Mincle-GSDMD-mediated release of IL-1ß small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

BACKGROUND: Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1ß production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1ß release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating ß-glucosylceramide (ß-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which ß-GluCer engages Mincle on hepatic macrophages to release IL-1ß in the absence of cell death and exacerbates ALD. METHOD AND RESULTS: Concentrations of ß-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and ß-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1ß and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1ß-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. CONCLUSION: ß-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1ß-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis.

BACKGROUND AND AIMS: Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. APPROACH AND RESULTS: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. CONCLUSION: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

PSA cancer screening: a case for shared decision-making.

Whether to screen for prostate cancer using PSA testing is a difficult decision for many men. Here's the information you need to help them make an educated choice.

HDL flux is higher in patients with nonalcoholic fatty liver disease.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS (n = 7), NASH (n = 8), and healthy controls (n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher (P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio (P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL-1·min-1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.

A 39-Year-Old Man With Diabetes, Pleuritic Chest Pain, and Multiple Cavitary Lung Nodules.

CASE PRESENTATION: A 39-year-old male presented to the ED with a 2-day history of fever (Temperature-Maximum 39°C), nonbloody productive cough, and worsening right-sided pleuritic chest pain. The patient denied shortness of breath, nausea, vomiting, sinus symptoms, and abdominal pain. His medical history included type 2 diabetes mellitus (glycated hemoglobin, 11.1), hyperlipidemia, and depression. He smoked marijuana but denied tobacco or illicit drug use. He reported no recent travels. He reported a 1-week history of left molar pain that began after he siphoned stagnant water with a straw from a refrigerator drip pan. He lived in Ohio all of his life. He denied any sick contacts. His medications include Lantus insulin at night, metformin, glimepiride, pravastatin, and Remeron.

Patients with Nonalcoholic Fatty Liver Disease Have a Low Response Rate to Vitamin D Supplementation.

Background: Hypovitaminosis D is associated with an increased severity of nonalcoholic fatty liver disease (NAFLD), but reports on the response to cholecalciferol (vitamin D3) supplementation are conflicting.Objective: The objective of this study was to determine if standard vitamin D3 supplementation is effective in NAFLD with hypovitaminosis D.Methods: Sixty-five well-characterized adults [age (mean ± SD): 51.6 ± 12.3 y] with biopsy-proven NAFLD were screened. Forty-two patients (the ratio of men to women was 13:29) had hypovitaminosis D (plasma 25-hydroxyvitamin D [25(OH)D] <30 ng/mL). An observational study was performed in NAFLD patients with hypovitaminosis D treated with 2000 IU cholecalciferol (vitamin D3) daily for 6 mo per clinical practice. Plasma 25(OH)D, hepatic and metabolic panels, and metabolic syndrome components were assessed before and after cholecalciferol supplementation. Body composition was measured by using bioelectrical impedance analysis. The primary outcome measure was plasma 25(OH)D ≥30 ng/mL at the end of the study. Secondary outcomes included change in serum transaminases, fasting plasma glucose, and insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Chi-square, Student's t tests, correlation coefficient, and multivariate analysis were performed.Results: Twenty-six (61.9%) patients had nonalcoholic steatohepatitis (NASH), and 16 (38.1%) had hepatic steatosis. After 6 mo of cholecalciferol supplementation, plasma 25(OH)D ≥30 ng/mL was observed in 16 subjects (38.1%; responders) whereas the remaining 26 patients (61.9%) were nonresponders with plasma 25(OH)D <30 ng/mL. Significantly fewer (P < 0.01) patients with NASH were responders (4 of 26, 15.4%) than those with hepatic steatosis (12 of 16, 75%). Baseline fasting serum alanine aminotransferase, plasma glucose, and HOMA-IR were similar in the responders and nonresponders, but the NASH score on the liver biopsy was lower (16.5%) in the responders (P < 0.001). Nonresponders had a higher fat mass (10.5%) and lower fat-free mass (10.4%) than responders did. End-of-treatment alanine aminotransferase and HOMA-IR improved only in responders. The baseline HOMA-IR and histological NASH score were independent predictors of nonresponse to cholecalciferol supplementation.Conclusions: Daily supplementation with 2000 IU cholecalciferol for 6 mo did not correct hypovitaminosis D in the majority of patients with NASH. Further studies are needed to determine if higher doses are effective. This trial was registered at clinicaltrials.gov as 13-00153.

The development of a non-invasive model to predict the presence of non-alcoholic steatohepatitis in patients with non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is an advanced and aggressive form of non-alcoholic fatty liver disease (NAFLD), which remains difficult to diagnose without a liver biopsy. Hyperferritinemia has increasingly been associated with the presence of NASH. Hence, we sought to explore the relationship between ferritin and NASH and to develop a composite model based on ferritin to predict the presence of NASH. METHODS: A total of 405 patients with biopsy-proven NAFLD were enrolled in the study. Comparison was explored to assess differences between patients with and without NASH, upon which a scoring model was established using variables found to be independent predictors of NASH. RESULTS: Among all patients with NAFLD, 291 (72%) had biopsy-proven NASH, and 114 (28%) had non-NASH. Mean age was 48 ± 12 years, and 56% were female. Ferritin was significantly higher in NASH compared with non-NASH patients (184 vs 126, respectively; P < 0.001) but lacked diagnostic accuracy for predicting NASH alone (area under the curve [AUC 0.62]). The addition of other significant variables such as aspartate aminotransferase, body mass index, platelet count, diabetes, and hypertension to ferritin improved the prediction of NASH with an AUC 0.81 (95% confidence interval: 0.76-0.86). Internal validation of the model using imputed data sets demonstrated that AUC did not change materially. CONCLUSIONS: While higher ferritin was significantly associated with NASH, ferritin alone lacked diagnostic accuracy to predict NASH. However, incorporating several easily obtainable variables with ferritin allowed the construction of a novel scoring system that can be easily applied in the clinical setting to guide management of NAFLD.

Clinical spectrum of non-alcoholic fatty liver disease in diabetic and non-diabetic patients.

BACKGROUND: While non-alcoholic fatty liver disease (NAFLD) has been well characterised in patients with diabetes mellitus (DM), less is known about NAFLD in non-DM patients. We investigated the clinical characteristics of NAFLD patients with and without DM and accuracy of the NAFLD fibrosis score (NFS) in these two NAFLD groups. METHODS: Clinical, biochemical and histological variables were evaluated in this prospective cross-sectional study of 503 patients with biopsy proven NAFLD. Comparisons between patients with and without DM were analysed. NFS was correlated with liver histology to assess its robustness in patients with and without DM. RESULTS: There were 503 biopsy proven NAFLD patients with 48% of the cohort being diabetic. Relative to patients without DM, patients with DM were older (52 vs. 46 years, p < 0.001), with higher proportion of females (70% vs. 54%, p < 0.001), higher BMI (37 vs. 35, p = 0.009), higher prevalence of hypertension (73% vs. 44%, p < 0.001), higher prevalence of NASH (80.2% vs. 64.4%; p < 0.001) and advanced fibrosis (40.3% vs. 17.0%; p < 0.001). A considerable amount of patients without DM still had NASH (64%) and advanced fibrosis (17%). The clinical utility of the NFS differed between NAFLD patients with and without DM, with sensitivity to exclude advanced fibrosis being 90% of NAFLD patients with DM but only 58% of patients without DM. CONCLUSION: Patients with DM have more severe NAFLD based on histology. However, NASH and advanced fibrosis also occur in a considerable proportion of NAFLD patients without DM. The lower utility of the NFS in NAFLD patients without DM emphasises the heterogeneous nature of the NAFLD phenotype.

Age impacts ability of aspartate-alanine aminotransferase ratio to predict advanced fibrosis in nonalcoholic Fatty liver disease.

BACKGROUND AND AIM: While histological differences have been reported between pediatric and adult nonalcoholic fatty liver disease (NAFLD), potential age-related changes in serum transaminases and liver histology remain largely unexplored. Our study sought to investigate the clinical and histological characteristics of NAFLD across age. METHODS: This was a prospective cross-sectional study of 502 biopsy-proven NAFLD patients. Clinical data were evaluated and compared among different age groups; group A (ages 18-44), B (ages 45-64), and C (≥ ages 65). RESULTS: 34.9, 56.0, and 9.1 % of the cohort were distributed among group A, B, and C, respectively. While the prevalence of nonalcoholic steatohepatitis (NASH) was comparable across age groups, the prevalence of advanced fibrosis increased with age (p = 0.000). Although the mean ALT progressively decreased with age; 87, 64, 56 U/L in group A, B, and C, respectively (p = 0.000), there was no difference in mean AST (p = 0.939) across age. The AST:ALT ratio (AAR) progressively increased from 0.7, 0.9, and 1.1 in group A, B, and C, respectively (p = 0.000). In group C, an AAR ≥ 1 was found in 74 and 40 % of patients with and without advanced fibrosis. CONCLUSION: With advancing age, ALT levels progressively declined while AST levels remained stable, leading to a higher AAR. Although higher AAR is often used as a surrogate measure of advanced fibrosis, advancing age can also contribute to increased AAR. In fact, an AAR ≥ 1 was found in significant number of elderly patients without advanced fibrosis. Consequently, an increased AAR may be a function of decreasing ALT with age in addition to progressive fibrosis.

Double-blind randomized placebo-controlled clinical trial of omega 3 fatty acids for the treatment of diabetic patients with nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is common and severe in patients with diabetes mellitus. Although, there are no effective treatments for NASH in diabetic patients, preliminary reports suggest that polyunsaturated fatty acids (PUFA) may be beneficial in these patients. AIM: A prospective, randomized, double-blind placebo-controlled study (NCT 00323414) was performed in NASH patients with diabetes. Clinicaltrials.gov (NCT 00323414). SUBJECTS AND METHODS: A total of 37 patients (50.6 ± 9.8 y) with well-controlled diabetes (HbA1C<8.5%) were randomized to receive either PUFA containing eicosapentaenoic acid (2160 mg) and docosahexaenoic acid (1440 mg) daily or an isocaloric, identical placebo containing corn oil for 48 weeks under CONSORT guidelines. Clinical, demographics, biochemical laboratory tests, body composition using DEXA, and liver biopsy were performed at randomization and at the end of treatment. Liver biopsy was scored by the NASH CRN criteria. An intention-to-treat analysis was performed. RESULTS: At inclusion, sex, age, body weight, biochemical tests, glucose control, and liver histology were similar in the 2 treatment groups. There was no change in liver enzymes, body weight, or body composition during the study in either group. At the end of the treatment, hepatic steatosis and the activity score improved (P<0.05) and lobular inflammation worsened (P<0.001) with placebo but was unchanged with PUFA. At the end of the treatment, insulin resistance (serum glucose and HOMA) worsened with PUFA but not placebo. CONCLUSIONS: PUFA provided no benefit over placebo in NASH patients with diabetes. The effects of PUFA on histology and insulin resistance were inferior to placebo. These data provide no support for PUFA supplements in NASH.

Renin-angiotensin system and fibrosis in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Therapeutic options are limited for patients with non-alcoholic fatty liver disease (NAFLD). One promising approach is the attenuation of necroinflammation and fibrosis by inhibition of the renin-angiotensin system (RAS). We explored whether the risk of fibrosis was associated with the use of commonly used medications in NAFLD patients with hypertension. Specifically, we sought to determine the association between RAS blocking agents and severity of hepatic fibrosis in NAFLD patients with hypertension. METHODS: Cross-sectional study where clinical information including demographics, anthropometry, medical history, concomitant medication use, biochemical and histological features were ascertained in 290 hypertensive patients with biopsy proven NAFLD followed at two hepatology outpatient clinics. Stage of hepatic fibrosis was compared in patients with and without RAS blocker use. Other risk factors for fibrosis were evaluated from the electronic medical records and patient follow-up. RESULTS: Baseline characteristics of hypertensive patients treated with and without RAS blockers were similar except for less ballooning (1.02 vs. 1.31, P = 0.001) and lower fibrosis stage (1.63 vs. 2.16, P = 0.002) in patients on RAS blockers On multivariate analysis, advancing age (OR: 1.04; 95%CI: 1.01-1.06, P = 0.012) and presence of diabetes (OR: 2.55; 95%CI: 1.28-5.09, P = 0.008) had an independent positive association, while use of RAS blockers (OR: 0.37; 95%CI: 0.21-0.65, P = 0.001) and statins (OR: 0.52; 95%CI: 0.29-0.93, P = 0.029) had a negative association with advanced fibrosis. CONCLUSION: Hypertensive patients with NAFLD on baseline RAS blockers had less advanced hepatic fibrosis suggesting a beneficial effect of RAS blockers in NAFLD.

Hypovitaminosis D is associated with increased whole body fat mass and greater severity of non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Hypovitaminosis D is common in obesity and insulin-resistant states. Increased fat mass in patients with non-alcoholic fatty liver disease (NAFLD) may contribute to hypovitaminosis D. To determine the relation among plasma vitamin D concentration, severity of disease and body composition in NAFLD. METHODS: Plasma vitamin D concentration was quantified in 148 consecutive biopsy-proven patients with NAFLD (non-alcoholic steatohepatitis - NASH: n = 81; and hepatic steatosis: n = 67) and healthy controls (n = 39). NAFLD was scored using the NASH CRN criteria. Body composition was quantified by bioelectrical impedance analysis and abdominal CT image analysis. RESULTS: Plasma vitamin D concentration was significantly lower in NAFLD (21.2 ± 10.4 ng/ml) compared with healthy controls (35.7 ± 6.0 ng/ml). Higher NAFLD activity scores were associated with lower plasma concentration of vitamin D (r(2)  = 0.29; P < 0.001). Subgroup analysis among patients with NAFLD showed that patients with NASH had significantly lower (P < 0.01) vitamin D levels than those with steatosis alone (18.1 ± 8.4 vs. 25.0 ± 11.3 ng/ml). Low concentrations of vitamin D were associated with greater severity of steatosis, hepatocyte ballooning and fibrosis (P < 0.05).On multivariate regression analysis, only severity of hepatocyte ballooning was independently associated (P = 0.02) with low vitamin D concentrations. Plasma vitamin D (P = 0.004) and insulin concentrations (P = 0.03) were independent predictors of the NAFLD activity score on biopsy. Patients with NAFLD had higher fat mass that correlated with low vitamin D (r(2)  = 0.26; P = 0.008). CONCLUSIONS: Low plasma vitamin D concentration is an independent predictor of the severity of NAFLD. Further prospective studies demonstrating the impact of vitamin D replacement in NAFLD patients are required.

Methionine metabolism in human pregnancy.

BACKGROUND: Hyperhomocysteinemia during pregnancy, which is a consequence of perturbations in methionine and/or folate metabolism, has been implicated in adverse outcomes such as neural tube defects, preeclampsia, spontaneous abortion, and premature delivery. The adaptive changes in methionine metabolism during pregnancy in humans have not been determined. OBJECTIVE: Our objective was to examine the kinetics of methionine and its rate of transsulfuration and transmethylation in healthy women with advancing gestation. DESIGN: The whole-body rate of appearance (Ra) of methionine and phenylalanine was measured in healthy pregnant women during the first (n = 10), second (n = 5), and third (n = 10) trimesters of pregnancy. These data were compared with those for nonpregnant women (n = 8). Tracers [1-(13)C]methionine, [C(2)H(3)]methionine, and [(2)H(5)]phenylalanine were administered as prime-constant rate infusions. The effect of enteral high-protein, mixed-nutrient load on tracer-determined variables was also examined. RESULTS: In pregnant women, the Ra of phenylalanine was significantly (P < 0.05) lower in the first trimester than in the second and third trimesters and was significantly lower than that in nonpregnant women. A linear positive correlation was evident between gestational age and phenylalanine Ra. The fractional rate and total rate of transsulfuration of methionine was significantly (P < 0.05) higher during the first trimester, whereas the rate of transmethylation was higher during the third trimester. Plasma concentrations of total cysteine and homocysteine were lower during pregnancy. CONCLUSIONS: Uncomplicated pregnancy in humans is associated with a higher rate of transsulfuration early in gestation and a higher rate of transmethylation of methionine in late gestation. These data may have implications for understanding the role of methionine and homocysteine in complications of pregnancy and for the nutritional care of pregnant women.

Validity of real time ultrasound in the diagnosis of hepatic steatosis: a prospective study.

BACKGROUND/AIMS: Ultrasound is used to screen for hepatic steatosis, the most common liver disease in the United States. However, few studies have prospectively evaluated the accuracy of ultrasound to diagnose hepatic steatosis. Therefore, a double blinded prospective study was performed in consecutive patients undergoing liver biopsy to evaluate the accuracy of ultrasound to diagnose hepatic steatosis. METHODS: Real time ultrasound was performed just prior to the biopsy by a single investigator masked to the clinical diagnosis. The liver biopsy was reviewed by a pathologist masked to the clinical indication or sonographic findings. RESULTS: Of 73 consecutive patients studied, macrovesicular steatosis of any severity on biopsy was found in 46 (63%) and micro vesicular fat found in 51 (69.9%). The overall impression of the sonographer for the presence of macrovesicular hepatic steatosis of any degree had a sensitivity of 60.9% and a specificity of 100%. The sensitivity increased to 100% and the specificity to 90% when there was > or =20% of fat. The zonular distribution of the fat did not alter the diagnostic accuracy of ultrasound. Ultrasound had a poor yield in the diagnosis of microvesicular fat with an overall sensitivity of 43% and a specificity of 73%. The combination of increased echogenicity and portal vein blurring on ultrasound had the greatest sensitivity in the diagnosis of hepatic steatosis. CONCLUSION: Real time ultrasound using a combination of sonographic findings has a high specificity but underestimates the prevalence of hepatic steatosis when there is<20% fat.