Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

Spectral analysis-based risk score enables early prediction of mortality and cerebral performance in patients undergoing therapeutic hypothermia for ventricular fibrillation and comatose status

Background: Early prognosis in comatose survivors after cardiac arrest due to ventricular fibrillation (VF) is unreliable, especially in patients undergoing mild hypothermia. We aimed at developing a reliable risk-score to enable early prediction of cerebral performance and survival. Methods: Sixty-one out of 239 consecutive patients undergoing mild hypothermia after cardiac arrest, with eventual return of spontaneous circulation (ROSC), and comatose status on admission fulfilled the inclusion criteria. Background clinical variables, VF time and frequency domain fundamental variables were considered. The primary and secondary outcomes were a favorable neurological performance (FNP) during hospitalization and survival to hospital discharge, respectively. The predictive model was developed in a retrospective cohort (n = 32; September 2006 September 2011, 48.5 ± 10.5 months of follow-up) and further validated in a prospective cohort (n = 29; October 2011 July 2013, 5 ± 1.8 months of follow-up). Results: FNP was present in 16 (50.0%) and 21 patients (72.4%) in the retrospective and prospective cohorts, respectively. Seventeen (53.1%) and 21 patients (72.4%), respectively, survived to hospital discharge. Both outcomes were significantly associated (p < 0.001). Retrospective multivariate analysis provided a prediction model (sensitivity = 0.94, specificity = 1) that included spectral dominant frequency, derived power density and peak ratios between high and low frequency bands, and the number of shocks delivered before ROSC. Validation on the prospective cohort showed sensitivity = 0.88 and specificity = 0.91. A model-derived risk-score properly predicted 93% of FNP. Testing the model on follow-up showed a c-statistic ≥ 0.89. Conclusions: A spectral analysis-based model reliably correlates time-dependent VF spectral changes with acute cerebral injury in comatose survivors undergoing mild hypothermia after cardiac arrest.the CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation.Filgueiras-Rama, D.; Calvo Saiz, CJ.; Salvador-Montañés, Ó.; Cádenas, R.; Ruiz-Cantador, J.; Armada, E.; Rey, JR.... (2015). Spectral analysis-based risk score enables early prediction of mortality and cerebral performance in patients undergoing therapeutic hypothermia for ventricular fibrillation and comatose status. International Journal of Cardiology. 186:250-258. doi:10.1016/j.ijcard.2015.03.074S25025818

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting

26 p.-6 fig.-1 tab.-1 graph. abst.There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)—the principal methyl donor—acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, β-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive β-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.M.V.-R. is supported by Proyecto PID2020-119486RB-100 (funded by MCIN/AEI/10.13039/501100011033), Gilead Sciences International Research Scholars Program in Liver Disease, Acción Estratégica Ciberehd Emergentes 2018 (ISCIII), Fundación BBVA, HORIZON-TMA-MSCA-Doctoral Networks 2021 (101073094), and Redes de Investigación 2022 (RED2022-134485-T). M.L.M.-C. is supported by La CAIXA Foundation (LCF/PR/HP17/52190004), Proyecto PID2020-117116RB-I00 (funded by MCIN/AEI/10.13039/501100011033), Ayudas Fundación BBVA a equipos de investigación científica (Umbrella 2018), and AECC Scientific Foundation (Rare Cancers 2017). A.W. is supported by RTI2018-097503-B-I00 and PID2021-127169OB-I00, (funded by MCIN/AEI/10.13039/501100011033) and by “ERDF A way of making Europe,” Xunta de Galicia (Ayudas PRO-ERC), Fundación Mutua Madrileña, and European Community’s H2020 Framework Programme (ERC Consolidator grant no. 865157 and MSCA Doctoral Networks 2021 no. 101073094). C.M. is supported by CIBERNED. P.A. is supported by Ayudas para apoyar grupos de investigación del sistema Universitario Vasco (IT1476-22), PID2021-124425OB-I00 (funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe,” MCI/UE/ISCiii [PMP21/00080], and UPV/EHU [COLAB20/01]). M.F. and M.G.B. are supported by PID2019-105739GB-I00 and PID2020-115472GB-I00, respectively (funded by MCIN/AEI/10.13039/501100011033). M.G.B. is supported by Xunta de Galicia (ED431C 2019/013). C.A., T.L.-D., and J.B.-V. are recipients of pre-doctoral fellowships from Xunta de Galicia (ED481A-2020/046, ED481A-2018/042, and ED481A 2021/244, respectively). T.C.D. is supported by Fundación Científica AECC. A.T.-R. is a recipient of a pre-doctoral fellowship from Fundación Científica AECC. S.V.A. and C.R. are recipients of Margarita Salas postdoc grants under the “Plan de Recuperación Transformación” program funded by the Spanish Ministry of Universities with European Union’s NextGeneration EU funds (2021/PER/00020 and MU-21-UP2021-03071902373A, respectively). T.C.D., A.S.-R., and M.T.-C. are recipients of Ayuda RYC2020-029316-I, PRE2019/088960, and BES-2016/078493, respectively, supported by MCIN/AEI/10.13039/501100011033 and by El FSE invierte en tu futuro. S.L.-O. is a recipient of a pre-doctoral fellowship from the Departamento de Educación del Gobierno Vasco (PRE_2018_1_0372). P.A.-G. is recipient of a FPU pre-doctoral fellowship from the Ministry of Education (FPU19/02704). CIC bioGUNE is supported by Ayuda CEX2021-001136-S financiada por MCIN/AEI/10.13039/501100011033. A.B.-C. was funded by predoctoral contract PFIS (FI19/00240) from Instituto de Salud Carlos III (ISCIII) co-funded by Fondo Social Europeo (FSE), and A.D.-L. was funded by contract Juan Rodés (JR17/00016) from ISCIII. A.B.-C. is a Miguel Servet researcher (CPII22/00008) from ISCIII.Peer reviewe

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: diagnostic and mechanistic relevance

Background & Aims: Serum microRNAs (miRNAs) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages.Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 NAFLD cases representing the complete NAFLD spectrum and 10 population controls). MiRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional NAFLD cases and 15 population controls by quantitative reverse transcriptase-polymerase chain reaction.Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages but miR-193a-5p consistently the showed increased levels in all comparisons. Relative to NAFL/NASH with mild fibrosis (stage 0/1), three miRNAs (miR-193a-5p, miR-378d and miR378d) were increased in cases with NASH and clinically significant fibrosis (stage 2-4), seven (miR193a-5p, miR-378d, miR-378e, miR-320b, c, d & e) increased in cases with NAFLD Activity Score (NAS) 5-8 compared with lower NAS, and three (miR-193a-5p, miR-378d, miR-378e) increased but one (miR-19b-3p) decreased in steatosis, activity, and fibrosis "activity" (SAF-A) score 2-4 compared with lower SAF-A. The significant findings for miR-193a-5p were replicated in the additional NAFLD cohort. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n=80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases

Inhibition of carnitine palmitoyltransferase 1A in hepatic stellate cells protects against fibrosis

One of the most common types of fibrotic liver occurs in patients with non-alcoholic fatty liver disease (NAFLD), which may develop into non-alcoholic steatohepatitis (NASH). Hepatic stellate cells (HSCs) are the primary fibrogenic cell type activated following liver injury, moving from a quiescent phenotype rich in vitamin A into activated myofibroblast-like cells with proliferative and migratory properties.This work has been supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (DS and LH: SAF2017-83813-C3-1-R; MLMC: RTC2019-007125-1; CD: BFU2017-87721; ML: RTI2018–101840-B-I00; RN: RTI2018-099413-B-I00 and RED2018-102379-T; MLMC: SAF2017-87301-R; TCD: RTI2018-096759-A-100), Xunta de Galicia (ML: 2016-PG068; RN: 2015-CP080 and 2016-PG057), Fundación BBVA (RN and MLM), Proyectos Investigación en Salud (MLMC: DTS20/00138), Sistema Universitario Vasco (PA: IT971-16); Fundacion Araucaria (ML and RN), Gilead Sciences International Research Scholars Program in Liver Disease (MVR), Marató TV3 Foundation (DS: 201627), Government of Catalonia (DS: 2017SGR278) and European Foundation for the Study of Diabetes (RN). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH- 810331, to RN, VP and MS). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd). CIBERobn and CIBERehd are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644)S

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