Osteopontin-driven T-cell accumulation and function in adipose tissue and liver promoted insulin resistance and MAFLD.

OBJECTIVE: This study investigated the contribution of osteopontin/secreted phosphoprotein 1 (SPP1) to T-cell regulation in initiation of obesity-driven adipose tissue (AT) inflammation and macrophage infiltration and the subsequent impact on insulin resistance (IR) and metabolic-associated fatty liver disease (MAFLD) development. METHODS: SPP1 and T-cell marker expression was evaluated in AT and liver according to type 2 diabetes and MAFLD in human individuals with obesity. The role of SPP1 on T cells was evaluated in Spp1-knockout mice challenged with a high-fat diet. RESULTS: In humans with obesity, elevated SPP1 expression in AT was parallel to T-cell marker expression (CD4, CD8A) and IR. Weight loss reversed AT inflammation with decreased SPP1 and CD8A expression. In liver, elevated SPP1 expression correlated with MAFLD severity and hepatic T-cell markers. In mice, although Spp1 deficiency did not impact obesity, it did improve AT IR associated with prevention of proinflammatory T-cell accumulation at the expense of regulatory T cells. Spp1 deficiency also decreased ex vivo helper T cell, subtype 1 (Th1) polarization of AT CD4+ and CD8+ T cells. In addition, Spp1 deficiency significantly reduced obesity-associated liver steatosis and inflammation. CONCLUSIONS: Current findings highlight a critical role of SPP1 in the initiation of obesity-driven chronic inflammation by regulating accumulation and/or polarization of T cells. Early targeting of SPP1 could be beneficial for IR and MAFLD treatment.

Arterial Calcifications in Patients with Liver Cirrhosis Are Linked to Hepatic Deficiency of Pyrophosphate Production Restored by Liver Transplantation.

Liver fibrosis is associated with arterial calcification (AC). Since the liver is a source of inorganic pyrophosphate (PPi), an anti-calcifying compound, we investigated the relationship between plasma PPi ([PPi]pl), liver fibrosis, liver function, AC, and the hepatic expression of genes regulating PPi homeostasis. To that aim, we compared [PPi]pl before liver transplantation (LT) and 3 months after LT. We also assessed the expression of four key regulators of PPi in liver tissues and established correlations between AC, and scores of liver fibrosis and liver failure in these patients. LT candidates with various liver diseases were included. AC scores were assessed in coronary arteries, abdominal aorta, and aortic valves. Liver fibrosis was evaluated on liver biopsies and from non-invasive tests (FIB-4 and APRI scores). Liver functions were assessed by measuring serum albumin, ALBI, MELD, and Pugh−Child scores. An enzymatic assay was used to dose [PPi]pl. A group of patients without liver alterations from a previous cohort provided a control group. Gene expression assays were performed with mRNA extracted from liver biopsies and compared between LT recipients and the control individuals. [PPi]pl negatively correlated with APRI (r = −0.57, p = 0.001, n = 29) and FIB-4 (r = −0.47, p = 0.006, n = 29) but not with interstitial fibrosis index from liver biopsies (r = 0.07, p = 0.40, n = 16). Serum albumin positively correlated with [PPi]pl (r = 0.71; p < 0.0001, n = 20). ALBI, MELD, and Pugh−Child scores correlated negatively with [PPi]pl (r = −0.60, p = 0.0005; r = −0.56, p = 0.002; r = −0.41, p = 0.02, respectively, with n = 20). Liver fibrosis assessed on liver biopsies by FIB-4 and by APRI positively correlated with coronary AC (r = 0.51, p = 0.02, n = 16; r = 0.58, p = 0.009, n = 20; r = 0.41, p = 0.04, n = 20, respectively) and with abdominal aorta AC (r = 0.50, p = 0.02, n = 16; r = 0.67, p = 0.002, n = 20; r = 0.61, p = 0.04, n = 20, respectively). FIB-4 also positively correlated with aortic valve calcification (r = 0.40, p = 0.046, n = 20). The key regulator genes of PPi production in liver were lower in patients undergoing liver transplantation as compared to controls. Three months after surgery, serum albumin levels were restored to physiological levels (40 [37−44] vs. 35 [30−40], p = 0.009) and [PPi]pl was normalized (1.40 [1.07−1.86] vs. 0.68 [0.53−0.80] µmol/L, p = 0.0005, n = 12). Liver failure and/or fibrosis correlated with AC in several arterial beds and were associated with low plasma PPi and dysregulation of key proteins involved in PPi homeostasis. Liver transplantation normalized these parameters.

Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.

BACKGROUND & AIMS: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment. METHODS: We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations. RESULTS: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index. CONCLUSIONS: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis. LAY SUMMARY: The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.

SYK-3BP2 Pathway Activity in Parenchymal and Myeloid Cells Is a Key Pathogenic Factor in Metabolic Steatohepatitis.

BACKGROUND & AIMS: Spleen tyrosine kinase (SYK) signaling pathway regulates critical processes in innate immunity, but its role in parenchymal cells remains elusive in chronic liver diseases. We investigate the relative contribution of SYK and its substrate c-Abl Src homology 3 domain-binding protein-2 (3BP2) in both myeloid cells and hepatocytes in the onset of metabolic steatohepatitis. METHODS: Hepatic SYK-3BP2 pathway was evaluated in mouse models of metabolic-associated fatty liver diseases (MAFLD) and in obese patients with biopsy-proven MAFLD (n = 33). Its role in liver complications was evaluated in Sh3bp2 KO and myeloid-specific Syk KO mice challenged with methionine and choline deficient diet and in homozygous Sh3bp2KI/KI mice with and without SYK expression in myeloid cells. RESULTS: Here we report that hepatic expression of 3BP2 and SYK correlated with metabolic steatohepatitis severity in mice. 3BP2 deficiency and SYK deletion in myeloid cells mediated the same protective effects on liver inflammation, injury, and fibrosis priming upon diet-induced steatohepatitis. In primary hepatocytes, the targeting of 3BP2 or SYK strongly decreased the lipopolysaccharide-mediated inflammatory mediator expression and 3BP2-regulated SYK expression. In homozygous Sh3bp2KI/KI mice, the chronic inflammation mediated by the proteasome-resistant 3BP2 mutant promoted severe hepatitis and liver fibrosis with augmented liver SYK expression. In these mice, the deletion of SYK in myeloid cells was sufficient to prevent these liver lesions. The hepatic expression of SYK is also up-regulated with metabolic steatohepatitis and correlates with liver macrophages in biopsy-proven MAFLD patients. CONCLUSIONS: Collectively, these data suggest an important role for the SYK-3BP2 pathway in the pathogenesis of chronic liver inflammatory diseases and highlight its targeting in hepatocytes and myeloid cells as a potential strategy to treat metabolic steatohepatitis.

Prevalence of NASH/NAFLD in people with obesity who are currently classified as metabolically healthy.

BACKGROUND: While metabolic health in obesity may confer a protective status, recent studies indicate that nonalcoholic fatty liver disease (NAFLD) or even nonalcoholic steatohepatitis (NASH) may exist in this category of individuals. Although cardiovascular and diabetic risks have been well described, the risk of NAFLD and NASH among this population requires further investigation. OBJECTIVE: Our goal was to compare the prevalence of steatosis, NAFLD, and NASH between individuals with metabolically healthy obesity (MHO) and individuals with metabolically abnormal obesity (MAO) and to identify preoperative risk factors for these conditions in a prospective cohort with morbid obesity scheduled for bariatric surgery. SETTINGS: Tertiary referral university hospital in France. METHODS: The prospective cohort included 837 bariatric patients who also had an intraoperative liver biopsy between 2002 and 2015. Obese individuals fulfilling none of the criteria in the strict definition of metabolic syndrome were considered metabolically healthy. Preoperative blood samples and liver pathology examinations were reviewed. Steatosis, NAFLD, and NASH were carefully identified allowing comparison of prevalence and risk factors between the 2 cohorts. RESULTS: In total, 149 patients (17.8%) had MHO and the remaining 688 (82.2%) had MAO. The cohort with MHO was significantly younger, had a significantly lower glycosylated hemoglobin, a lower homeostasis model assessment of insulin resistance, and increased C-reactive protein. In individuals with MHO, 44 patients (29.5%) had at least moderate steatosis (>33% macrovesicular steatosis) and 5.4% had NASH. Using logistic regression, waist circumference was positively associated with NASH, whereas body mass index and alanine aminotransferase were significantly associated with severe steatosis (>66%). CONCLUSION: Our study indicates that obese individuals without metabolic syndrome may develop subclinical liver involvement. Therefore, the occurrence of NAFLD and NASH in this population needs further investigation.

Accurate assessment of nonalcoholic fatty liver disease lesions in liver allograft biopsies by a smartphone platform: A proof of concept.

Macrovesicular steatosis (MS) is a major risk factor for liver graft failure after transplantation and pathological microscopic examination of a frozen tissue section remains the gold standard for its assessment. However, the latter requires an experienced in-house pathologist for correct and rapid diagnosis as well as specific equipment that is not always available. Smartphones, which are must-have tools for everyone, are very suitable for incorporation into promising technology to generate moveable diagnostic tools as for telepathology. The study aims to compare the microscopic assessment of nonalcoholic fatty liver disease (NAFLD) spectrum in liver allograft biopsies by a smartphone microscopy platform (DIPLE device) to standard light microscopy. Forty-two liver graft biopsies were evaluated in transmitted light, using an iPhone X and the microscopy platform. A significant correlation was reported between the two different approaches for graft MS assessment (Spearman's correlation coefficient: r = .93; p < .001) and for steatohepatitis feature (r = .56; p < .001; r = .45; p < .001). Based on these findings, a smartphone integrated with a cheap microscopy platform can achieve adequate accuracy in the assessment of NAFLD in liver graft and could be used as an alternative to standard light microscopy when the latter is unavailable.

MCD diet-induced steatohepatitis generates a diurnal rhythm of associated biomarkers and worsens liver injury in Klf10 deficient mice.

A large number of hepatic functions are regulated by the circadian clock and recent evidence suggests that clock disruption could be a risk factor for liver complications. The circadian transcription factor Krüppel like factor 10 (KLF10) has been involved in liver metabolism as well as cellular inflammatory and death pathways. Here, we show that hepatic steatosis and inflammation display diurnal rhythmicity in mice developing steatohepatitis upon feeding with a methionine and choline deficient diet (MCDD). Core clock gene mRNA oscillations remained mostly unaffected but rhythmic Klf10 expression was abolished in this model. We further show that Klf10 deficient mice display enhanced liver injury and fibrosis priming upon MCDD challenge. Silencing Klf10 also sensitized primary hepatocytes to apoptosis along with increased caspase 3 activation in response to TNFα. This data suggests that MCDD induced steatohepatitis barely affects the core clock mechanism but leads to a reprogramming of circadian gene expression in the liver in analogy to what is observed in other experimental disease paradigms. We further identify KLF10 as a component of this transcriptional reprogramming and a novel hepato-protective factor.

No association between binge eating disorder and severity of non-alcoholic fatty liver disease in severely obese patients.

BACKGROUND AND AIM: The main aim of this study was to evaluate if the binge eating disorders (BEDs) related to obesity were associated with the severity of non-alcoholic fatty liver disease (NAFLD). METHODS: Severely obese patients who had been referred for bariatric surgery were included in this study at the Nice University Hospital. All patients underwent a liver biopsy at the time of surgery. Between 2008 and 2015, 388 patients had an assessable Bulimia Test (BULIT) self-questionnaire at the time of surgery. A subgroup (n = 183), between 2011 and 2015, also responded to a Beck Depression Inventory, Hospital Anxiety and Depression Scale, and a Fatigue Impact Scale autoquestionnaire. A control group of 29 healthy people matched by age and gender was included. RESULTS: Among the 388 obese patients (median age 40 years, body mass index 41.7 kg/m2, 81% women), 14 patients had a "probable diagnosis" of BED, and 47 patients had a "high risk" of developing a BED according to the BULIT. Obese patients had significantly more severe BED, depression, anxiety, and fatigue compared to controls. Steatosis, non-alcoholic steatohepatitis, or fibrosis was not associated with BED. Similarly, the severity of NAFLD was not associated with depression, anxiety, or fatigue. CONCLUSIONS: Severely obese patients had more severe BED, depression, anxiety, and fatigue than lean subjects independent of the severity of NAFLD.

REDD1 deficiency protects against nonalcoholic hepatic steatosis induced by high-fat diet.

Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

Hepatic FNDC5 is a potential local protective factor against Non-Alcoholic Fatty Liver.

The proteolytic cleavage of Fibronectin type III domain-containing 5 (FNDC5) generates soluble irisin. Initially described as being mainly produced in muscle during physical exercise, irisin mediates adipose tissue thermogenesis and also regulates carbohydrate and lipid metabolism. The aim of this study was to evaluate the hepatic expression of FNDC5 and its role in hepatocytes in Non-Alcoholic Fatty Liver (NAFL). Here we report that hepatic expression of FNDC5 increased with hepatic steatosis and liver injury without impacting the systemic level of irisin in mouse models of NAFLD (HFD and MCDD) and in obese patients. The increased Fndc5 expression in fatty liver resulted from its upregulation in hepatocytes and non-parenchymal cells in mice. The local production of Fndc5 in hepatocytes was influenced by genotoxic stress and p53-dependent pathways. The down-regulation of FNDC5 in human HepG2 cells and in primary mouse hepatocytes increased the expression of PEPCK, a key enzyme involved in gluconeogenesis associated with a decrease in the expression of master genes involved in the VLDL synthesis (CIDEB and APOB). These alterations in FNDC5-silenced cells resulted to increased steatosis and insulin resistance in response to oleic acid and N-acetyl glucosamine, respectively. The downregulation of Fndc5 also sensitized primary hepatocytes to apoptosis in response to TNFα, which has been associated with decreased hepatoprotective autophagic flux. In conclusion, our human and experimental data strongly suggest that the hepatic expression of FNDC5 increased with hepatic steatosis and its upregulation in hepatocytes could dampen the development of NAFLD by negatively regulating steatogenesis and hepatocyte death.

The Differential Expression of Cide Family Members is Associated with Nafld Progression from Steatosis to Steatohepatitis.

Improved understanding of the molecular mechanisms responsible for the progression from a "non-pathogenic" steatotic state to Non-Alcoholic Steatohepatitis is an important clinical requirement. The cell death-inducing DFF45 like effector (CIDE) family members (A, B and FSP27) regulate hepatic lipid homeostasis by controlling lipid droplet growth and/or VLDL production. However, CIDE proteins, particularly FSP27, have a dual role in that they also regulate cell death. We here report that the hepatic expression of CIDEA and FSP27 (α/ß) was similarly upregulated in a dietary mouse model of obesity-mediated hepatic steatosis. In contrast, CIDEA expression decreased, but FSP27-ß expression strongly increased in a dietary mouse model of steatohepatitis. The inverse expression pattern of CIDEA and FSP27ß was amplified with the increasing severity of the liver inflammation and injury. In obese patients, the hepatic CIDEC2 (human homologue of mouse FSP27ß) expression strongly correlated with the NAFLD activity score and liver injury. The hepatic expression of CIDEA tended to increase with obesity, but decreased with NAFLD severity. In hepatic cell lines, the downregulation of FSP27ß resulted in the fractionation of lipid droplets, whereas its overexpression decreased the expression of the anti-apoptotic BCL2 marker. This, in turn, sensitized cells to apoptosis in response to TNF α and saturated fatty acid. Considered together, our animal, human and in vitro studies indicate that differential expression of FSP27ß/CIDEC2 and CIDEA is related to NAFLD progression and liver injury.

The mid-infrared spectroscopy: A novel non-invasive diagnostic tool for NASH diagnosis in severe obesity.

There is an urgent medical need to develop non-invasive tests for non-alcoholic steatohepatitis (NASH). This study evaluates the diagnostic performance of an innovative model based on mid-infrared (MIR) spectroscopy for the diagnosis of NASH. METHODS: Severely obese patients who underwent a bariatric procedure at the University Hospital of Nice, France (n = 395) were prospectively recruited. The clinico-biological characteristics were measured prior to surgery. Liver biopsies were collected during the surgical procedure and assessed by a pathologist. A training group (316 patients, NASH: 16.8%) and a validation group (79 patients, NASH: 16.5%) were randomly defined. MIR spectra were acquired by fiber evanescent wave spectroscopy, using chalcogenide glass fiber optic sensors and a spectrometer. This absorption spectroscopic technique delivers a spectrum that identifies the molecular composition of a sample, defining a patient's metabolic fingerprint. RESULTS: The areas under the receiver operating curve (AUROC) for the diagnosis of NASH were 0.82 and 0.77 in the training and validation groups, respectively. The best threshold was 0.15, which was associated with a sensitivity of 0.75 and 0.69, and a specificity of 0.72 and 0.76. Negative predictive values of 0.94 and 0.93 and positive predictive values of 0.35 and 0.36, as well as correctly classified patient rates of 72% and 75% were obtained in the training and validation groups, respectively. A composite model using aspartate aminotransferase level, triglyceride level and waist circumference alongside the MIR spectra led to an increase in AUROC (0.88 and 0.84 for the training and validations groups, respectively). CONCLUSIONS: MIR spectroscopy provides good sensitivity and negative predictive values for NASH screening in patients with severe obesity. LAY SUMMARY: There is an urgent need for tools to non-invasively diagnose and monitor non-alcoholic steatohepatitis (NASH). This study evaluates the performance of a new tool for fast NASH diagnosis based on mid-infrared (MIR) spectroscopy. Using serum samples from severely obese patients who underwent a bariatric procedure, which enabled a concomitant liver biopsy to be performed, the MIR spectroscopy model performed well in screening patients for NASH compared to a traditional, histological diagnosis.

Bax inhibitor-1 protects from nonalcoholic steatohepatitis by limiting inositol-requiring enzyme 1 alpha signaling in mice.

Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress-dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B-cell lymphoma 2 (BCL2)-associated X protein (Bax) inhibitor-1 (BI-1), a negative regulator of the ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI-1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild-type and BI-1-/- mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high-fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM-treated BI-1-/- mice showed IRE1α-dependent NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI-1 down-regulation parallel to the up-regulation of IRE1α endoribonuclease (RNase) signaling. In HFD-fed BI-1-/- mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase-1/-11. Rises in interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF-083010 or 4µ8c, was evaluated in HFD-induced NAFLD. In BI-1-/- mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte-specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF-083010 or 4µ8c. CONCLUSION: Targeting IRE1α-dependent NLRP3 inflammasome signaling with pharmacological agents or by BI-1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).

Non-invasive Evaluation of NAFLD with Indocyanine Green Clearance Test: a Preliminary Study in Morbidly Obese Patients Undergoing Bariatric Surgery.

BACKGROUND: Overweight and obesity dramatically increased in the last years. Hepatic complication of obesity, integrated in the term of non-alcoholic fatty liver disease (NAFLD), is a spectrum of abnormality ranging from steatosis to non-alcoholic steatohepatitis (NASH), potentially leading to cirrhosis. Liver biopsy remains the gold standard to evaluate the stage of NAFLD; however, the procedure is invasive. The indocyanine green (ICG) clearance test is performed since years to assess hepatic function before partial hepatectomy, or after liver transplantation. This study was designed to detect liver complications with the ICG clearance test in a population of obese patients scheduled for bariatric surgery. METHODS: In a prospective cohort study, morbidly obese individuals receiving bariatric surgery with scheduled hepatic biopsies were investigated. Liver function was determined by the ICG test preoperatively, and blood samples were collected. Liver biopsy specimens were obtained for each patient and classified according to the NAFLD activity score (NAS) by a single pathologist that was blinded to the results of the ICG test. RESULTS: Twenty-six patients were included (7 male and 19 female). The mean age of participants was 45.8 years; the mean body mass index was 41.4 kg/m2. According to the NAS, 6 (23.1%) patients revealed manifest NASH, and 5 patients were considered borderline (19.2%). A closed correlation was observed between the ICG clearance test and hepatic steatosis (r = 0.43, p = 0.03), NAS (r = 0.44, p = 0.025), and fibrosis (r = 0.49, p = 0.01). CONCLUSIONS: In obese patients, non-invasive evaluation of liver function with the indocyanine green clearance test correlated with histological features of NAFLD. This may detect non-invasively hepatopathy in obese population and could motive biopsy.

CD44 is a key player in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Cluster of differentiation (CD)44 regulates adipose tissue inflammation in obesity and hepatic leukocyte recruitment in a lithogenic context. However, its role in hepatic inflammation in a mouse model of steatohepatitis and its relevance in humans have not yet been investigated. We aimed to evaluated the contribution of CD44 to non-alcoholic steatohepatitis (NASH) development and liver injury in mouse models and in patients at various stages of non-alcoholic fatty liver disease (NAFLD) progression. METHODS: The role of CD44 was evaluated in CD44-/- mice and after injections of an αCD44 antibody in wild-type mice challenged with a methionine- and choline-deficient diet (MCDD). In obese patients, hepatic CD44 (n=30 and 5 NASH patients with a second liver biopsy after bariatric surgery) and serum sCD44 (n=64) were evaluated. RESULTS: Liver inflammation (including inflammatory foci number, macrophage and neutrophil infiltration and CCL2/CCR2 levels), liver injury and fibrosis strongly decreased in CD44-/- mice compared to wild-type mice on MCDD. CD44 deficiency enhanced the M2 polarization and strongly decreased the activation of macrophages by lipopolysaccharide (LPS), hepatocyte damage-associated molecular patterns (DAMPs) and saturated fatty acids. Neutralization of CD44 in mice with steatohepatitis strongly decreased the macrophage infiltration and chemokine ligand (CCL)2 expression with a partial correction of liver inflammation and injury. In obese patients, hepatic CD44 was strongly upregulated in NASH patients (p=0.0008) and correlated with NAFLD activity score (NAS) (p=0.001), ballooning (p=0.003), alanine transaminase (p=0.005) and hepatic CCL2 (p<0.001) and macrophage marker CD68 (p<0.001) expression. Correction of NASH was associated with a strong decrease in liver CD44+ cells. Finally, the soluble form of CD44 increased with severe steatosis (p=0.0005) and NASH (p=0.007). CONCLUSION: Human and experimental data suggest that CD44 is a marker and key player of hepatic inflammation and its targeting partially corrects NASH. LAY SUMMARY: Human and experimental data suggest that CD44, a cellular protein mainly expressed in immune cells, is a marker and key player of non-alcoholic steatohepatitis (NASH). Indeed, CD44 enhances the non-alcoholic fatty liver (NAFL) (hepatic steatosis) to NASH progression by regulating hepatic macrophage polarization (pro-inflammatory phenotype) and infiltration (macrophage motility and the MCP1/CCL2/CCR2 system). Targeting CD44 partially corrects NASH, making it a potential therapeutic strategy.

Acquired Localized (Monodactylous) Longitudinal Pachyonychia and Onychocytic Carcinoma In Situ (2 Cases): Part II.

The authors have recently proposed to use the name pachyonychia as an umbrella term that includes 2 distinctly different entities in the form of congenital pachyonychia and acquired longitudinal pachyonychia. Congenital pachyonychia is defined by a diffuse hyperkeratosis of the nail bed. Acquired longitudinal pachyonychia correspond to the true definition of pachyonychia, that is, a thickened nail plate without subungual keratosis. Acquired (monodactylous) pachyonychia can be diffuse typifying onychomatricoma or localized as a longitudinal band with three etiological possibilities: onychomatricoma, onychocytic matricoma, and onychocytic carcinoma. On histopathological examination, onychomatricoma is a fibroepithelial tumor; conversely, onychocytic matricoma and carcinoma are purely epithelial tumors. The purpose of this study was to present the clinical, pathological, and immunohistochemical profiles of 2 cases of in situ onychocytic carcinoma, to discuss the role of high-risk human papillomavirus in the pathogenesis of this tumor, and to confirm the interest value of nail clipping microscopy in the surgical planning of the acquired (monodactylous) pachyonychia band pattern. Two men were referred for a longitudinal thickening of the lateral part of the nail plate of their fingers. The bands were yellowish with a faint to dominant black coloration. Nail clippings were obtained before excision in one case; the nail plate was thickened with minute holes in a honeycomb pattern. Nail biopsy specimens revealed an onychocytic carcinoma with epithelial projections inducing active production of the nail plate and multiple minute cavitations into the thick nail plate. Hair-related keratins were expressed in all specimens. One tumor expressed p16 in a diffuse pattern, whereas the other showed an unspecific faint patchy staining. p53 was negative. A clinical finding of longitudinal pachyonychia, which by nail clipping microscopy contains small cavities in a honeycomb-like pattern, should be considered an alert to the possibility of a malignant lesions.

Roux-En Y Gastric Bypass Results in Long-Term Remission of Hepatocyte Apoptosis and Hepatic Histological Features of Non-alcoholic Steatohepatitis.

The long-term effects of bariatric surgery on non-alcoholic steatohepatitis (NASH), focusing on liver injury and hepatocyte apoptosis, are not well-established. We here performed a longitudinal study with paired liver biopsies of nine morbidly obese women (median BMI: 42 [38.7; 45.1] kg/m(2)) with NASH with a median follow-up of 55 [44; 75] months after laparoscopic Roux-en-Y gastric bypass (LRYGB) surgery. LRYGB surgery was associated with significant weight loss (median BMI loss -13.7 [-16.4; -9.5] kg/m(2)), improved hepatic steatosis in all patients (55.5% with total resolution), and resolution of hepatic inflammation and hepatocyte ballooning in 100 and 88.8% of cases, respectively. Alanine aminotransferase levels dropped to normal values while hepatic activated cleaved caspase-3 levels strongly decreased after a median follow-up of 55 months. Hepatocyte apoptosis, as evaluated by serum caspase-generated keratin-18 fragment, improved within the first year following LRYGB and these improvements persisted for at least 55 months. LRYGB in morbidly obese patients with NASH is thus associated with a long-lasting beneficial impact on hepatic steatohepatitis and hepatocyte death.

Study of LAT1 Expression in Brain Metastases: Towards a Better Understanding of the Results of Positron Emission Tomography Using Amino Acid Tracers.

Positron emission tomography using radiolabeled amino acid (PET-AA) appears to be promising in distinguishing between recurrent tumour and radionecrosis in the follow-up of brain metastasis (BM). The amino acid transporter LAT1 and its cofactor CD98, which are involved in AA uptake, have never been investigated in BM. The aim of our study was to determine and compare the expression of LAT1 and CD98 in BM and in non-tumoral brain tissue (NT). The expression of LAT1 and CD98 were studied by immunohistochemistry in 67 BM, including 18 BM recurrences after radiotherapy, in 53 NT, and in 13 cases of patients with previously irradiated brain tumor and investigated by [18F] FDOPA-PET. LAT1 and CD98 expression were detected in 98.5% and 59.7% of BM respectively and were significantly associated with BM tissue as compared to NT (p<0.001). LAT1 expression in recurrent BM was significantly increased as compared to newly occurring BM. Ten cases investigated by [18F] FDOPA-PET corresponding to recurrent BM displayed significant [18F] FDOPA uptake and LAT1 overexpression whereas three cases corresponding to radionecrosis showed no or low uptake and LAT1 expression. LAT1 expression level and [18F] FDOPA uptake were significantly correlated. In conclusion, we hypothesized that BM may overexpress the AA transporter LAT1. We have shown that LAT1 overexpression was common in BM and was specific for BM as compared to healthy brain. These results could explain the specific BM uptake on PET-AA.

Severe Vitamin D Deficiency Is Not Associated with Liver Damage in Morbidly Obese Patients.

BACKGROUND AND AIMS: A deficiency in vitamin D could be deleterious during chronic liver diseases. However, contradictory data have been published in patients with non-alcoholic fatty liver disease. The aim of the study was to compare the blood level of 25 hydroxy vitamin D (25-OH vitamin D) with the severity of liver lesions, in a large cohort of morbidly obese patients. PATIENTS AND METHOD: Three hundred ninety-eight morbidly obese patients had a liver biopsy. The non-alcoholic steatohepatitis (NASH) Clinical Research Network Scoring System Definition and Scores were used. 25-OH vitamin D was evaluated with a Diasorin®Elisa Kit. Logistic regression analyses were performed to obtain predictive factors of the severity of liver histology. RESULTS: 20.6 % of patients had NASH. The stage of fibrosis was F0 12.9 %, F1 57.36 %, F2 25.32 %, F3 (bridging fibrosis) 3.88 %, and F4 (cirrhosis) 0.52 %. The 25-OH vitamin D level inversely correlated to the NAS (r = 0.12 and p = 0.01) and to steatosis (r = 0.14 and p = 0.007); however, it was not associated with the presence of NASH. The level of vitamin D was significantly lower in patients with significant fibrosis compared to those without (15.9 (11.1-23.5) vs 19.6 (13.7-24.7) ng/ml, p = 0.02). There was an inverse correlation between the severity of fibrosis and the values of 25-OH vitamin D (r = 0.12 and p = 0.01). In a logistic regression analysis, no parameters were independently associated with the severity of fibrosis except the presence of steatohepatitis (1.94 (1.13-3.35) p = 0.017). CONCLUSION: Low levels of 25-OH vitamin D were not independently associated with liver damage in morbidly obese patients with non-alcoholic fatty liver diseases (NAFLD).