A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in ß-thalassemic mice.

ABSTRACT: Iron plays a major role in the deterioration of ß-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in ß-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in ß-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the ß-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of ß-thalassemia.

Development, validation, and prognostic evaluation of a risk score for long-term liver-related outcomes in the general population: a multicohort study.

BACKGROUND: Liver cirrhosis is a major cause of death worldwide. Cirrhosis develops after a long asymptomatic period of fibrosis progression, with the diagnosis frequently occurring late, when major complications or cancer develop. Few reliable tools exist for timely identification of individuals at risk of cirrhosis to allow for early intervention. We aimed to develop a novel score to identify individuals at risk for future liver-related outcomes. METHODS: We derived the LiverRisk score from an international prospective cohort of individuals from six countries without known liver disease from the general population, who underwent liver fibrosis assessment by transient elastography. The score included age, sex, and six standard laboratory variables. We created four groups: minimal risk, low risk, medium risk, and high risk according to selected cutoff values of the LiverRisk score (6, 10, and 15). The model's discriminatory accuracy and calibration were externally validated in two prospective cohorts from the general population. Moreover, we ascertained the prognostic value of the score in the prediction of liver-related outcomes in participants without known liver disease with median follow-up of 12 years (UK Biobank cohort). FINDINGS: We included 14 726 participants: 6357 (43·2%) in the derivation cohort, 4370 (29·7%) in the first external validation cohort, and 3999 (27·2%) in the second external validation cohort. The score accurately predicted liver stiffness in the development and external validation cohorts, and was superior to conventional serum biomarkers of fibrosis, as measured by area under the receiver-operating characteristics curve (AUC; 0·83 [95% CI [0·78-0·89]) versus the fibrosis-4 index (FIB-4; 0·68 [0·61-0·75] at 10 kPa). The score was effective in identifying individuals at risk of liver-related mortality, liver-related hospitalisation, and liver cancer, thereby allowing stratification to different risk groups for liver-related outcomes. The hazard ratio for liver-related mortality in the high-risk group was 471 (95% CI 347-641) compared with the minimal risk group, and the overall AUC of the score in predicting 10-year liver-related mortality was 0·90 (0·88-0·91) versus 0.84 (0·82-0·86) for FIB-4. INTERPRETATION: The LiverRisk score, based on simple parameters, predicted liver fibrosis and future development of liver-related outcomes in the general population. The score might allow for stratification of individuals according to liver risk and thus guide preventive care. FUNDING: European Commission under the H20/20 programme; Fondo de Investigación Sanitaria de Salud; Instituto de Salud Carlos III; Spanish Ministry of Economy, Industry, and Competitiveness; the European Regional Development Fund; and the German Ministry of Education and Research (BMBF).

Double-Edged Effects of Venglustat on Behavior and Pathology in Mice Overexpressing α-Synuclein.

BACKGROUND: Venglustat is a brain-penetrant, small molecule inhibitor of glucosylceramide synthase used in clinical testing for treatment of Parkinson's disease (PD). Despite beneficial effects in certain cellular and rodent models, patients with PD with mutations in GBA, the gene for lysosomal glucocerebrosidase, experienced worsening of their motor function under venglustat treatment (NCT02906020, MOVES-PD, phase 2 trial). OBJECTIVE: The objective of this study was to evaluate venglustat in mouse models of PD with overexpression of wild-type α-synuclein. METHODS: Mice overexpressing α-synuclein (Thy1-aSyn line 61) or Gba-mutated mice with viral vector-induced overexpression of α-synuclein in the substantia nigra were administered venglustat as food admixture. Motor and cognitive performance, α-synuclein-related pathology, and microgliosis were compared with untreated controls. RESULTS: Venglustat worsened motor function in Thy1-aSyn transgenics on the challenging beam and the pole test. Although venglustat did not alter the cognitive deficit in the Y-maze test, it alleviated anxiety-related behavior in the novel object recognition test. Venglustat reduced soluble and membrane-bound α-synuclein in the striatum and phosphorylated α-synuclein in limbic brain regions. Although venglustat reversed the loss of parvalbumin immunoreactivity in the basolateral amygdala, it tended to increase microgliosis and phosphorylated α-synuclein in the substantia nigra. Furthermore, venglustat also partially worsened motor performance and tended to increase neurofilament light chain in the cerebrospinal fluid in the Gba-deficient model with nigral α-synuclein overexpression and neurodegeneration. CONCLUSIONS: Venglustat treatment in two mouse models of α-synuclein overexpression showed that glucosylceramide synthase inhibition had differential detrimental or beneficial effects on behavior and neuropathology possibly related to brain region-specific effects. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study.

BACKGROUND AND AIMS: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. APPROACH AND RESULTS: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). CONCLUSIONS: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis.

LiverScreen project: study protocol for screening for liver fibrosis in the general population in European countries.

BACKGROUND: The development of liver cirrhosis is usually an asymptomatic process until late stages when complications occur. The potential reversibility of the disease is dependent on early diagnosis of liver fibrosis and timely targeted treatment. Recently, the use of non-invasive tools has been suggested for screening of liver fibrosis, especially in subjects with risk factors for chronic liver disease. Nevertheless, large population-based studies with cost-effectiveness analyses are still lacking to support the widespread use of such tools. The aim of this study is to investigate whether non-invasive liver stiffness measurement in the general population is useful to identify subjects with asymptomatic, advanced chronic liver disease. METHODS: This study aims to include 30,000 subjects from eight European countries. Subjects from the general population aged ≥ 40 years without known liver disease will be invited to participate in the study either through phone calls/letters or through their primary care center. In the first study visit, subjects will undergo bloodwork as well as hepatic fat quantification and liver stiffness measurement (LSM) by vibration-controlled transient elastography. If LSM is ≥ 8 kPa and/or if ALT levels are ≥1.5 x upper limit of normal, subjects will be referred to hospital for further evaluation and consideration of liver biopsy. The primary outcome is the percentage of subjects with LSM ≥ 8kPa. In addition, a health economic evaluation will be performed to assess the cost-effectiveness and budget impact of such an intervention. The project is funded by the European Commission H2020 program. DISCUSSION: This study comes at an especially important time, as the burden of chronic liver diseases is expected to increase in the coming years. There is consequently an urgent need to change our current approach, from diagnosing the disease late when the impact of interventions may be limited to diagnosing the disease earlier, when the patient is asymptomatic and free of complications, and the disease potentially reversible. Ultimately, the LiverScreen study will serve as a basis from which diagnostic pathways can be developed and adapted to the specific socio-economic and healthcare conditions in each country. TRIAL REGISTRATION: This study is registered on Clinicaltrials.gov ( NCT03789825 ).

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.

OBJECTIVE: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. DESIGN: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. RESULTS: Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63-68) and 86% (84-87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37-39) and specificity of 90% (89-91) with 19% needing biopsy. CONCLUSION: Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies.

Improved Ultrasound Attenuation Measurement Method for the Non-invasive Evaluation of Hepatic Steatosis Using FibroScan.

Controlled attenuation parameter (CAP) is a measurement of ultrasound attenuation used to assess liver steatosis non-invasively. However, the standard method has some limitations. This study assessed the performance of a new CAP method by ex vivo and in vivo assessments. The major difference with the new method is that it uses ultrasound data continuously acquired during the imaging phase of the FibroScan examination. Seven reference tissue-mimicking phantoms were used to test the performance. In vivo performance was assessed in two cohorts (in total 195 patients) of patients using magnetic resonance imaging proton density fat fraction (MRI-PDFF) as a reference. The precision of CAP was improved by more than 50% on tissue-mimicking phantoms and 22%-41% in the in vivo cohort studies. The agreement between both methods was excellent, and the correlation between CAP and MRI-PDFF improved in both studies (0.71 to 0.74; 0.70 to 0.76). Using MRI-PDFF as a reference, the diagnostic performance of the new method was at least equal or superior (area under the receiver operating curve 0.889-0.900, 0.835-0.873). This study suggests that the new continuous CAP method can significantly improve the precision of CAP measurements ex vivo and in vivo.

Prospective evaluation of the prevalence of non-alcoholic fatty liver disease and steatohepatitis in a large middle-aged US cohort.

BACKGROUND & AIMS: Large prospective studies to establish the prevalence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), are lacking. We prospectively assessed the prevalence and severity of NAFLD/NASH in a cohort of asymptomatic middle-aged Americans attending a colonoscopy class at a gastroenterology clinic. METHODS: Screening for NAFLD was performed using magnetic resonance (MR)-based LiverMultiScan® proton density fat fraction (LMS-PDFF). MR exams also included corrected T1 and elastography for liver stiffness measurement (LSM). FibroScan® was also used to measure LSM. Participants with predetermined abnormal imaging parameters were offered a liver biopsy. Biopsies were read in a blinded fashion with results based on the consensus by 2 expert pathologists. The prevalence of NAFLD was determined by PDFF ≥5% or by histological diagnosis of NAFLD (if biopsy data were available). The prevalence of NASH was defined by biopsy. RESULTS: Of 835 participants, 664 met the inclusion and exclusion criteria. The mean age was 56 ± 6.4 years, 50% were male, the mean BMI was 30.48 ± 5.46 kg/m2, and 52% were obese. The prevalence of NAFLD was 38% (95% CI 34-41%) and the prevalence of NASH was 14% (95% CI 12-17%). While no patient had cirrhosis on biopsy, significant fibrosis (F ≥2) was present in 5.9% (95% CI 4-8%) and bridging fibrosis in 1.6% (95% CI 1-3%). In a multivariable analysis, factors associated with the presence of NASH were race, obesity, and diabetes. CONCLUSION: Using state-of-the-art liver imaging modalities and reference biopsy, this study establishes an overall prevalence of NAFLD of 38% and NASH by biopsy of 14% in this cohort of asymptomatic middle-aged US adults. LAY SUMMARY: There are no prospective studies to determine how common is nonalcoholic steatohepatitis (NASH), the severe form of non-alcoholic fatty liver disease (NAFLD). In a large number of asymptomatic middle-aged Americans, we used a combination of state-of-the-art liver imaging methods and liver biopsy to prospectively determine the prevalence of NAFLD and NASH. NAFLD was diagnosed in 38%, NASH in 14%, and significant liver fibrosis in 6% of asymptomatic middle-aged Americans.

Deletion of BMP6 worsens the phenotype of HJV-deficient mice and attenuates hepcidin levels reached after LPS challenge.

Lack of either bone morphogenetic protein 6 (BMP6) or the BMP coreceptor hemojuvelin (HJV) in mice leads to a similar phenotype with hepcidin insufficiency, hepatic iron loading, and extrahepatic iron accumulation in males. This is consistent with the current views that HJV is a coreceptor for BMP6 in hepatocytes. To determine whether BMP6 and HJV may also signal to hepcidin independently of each other, we intercrossed Hjv-/- and Bmp6-/- mice and compared the phenotype of animals of the F2 progeny. Loss of Bmp6 further repressed Smad signaling and hepcidin expression in the liver of Hjv-/- mice of both sexes, and led to iron accumulation in the pancreas and the heart of females. These data suggest that, in Hjv-/- females, Bmp6 can provide a signal adequate to maintain hepcidin to a level sufficient to avoid extrahepatic iron loading. We also examined the impact of Bmp6 and/or Hjv deletion on the regulation of hepcidin by inflammation. Our data show that lack of 1 or both molecules does not prevent induction of hepcidin by lipopolysaccharide (LPS). However, BMP/Smad signaling in unchallenged animals is determinant for the level of hepcidin reached after stimulation, which is consistent with a synergy between interleukin 6/STAT3 and BMP/SMAD signaling in regulating hepcidin during inflammation.

Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice.

Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling.

Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

UNLABELLED: Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and ß2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. CONCLUSION: These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice.

Liver Steatosis Assessed by Controlled Attenuation Parameter (CAP) Measured with the XL Probe of the FibroScan: A Pilot Study Assessing Diagnostic Accuracy.

To assess liver steatosis, the controlled attenuation parameter (CAP; giving an estimate of ultrasound attenuation ∼3.5 MHz) is available with the M probe of the FibroScan. We report on the adaptation of the CAP for the FibroScan XL probe (center frequency 2.5 MHz) without modifying the range of values (100-400 dB/m). CAP validation was successfully performed on Field II simulations and on tissue-mimicking phantoms. In vivo performance was assessed in a cohort of 59 patients spanning the range of steatosis. In vivo reproducibility was good and similar with both probes. The area under receiver operative characteristic curve was equal to 0.83/0.84 and 0.92/0.91 for the M/XL probes to detect >2% and >16% liver fat, respectively, as assessed by magnetic resonance imaging. Patients can now be assessed simultaneously for steatosis and fibrosis using the FibroScan, regardless of their morphology.

Hippocampal neuroligin-2 links early-life stress with impaired social recognition and increased aggression in adult mice.

Early-life stress is a key risk factor for the development of neuropsychiatric disorders later in life. Neuronal cell adhesion molecules have been strongly implicated in the pathophysiology of psychiatric disorders and in modulating social behaviors associated with these diseases. Neuroligin-2 is a synaptic cell adhesion molecule, located at the postsynaptic membrane of inhibitory GABAergic synapses, and is involved in synaptic stabilization and maturation. Alterations in neuroligin-2 expression have previously been associated with changes in social behavior linked to psychiatric disorders, including schizophrenia and autism. In this study, we show that early-life stress, induced by limited nesting and bedding material, leads to impaired social recognition and increased aggression in adult mice, accompanied by increased expression levels of hippocampal neuroligin-2. Viral overexpression of hippocampal neuroligin-2 in adulthood mimics early-life stress-induced alterations in social behavior and social cognition. Moreover, viral knockdown of neuroligin-2 in the adult hippocampus attenuates the early-life stress-induced behavioral changes. Our results highlight the importance of neuroligin-2 in mediating early-life stress effects on social behavior and social cognition and its promising role as a novel therapeutic target for neuropsychiatric disorders.

Role for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations.

Chronic stress is a risk factor for the development of psychopathologies characterized by cognitive dysfunction and deregulated social behaviours. Emerging evidence suggests a role for cell adhesion molecules, including nectin-3, in the mechanisms that underlie the behavioural effects of stress. We tested the hypothesis that proteolytic processing of nectins by matrix metalloproteinases (MMPs), an enzyme family that degrades numerous substrates, including cell adhesion molecules, is involved in hippocampal effects induced by chronic restraint stress. A reduction in nectin-3 in the perisynaptic CA1, but not in the CA3, compartment is observed following chronic stress and is implicated in the effects of stress in social exploration, social recognition and a CA1-dependent cognitive task. Increased MMP-9-related gelatinase activity, involving N-methyl-D-aspartate receptor, is specifically found in the CA1 and involved in nectin-3 cleavage and chronic stress-induced social and cognitive alterations. Thus, MMP-9 proteolytic processing emerges as an important mediator of stress effects in brain function and behaviour.