HIV/antiretroviral therapy–related lipodystrophy syndrome (HALS) is associated with higher RBP4 and lower omentin in plasma

AbstractVery little information is available on the involvement of newly characterized adipokines in human immunodeficiency virus (HIV)/antiretroviral therapy (ART)-associated lipodystrophy syndrome (HALS). Our aim was to determine whether apelin, apelin receptor, omentin, RBP4, vaspin and visfatin genetic variants and plasma levels are associated with HALS. We performed a cross-sectional multicentre study that involved 558 HIV type 1–infected patients treated with a stable highly active ART regimen, 240 of which had overt HALS and 318 who did not have HALS. Epidemiologic and clinical variables were determined. Polymorphisms in the apelin, omentin, RBP4, vaspin and visfatin genes were assessed by genotyping. Plasma apelin, apelin receptor, omentin, RBP4, vaspin and visfatin levels were determined by enzyme-linked immunosorbent assay in 163 patients (81 with HALS and 82 without HALS) from whom stored plasma samples were available. Student's t test, one-way ANOVA, chi-square test, Pearson and Spearman correlations and linear regression analysis were used for statistical analyses. There were no associations between the different polymorphisms assessed and the HALS phenotype. Circulating RBP4 was significantly higher (p < 0.001) and plasma omentin was significantly lower (p 0.001) in patients with HALS compared to those without HALS; differences in plasma levels of the remaining adipokines were nonsignificant between groups. Circulating RBP4 concentration was predicted independently by the presence of HALS. Apelin and apelin receptor levels were independently predicted by body mass index. Visfatin concentration was predicted independently by the presence of acquired immunodeficiency syndrome. HALS is associated with higher RBP4 and lower omentin in plasma. These two adipokines, particularly RBP4, may be a link between HIV/ART and fat redistribution syndromes

Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients

Altres ajuts: Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36610, 36572/06); Red de Investigación en SIDA (RIS RD12/0017/0005, RD12/0017/0014).To assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS). Three-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method. HALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53-3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/10 6 cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): 'A+A'; OR for 'A+B' vs. ref.: 1.39 [0.69-2.80]; OR for 'B+A' vs. ref.: 2.16 [1.22-3.83]; OR for 'B+B' vs. ref.: 3.13, 95%CI: 1.54-6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56-4.14, P = 0.001), (c) use of EFV (1.10 [1.00-1.21], P = 0.008, per year of use). HALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7473G>A (p.=) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of 3 mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that 4 of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease.info:eu-repo/semantics/publishedVersio

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074

Differential body composition effects of protease inhibitors recommended for initial treatment of HIV infection: A randomized clinical trial

This article has been accepted for publication in Clinical Infectious Diseases ©2014 The Authors .Published by Oxford University Press on Clinical Infectious Disease 60.5. DOI: 10.1093/cid/ciu898Background. It is unclear whether metabolic or body composition effects may differ between protease inhibitor-based regimens recommended for initial treatment of HIV infection. Methods. ATADAR is a phase IV, open-label, multicenter randomized clinical trial. Stable antiretroviral-naive HIV-infected adults were randomly assigned to atazanavir/ritonavir 300/100 mg or darunavir/ritonavir 800/100 mg in combination with tenofovir/emtricitabine daily. Pre-defined end-points were treatment or virological failure, drug discontinuation due to adverse effects, and laboratory and body composition changes at 96 weeks. Results. At 96 weeks, 56 (62%) atazanavir/ritonavir and 62 (71%) darunavir/ritonavir patients remained free of treatment failure (estimated difference 8.2%; 95%CI -0.6 to 21.6); and 71 (79%) atazanavir/ritonavir and 75 (85%) darunavir/ritonavir patients remained free of virological failure (estimated difference 6.3%; 95%CI -0.5 to 17.6). Seven vs. five patients discontinued atazanavir/ritonavir or darunavir/ritonavir due to adverse effects. Total and HDL cholesterol similarly increased in both arms, but triglycerides increased more in atazanavir/ritonavir arm. At 96 weeks, body fat (estimated difference 2862.2 gr; 95%CI 726.7 to 4997.7; P=0.0090), limb fat (estimated difference 1403.3 gr; 95%CI 388.4 to 2418.2; P=0.0071), and subcutaneous abdominal adipose tissue (estimated difference 28.4 cm2; 95%CI 1.9 to 55.0; P=0.0362) increased more in atazanavir/ritonavir than in darunavir/ritonavir arm. Body fat changes in atazanavir/ritonavir arm were associated with higher insulin resistance. Conclusions. We found no major differences between atazanavir/ritonavir and darunavir/ritonavir in efficacy, clinically-relevant side effects, or plasma cholesterol fractions. However, atazanavir/ritonavir led to higher triglycerides and total and subcutaneous fat than darunavir/ritonavir and fat gains with atazanavir/ritonavir were associated with insulin resistanceThis is an Investigator Sponsored Research study. It was supported in part by research grants from Bristol‐Myers Squibb and Janssen‐Cilag; Instituto de Salud Carlos III (PI12/01217) and Red Temática Cooperativa de Investigación en SIDA G03/173 (RIS‐EST11), Ministerio de Ciencia e Innovación, Spain. (Registration number: NCT01274780; registry name: ATADAR; EUDRACT; 2010‐021002‐38)

Uridine Metabolism in HIV-1-Infected Patients: Effect of Infection, of Antiretroviral Therapy and of HIV-1/ART-Associated Lipodystrophy Syndrome

Background Uridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood. Methods Plasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR. Results Median plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40-4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55-4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls. Conclusions HIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

Background: The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. Methods: We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero (2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. Findings: 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16-0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26-0·57; p<0·0001, compared with the VIR-N1-Storm group). Interpretation: The presence of a so-called viral storm is associated with increased all-cause death in patients admitted to the intensive care unit with severe COVID-19. Preventing this viral storm could help to reduce poor outcomes. Viral storm could be an enrichment marker for treatment with antivirals or purification devices to remove viral components from the blood.This work was supported by grants from the Instituto de Salud Carlos III (FONDO-COVID19, COV20/00110, CIBERES, 06/06/0028; AT), Proyectos de Investigación en Salud (PI19/00590; JFB-M), Miguel Servet (CP20/00041; DdG-C), Sara Borrell (CD018/0123; APT), and Predoctorales de Formación en Investigación en Salud (FI20/00278; AdF). We also received funds from Programa de Donaciones Estar Preparados, UNESPA (Madrid, Spain), and from the Canadian Institutes of Health Research (CIHR OV2–170357; DJK and JFB-M), Research Nova Scotia, Li-Ka Shing Foundation (DJK), and finally by a Research Grant 2020 from ESCMID (APT). COV20/00110, PI19/00590, CP20/00041, CD018/0123, FI20/00278 were co-funded by European Regional Development Fund and European Social Fund (A way to make Europe, and Investing in your future). We thank the IRB-Lleida Biobank 119 (B.0000682) and Plataforma Biobancos PT17/0015/0027 in Lleida, the Hospital Clinic Barcelona (HCB)-IDIBAPS biobank in Barcelona, and the National DNA Bank and the Hospital Universitario de Salamanca biobank (both in Salamanca) for their logistical support with sample processing and storage. We are indebted to the Fundació Glòria Soler for its contribution and support to the COVIDBANK of HCBIDIBAPS Biobank. This work was not supported by any pharmaceutical company or other agency.S

Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies

<p>Abstract</p> <p>Background</p> <p>All polypeptide backbones have the potential to form amyloid fibrils, which are associated with a number of degenerative disorders. However, the likelihood that amyloidosis would actually occur under physiological conditions depends largely on the amino acid composition of a protein. We explore using a naive Bayesian classifier and a weighted decision tree for predicting the amyloidogenicity of immunoglobulin sequences.</p> <p>Results</p> <p>The average accuracy based on leave-one-out (LOO) cross validation of a Bayesian classifier generated from 143 amyloidogenic sequences is 60.84%. This is consistent with the average accuracy of 61.15% for a holdout test set comprised of 103 AM and 28 non-amyloidogenic sequences. The LOO cross validation accuracy increases to 81.08% when the training set is augmented by the holdout test set. In comparison, the average classification accuracy for the holdout test set obtained using a decision tree is 78.64%. Non-amyloidogenic sequences are predicted with average LOO cross validation accuracies between 74.05% and 77.24% using the Bayesian classifier, depending on the training set size. The accuracy for the holdout test set was 89%. For the decision tree, the non-amyloidogenic prediction accuracy is 75.00%.</p> <p>Conclusions</p> <p>This exploratory study indicates that both classification methods may be promising in providing straightforward predictions on the amyloidogenicity of a sequence. Nevertheless, the number of available sequences that satisfy the premises of this study are limited, and are consequently smaller than the ideal training set size. Increasing the size of the training set clearly increases the accuracy, and the expansion of the training set to include not only more derivatives, but more alignments, would make the method more sound. The accuracy of the classifiers may also be improved when additional factors, such as structural and physico-chemical data, are considered. The development of this type of classifier has significant applications in evaluating engineered antibodies, and may be adapted for evaluating engineered proteins in general.</p