASH Patients versus NAFL Patients: pNRF2 Expression Is Concomitantly Associated with Elevated Fasting Glucose Levels.

Non-alcoholic fatty liver disease (NAFLD) embraces simple steatosis in non-alcoholic fatty liver (NAFL) to advanced non-alcoholic steatohepatitis (NASH) associated with inflammation, fibrosis, and cirrhosis. NAFLD patients often have metabolic syndrome and high risks of cardiovascular and liver-related mortality. Our aim was to clarify which proteins play a role in the progression of NAFL to NASH. The study investigates paraffin-embedded samples of 22 NAFL and 33 NASH patients. To detect potential candidates, samples were analyzed by immunohistochemistry for the proteins involved in innate immune regulation, autophagy, apoptosis, and antioxidant defense: IRF3, RIG-1, SOCS3, pSTAT3, STX17, SGLT2, Ki67, M30, Caspase 3, and pNRF2. The expression of pNRF2 immunopositive nuclei and SOCS3 cytoplasmic staining were higher in NASH than in NAFL (p = 0.001); pNRF2 was associated with elevated fasting glucose levels. SOCS3 immunopositivity correlated positively with RIG1 (r = 0.765; p = 0.001). Further, in NASH bile ducts showed stronger IRF3 immunostaining than in NAFL (p = 0.002); immunopositive RIG1 tissue was higher in NASH than in NAFL (p = 0.01). Our results indicate that pNRF2, SOCS3, IRF3, and RIG1 are involved in hepatic lipid metabolism. We suggest that they may be suitable for further studies to assess their potential as therapeutics.

Influence of the Bile Acid Transporter Genes ABCB4, ABCB8, and ABCB11 and the Farnesoid X Receptor on the Response to Ursodeoxycholic Acid in Patients with Nonalcoholic Steatohepatitis.

The prevalence of NAFLD and NASH is increasing worldwide, and there is no approved medical treatment until now. Evidence has emerged that interfering with bile acid metabolism may lead to improvement in NASH. In this study, 28 patients with elevated cholestatic liver function tests (especially GGT) were screened for bile acid gene polymorphisms and treated with UDCA. All patients had a bile acid gene polymorphism in ABCB4 or ABCB11. Treatment with UDCA for 12 months significantly reduced GGT in all patients and ALT in homozygous patients. No difference in fibrosis was observed using FIb-4, NFS, and transient elastography (TE). PNPLA3 and TM6SF2 were the most common NASH-associated polymorphisms, and patients with TM6SF2 showed a significant reduction in GGT and ALT with the administration of UDCA. In conclusion, NASH patients with elevated GGT should be screened for bile acid gene polymorphisms, as UDCA therapy may improve liver function tests. However, no difference in clinical outcomes, such as progression to cirrhosis, has been observed using non-invasive tests (NITs).

Long-term exposure to air pollution and prevalent nonalcoholic fatty liver disease.

Background: Nonalcoholic fatty liver disease (NAFLD) is a disease characterized by lipid accumulation within hepatocytes, ranging from simple steatosis to steatohepatitis, in the absence of secondary causes of hepatic fat accumulation. Although air pollution (AP) has been associated with several conditions related to NAFLD (e.g., metabolic syndrome, type 2 diabetes mellitus), few studies have explored an association between AP and NAFLD. The aim of the study was to investigate whether exposure to AP is associated with NAFLD prevalence. Methods: We used baseline cross-sectional data (2000-2003) of the Heinz-Nixdorf-Recall cohort study in Germany (baseline n = 4,814), a prospective population-based cohort study in the urbanized Ruhr Area. Mean annual exposure to size-fractioned particulate matter (PM10, PM2.5, PMcoarse, and PM2.5abs), nitrogen dioxide, and particle number was assessed using two different exposure models: a chemistry transport dispersion model, which captures urban background AP exposure on a 1 km2 grid at participant's residential addresses, and a land use regression model, which captures point-specific AP exposure at participant's residential addresses. NAFLD was assessed with the fatty liver index (n = 4,065), with NAFLD defined as fatty liver index ≥60. We estimated ORs of NAFLD per interquartile range of exposure using logistic regression, adjusted for socio-demographic and lifestyle variables. Results: We observed a NAFLD prevalence of 31.7% (n = 1,288). All air pollutants were positively associated with NAFLD prevalence, with an OR per interquartile range for PM2.5 of 1.11 (95% confidence interval [CI] = 1.00, 1.24) using chemistry transport model, and 1.06 (95% CI = 0.94, 1.19) using the land use regression model, respectively. Conclusion: There was a positive association between long-term AP exposure and NAFLD.

Effects of Moderate Alcohol Consumption in Non-Alcoholic Fatty Liver Disease.

Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) have emerged as leading causes of chronic liver diseases worldwide. ALD and NAFLD share several pathophysiological patterns as well as histological features, while clinically, they are distinguished by the amount of alcohol consumed daily. However, NAFLD coexists with moderate alcohol consumption in a growing proportion of the population. Here, we investigated the effects of moderate alcohol consumption on liver injury, lipid metabolism, and gut microbiota in 30 NAFLD-patients. We anonymously assessed drinking habits, applying the AUDIT- and CAGE-questionnaires and compared subgroups of abstainers vs. low to harmful alcohol consumers (AUDIT) and Cage 0-1 vs. Cage 2-4. Patients who did not drink any alcohol had lower levels of γGT, ALT, triglycerides, and total cholesterol. While the abundance of Bacteroidaceae, Bifidobacteriaceae, Streptococcaceae, and Ruminococcaceae was higher in the low to harmful alcohol drinking cohort, the abundance of Rikenellaceae was higher in the abstainers. Our study suggests that even moderate alcohol consumption has an impact on the liver and lipid metabolism, as well as on the composition of gut microbiota.

A Potential Role for Bile Acid Signaling in Celiac Disease-Associated Fatty Liver.

Celiac disease (CeD) is a chronic autoimmune disorder characterized by an intolerance to storage proteins of many grains. CeD is frequently associated with liver damage and steatosis. Bile acid (BA) signaling has been identified as an important mediator in gut-liver interaction and the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we aimed to analyze BA signaling and liver injury in CeD patients. Therefore, we analyzed data of 20 CeD patients on a gluten-free diet compared to 20 healthy controls (HC). We furthermore analyzed transaminase levels, markers of cell death, BA, and fatty acid metabolism. Hepatic steatosis was determined via transient elastography, by MRI and non-invasive scores. In CeD, we observed an increase of the apoptosis marker M30 and more hepatic steatosis as compared to HC. Fibroblast growth factor 19 (FGF19) was repressed in CeD, while low levels were associated with steatosis, especially in patients with high levels of anti-tissue transglutaminase antibodies (anti-tTG). When comparing anti-tTG-positive CeD patients to individuals without detectable anti-tTG levels, hepatic steatosis was accentuated. CeD patients with significant sonographic steatosis (defined by CAP ≥ 283 db/m) were exclusively anti-tTG-positive. In summary, our results suggest that even in CeD patients in clinical remission under gluten-free diet, alterations in gut-liver axis, especially BA signaling, might contribute to steatotic liver injury and should be further addressed in future studies and clinical practice.

Risk of recurrent hepatic encephalopathy in patients with liver cirrhosis: a German registry study.

BACKGROUND AND AIMS: Patients with hepatic encephalopathy (HE) show low quality of life, recurrent hospitalizations and an increased mortality. We aimed to assess the natural course of patients after a recent episode of overt HE and to identify risk factors for HE recurrence in Germany. METHODS: Fifteen sites took part in a prospective, observational study including patients with liver cirrhosis who had been hospitalized for HE within 3 months before recruitment. Clinical data, psychometric hepatic encephalopathy score (PHES) and critical flicker frequency were assessed quarterly for 1 year. Primary endpoint was HE recurrence requiring hospitalization, all-cause-mortality was treated as a competing risk factor. RESULTS: From January 2014 to March 2016, a total of 115 patients were recruited. Overall 14 premature deaths were documented. For 78 subjects follow-up data were available in accordance with the protocol. After a median of 118 days, more than half of the per-protocol cohort was readmitted to hospital due to HE (N = 34) or died (N = 11). The risk for hospitalization was significantly increased in patients who had been recruited by liver transplant centers (P = 0.003), had had frequent HE relapses prior to recruitment (P = <0.0001) or an abnormal PHES result of <-4 (P = 0.044). Abnormal PHES results barely missed level of significance as an independent risk factor for re-hospitalization in a multivariable competing risk model (P = 0.093). CONCLUSION: Patients with a history of HE are at high risk for the development of recurrent overt HE demanding hospitalization. The PHES test may aid in detection, monitoring and risk stratification of recurrent HE.

Liver parameters as part of a non-invasive model for prediction of all-cause mortality after myocardial infarction.

INTRODUCTION: Liver parameters are associated with cardiovascular disease risk and severity of stenosis. It is unclear whether liver parameters could predict the long-term outcome of patients after acute myocardial infarction (AMI). We performed an unbiased analysis of the predictive value of serum parameters for long-term prognosis after AMI. MATERIAL AND METHODS: In a retrospective, observational, single-center, cohort study, 569 patients after AMI were enrolled and followed up until 6 years for major adverse cardiovascular events, including cardiac death. Patients were classified into non-survivors (n = 156) and survivors (n = 413). Demographic and laboratory data were analyzed using ensemble feature selection (EFS) and logistic regression. Correlations were performed for serum parameters. RESULTS: Age (73; 64; p < 0.01), alanine aminotransferase (ALT; 93 U/l; 40 U/l; p < 0.01), aspartate aminotransferase (AST; 162 U/l; 66 U/l; p < 0.01), C-reactive protein (CRP; 4.7 U/l; 1.6 U/l; p < 0.01), creatinine (1.6; 1.3; p < 0.01), γ-glutamyltransferase (GGT; 71 U/l; 46 U/l; p < 0.01), urea (29.5; 20.5; p < 0.01), estimated glomerular filtration rate (eGFR; 49.6; 61.4; p < 0.01), troponin (13.3; 7.6; p < 0.01), myoglobin (639; 302; p < 0.01), and cardiovascular risk factors (hypercholesterolemia p < 0.02, family history p < 0.01, and smoking p < 0.01) differed significantly between non-survivors and survivors. Age, AST, CRP, eGFR, myoglobin, sodium, urea, creatinine, and troponin correlated significantly with death (r = -0.29; 0.14; 0.31; -0.27; 0.20; -0.13; 0.33; 0.24; 0.12). A prediction model was built including age, CRP, eGFR, myoglobin, and urea, achieving an AUROC of 77.6% to predict long-term survival after AMI. CONCLUSIONS: Non-invasive parameters, including liver and renal markers, can predict long-term outcome of patients after AMI.

Characterization of two types of intranuclear hepatocellular inclusions in NAFLD.

Nuclear inclusions (NI) are a common finding in hepatocytes from patients with liver disease especially in diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) but studies examining the shape and content of these inclusions in detail are lacking. In this study we define two distinct types of NI in NAFLD: inclusions bounded by the nuclear membrane, containing degenerative cell organelles and heterolysosomes (type1) and inclusions with deposits of glycogen but without any kind of organelles and delimiting membrane (type2). NI in 77 paraffin-embedded patients of NAFLD including NAFL and non-alcoholic steatohepatitis (NASH) were analyzed. In 4-12% of type1 NI immunopositivity for the autophagy-associated proteins LC3B, ubiquitin, p62/sequestosome1, cathepsin D and cathepsin B were detected with co-localizations of ubiquitin and p62; type2 NI showed no immunoreactivity. Three-dimensional reconstructions of isolated nuclei revealed that NI type1 are completely enclosed within the nucleus, suggesting that NI, although probably derived from cytoplasmic invaginations, are not just simple invaginations. Our study demonstrates two morphologically different types of inclusions in NAFLD, whereby both gained significantly in number in advanced stages. We suggest that the presence of autophagy-associated proteins and degenerated organelles within type1 NI plays a role in disease progression.

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Non-invasive assessment of NAFLD as systemic disease-A machine learning perspective.

BACKGROUND & AIMS: Current non-invasive scores for the assessment of severity of non-alcoholic fatty liver disease (NAFLD) and identification of patients with non-alcoholic steatohepatitis (NASH) have insufficient performance to be included in clinical routine. In the current study, we developed a novel machine learning approach to overcome the caveats of existing approaches. METHODS: Non-invasive parameters were selected by an ensemble feature selection (EFS) from a retrospectively collected training cohort of 164 obese individuals (age: 43.5±10.3y; BMI: 54.1±10.1kg/m2) to develop a model able to predict the histological assessed NAFLD activity score (NAS). The model was evaluated in an independent validation cohort (122 patients, age: 45.2±11.75y, BMI: 50.8±8.61kg/m2). RESULTS: EFS identified age, γGT, HbA1c, adiponectin, and M30 as being highly associated with NAFLD. The model reached a Spearman correlation coefficient with the NAS of 0.46 in the training cohort and was able to differentiate between NAFL (NAS≤4) and NASH (NAS>4) with an AUC of 0.73. In the independent validation cohort, an AUC of 0.7 was achieved for this separation. We further analyzed the potential of the new model for disease monitoring in an obese cohort of 38 patients under lifestyle intervention for one year. While all patients lost weight under intervention, increasing scores were observed in 15 patients. Increasing scores were associated with significantly lower absolute weight loss, lower reduction of waist circumference and basal metabolic rate. CONCLUSIONS: A newly developed model (http://CHek.heiderlab.de) can predict presence or absence of NASH with reasonable performance. The new score could be used to detect NASH and monitor disease progression or therapy response to weight loss interventions.

Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival.

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival.