Efforts to manage non-alcoholic fatty liver disease (NAFLD) are limited by the incomplete understanding of the pathogenic mechanisms and the absence of accurate non-invasive biomarkers. The aim of ...this study was to identify novel NAFLD therapeutic targets andbiomarkers by conducting liver transcriptomic analysis in patients stratified by the presence of the
I148M genetic risk variant.
We sequenced the hepatic transcriptome of 125 obese individuals. 'Severe NAFLD' was defined as the presence of steatohepatitis, NAFLD activity score ≥4 or fibrosis stage ≥2. The circulating levels of the most upregulated transcript, interleukin-32 (IL32), were measured by ELISA.
Carriage of the
I148M variant correlated with the two major components of hepatic transcriptome variability and broadly influenced gene expression. In patients with severe NAFLD, there was an upregulation of inflammatory and lipid metabolism pathways. IL32 was the most robustly upregulated gene in the severe NAFLD group (adjusted p=1×10
), and its expression correlated with steatosis severity, both in I148M variant carriers and non-carriers. In 77 severely obese, and in a replication cohort of 160 individuals evaluated at the hepatology service, circulating IL32 levels were associated with both NAFLD and severe NAFLD independently of aminotransferases (p<0.01 for both). A linear combination of IL32-ALT-AST showed a better performance than ALT-AST alone in NAFLD diagnosis (area under the curve=0.92 vs 0.81, p=5×10
).
Hepatic IL32 is overexpressed in NAFLD, correlates with hepatic fat and liver damage, and is detectable in the circulation, where it is independently associated with the presence and severity of NAFLD.
Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD ...by examining rare variants.
We performed whole-exome sequencing in individuals with NAFLD and advanced fibrosis or hepatocellular carcinoma (n = 301) and examined the enrichment of likely pathogenic rare variants vs. the general population. This was followed by validation at the gene level.
In patients with severe NAFLD, we observed an enrichment of the p.P426L variant (rs143545741 C>T; odds ratio OR 5.26, 95% CI 2.1-12.6; p = 0.003) of autophagy-related 7 (ATG7), which we characterized as a loss-of-function, vs. the general population, and an enrichment in rare variants affecting the catalytic domain (OR 13.9; 95% CI 1.9-612; p = 0.002). In the UK Biobank cohort, loss-of-function ATG7 variants increased the risk of cirrhosis and hepatocellular carcinoma (OR 3.30; 95% CI 1.1-7.5 and OR 12.30, 95% CI 2.6-36, respectively; p <0.001 for both). The low-frequency loss-of-function p.V471A variant (rs36117895 T>C) was also associated with severe NAFLD in the clinical cohort (OR 1.7; 95% CI 1.2-2.5; p = 0.003), predisposed to hepatocellular ballooning (p = 0.007) evolving to fibrosis in the Liver biopsy cohort (n = 2,268), and was associated with liver injury in the UK Biobank (aspartate aminotransferase levels, p <0.001), with a larger effect in severely obese individuals in whom it was linked to hepatocellular carcinoma (p = 0.009). ATG7 protein localized to periportal hepatocytes, particularly in the presence of ballooning. In the Liver Transcriptomic cohort (n = 125), ATG7 expression correlated with suppression of the TNFα pathway, which was conversely upregulated in p.V471A carriers.
We identified rare and low-frequency ATG7 loss-of-function variants that promote NAFLD progression by impairing autophagy and facilitating ballooning and inflammation.
We found that rare mutations in a gene called autophagy-related 7 (ATG7) increase the risk of developing severe liver disease in individuals with dysmetabolism. These mutations cause an alteration in protein function and impairment of self-renewal of cellular content, leading to liver damage and inflammation.
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•NAFLD is the leading cause of liver disorders and has a strong heritable component.•Rare loss-of-function ATG7 gene mutations increase the risk of severe liver disease in patients with NAFLD.•ATG7 mutations cause altered protein function and impairment of autophagy, leading to hepatocellular ballooning and inflammation.•The most frequent variant is responsible for a meaningful fraction of predisposition to ballooning and hepatocellular carcinoma.
There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to ...understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD.
Widespread infections of avian species with avian influenza H5N1 virus and its limited spread to humans suggest that the virus has the potential to cause a human influenza pandemic. An urgent need ...exists for an H5N1 vaccine that is effective against divergent strains of H5N1 virus.
In a randomized, dose-escalation, phase 1 and 2 study involving six subgroups, we investigated the safety of an H5N1 whole-virus vaccine produced on Vero cell cultures and determined its ability to induce antibodies capable of neutralizing various H5N1 strains. In two visits 21 days apart, 275 volunteers between the ages of 18 and 45 years received two doses of vaccine that each contained 3.75 microg, 7.5 microg, 15 microg, or 30 microg of hemagglutinin antigen with alum adjuvant or 7.5 microg or 15 microg of hemagglutinin antigen without adjuvant. Serologic analysis was performed at baseline and on days 21 and 42.
The vaccine induced a neutralizing immune response not only against the clade 1 (A/Vietnam/1203/2004) virus strain but also against the clade 2 and 3 strains. The use of adjuvants did not improve the antibody response. Maximum responses to the vaccine strain were obtained with formulations containing 7.5 microg and 15 microg of hemagglutinin antigen without adjuvant. Mild pain at the injection site (in 9 to 27% of subjects) and headache (in 6 to 31% of subjects) were the most common adverse events identified for all vaccine formulations.
A two-dose vaccine regimen of either 7.5 microg or 15 microg of hemagglutinin antigen without adjuvant induced neutralizing antibodies against diverse H5N1 virus strains in a high percentage of subjects, suggesting that this may be a useful H5N1 vaccine. (ClinicalTrials.gov number, NCT00349141.)
Summary Background Lyme borreliosis is caused by Borrelia burgdorferi sensu stricto in the USA and by several Borrelia species in Europe and Asia, but no human vaccine is available. We investigated ...the safety and immunogenicity of adjuvanted and non-adjuvanted vaccines containing protective epitopes from Borrelia species outer surface protein A (OspA) serotypes in healthy adults. Methods Between March 1, 2011, and May 8, 2012, we did a double-blind, randomised, dose-escalation phase 1/2 study at four sites in Austria and Germany. Healthy adults aged 18–70 years who were seronegative for B burgdorferi sensu lato were eligible for inclusion. Participants were recruited sequentially and randomly assigned to one of six study groups in equal ratios via an electronic data capture system. Participants and investigators were masked to group allocation. Participants received three vaccinations containing 30 μg, 60 μg, or 90 μg OspA antigen with or without an adjuvant, with intervals of 28 days, and a booster 9–12 months after the first immunisation. The coprimary endpoints were the frequency and severity of injection-site and systemic reactions within 7 days of each vaccination, and the antibody responses to OspA serotypes 1–6, as established by ELISA. This study is registered with ClinicalTrials.gov , number NCT01504347. Findings 300 participants were randomly assigned: 151 to adjuvanted vaccines (50 to 30 μg, 51 to 60 μg, and 50 to 90 μg doses), and 149 to non-adjuvanted vaccines (50 to 30 μg, 49 to 60 μg, and 50 to 90 μg doses). Adverse reactions were predominantly mild, and no vaccine-related serious adverse events were reported. The risk of systemic reactions (risk ratio 0·54 95% CI 0·41–0·70; p<0·0001) and of moderate or severe systemic reactions (0·35 0·13–0·92; p=0·034) was significantly lower for adjuvanted than non-adjuvanted formulations. The 30 μg adjuvanted formulation had the best tolerability profile; only headache (five 10%, 95% CI 4–20 of 50), injection-site pain (16 32%, 21–45), and tenderness (17 34%, 23–47) affected more than 6% of patients. All doses and formulations induced substantial mean IgG antibody titres against OspA serotypes 1–6 after the first three vaccinations (range 6944–17 321) and booster (19 056–32 824) immunisations. The 30 μg adjuvanted formulation induced the highest antibody titres after the booster: range 26 143 (95% CI 18 906–36 151) to 42 381 (31 288–57 407). Interpretation The novel multivalent OspA vaccine could be an effective intervention for prevention of Lyme borreliosis in Europe and the USA, and possibly worldwide. Larger confirmatory formulation studies will need to be done that include individuals seropositive for Borrelia burgdorferi sensu lato before placebo-controlled phase 3 efficacy studies can begin. Funding Baxter.
Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, ...non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD.
Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8.
Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models
Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.
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•Hepatic neddylation is induced in clinical and pre-clinical NAFLD.•Neddylation inhibition reduces hepatic steatosis in pre-clinical models of NAFLD.•Neddylation inhibition boosts fatty acid oxidation through a DEPTOR/mTOR axis.•Neddylation inhibition reduces hepatic inflammation and lipid peroxidation in NAFLD.•Serum NEDD8 levels correlate positively with liver disease in patients and pre-clinical models of NAFLD.
Event-related changes of brain electrical rhythms are typically analysed as amplitude modulations of local field potential
(LFP) oscillations, like radio amplitude modulation broadcasting. In ...telecommunications, frequency modulation (FM) is less
susceptible to interference than amplitude modulation (AM) and is therefore preferred for high-fidelity transmissions. Here
we hypothesized that LFP rhythms detected from deep brain stimulation (DBS) electrodes implanted in the subthalamic nucleus
(STN) in patients with Parkinson's disease could represent movement-related activity not only in AM but also in FM. By combining
adaptive autoregressive identification with spectral power decomposition, we were able to show that FM of low-beta (13â20
Hz) and high-beta (20â35 Hz) rhythms significantly contributes to the involvement of the human STN in movement preparation,
execution and recovery, and that the FM patterns are regulated by the dopamine levels in the system. Movement-related FM of
beta oscillatory activity in the human subthalamic nucleus therefore provides a novel informational domain for rhythm-based
pathophysiological models of cortico-basal ganglia processing.
In patients with nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) is a risk factor for the development of fibrosis. However, fibrosis has been observed in livers of ...patients without NASH. We aimed to estimate the prevalence of fibrosis in patients without NASH and risk factors for fibrosis.
We analyzed data from 1738 subjects (44.9% with severe obesity) in a cross-sectional liver biopsy cohort enrolled at referral centers in Italy and Finland. Biopsy specimens were analyzed histologically by a blinded pathologist at each center, and a diagnosis of NASH was made based on steatosis (≥5% of hepatocytes), hepatocellular ballooning, and lobular inflammation. We also collected data on demographic features, metabolic comorbidities, and genetic factors, and performed logistic regression analyses. Findings were validated using data from 118 consecutive patients with NAFLD who underwent sequential liver biopsies at tertiary referral centers in Italy.
In the cross-sectional cohort, 132 of 389 patients (33.9%) with significant fibrosis had no NASH and 39 patients (10.0%) had no inflammation. The dissociation between NASH and fibrosis was significantly greater in patients with severe obesity (P < .005). Steatosis, ballooning, and lobular inflammation each were associated independently with significant fibrosis (P < .001); age, adiposity, fasting hyperglycemia, and the PNPLA3 I148M variant also were associated with fibrosis. In patients without, but not in those with NASH, significant fibrosis was associated with steatosis grade and the PNPLA3 I148M variant. In patients without NASH, age, fasting hyperglycemia, ballooning, and inflammation were associated with fibrosis. In the validation cohort, 16 of 47 patients (34.0%) with clinically significant fibrosis did not have NASH at baseline. In patients with fibrosis without baseline NASH, worsening of fibrosis (based on later biopsies) was associated with fasting hyperglycemia and more severe steatosis (P = .016).
In an analysis of biopsy specimens collected from patients with NAFLD at a single time point, one third of patients with significant fibrosis did not have NASH. We validated this finding in a separate cohort. In patients without NASH, fasting hyperglycemia, severe steatosis, mild inflammation or ballooning, and the PNPLA3 I148M variant identified those at risk of significant fibrosis.
Duchenne muscular dystrophy (DMD) is a progressive severe muscle‐wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory ...failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ‐free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.
Synopsis
The susceptibility of DMD patients to inflammatory events cannot solely be explained by skeletal muscle genetic defects but rather favors a new paradigm linking development of chronic inflammation with a strict regulation between epigenetics factors and degenerative environment.
Gut microbiota–specific alterations (dysbiosis) correlate with the dystrophic pathology in mdx mice, influencing muscle immunity and fibrosis.
Dysbiotic mdx microbiota induces a decreased innate immune response and altered muscle metabolism.
The study of the dysregulated immune system‐microbiota axis in mdx mice highlights the importance of microbiota as a potential target for therapeutic interventions.
The susceptibility of DMD patients to inflammatory events cannot solely be explained by skeletal muscle genetic defects but rather favors a new paradigm linking the development of chronic inflammation with a strict regulation between epigenetics factors and degenerative environment.
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