Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2 -) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M-1 s-1) of ONOO- with NADH (233 ± 27), ubiquinol-0 (485 ± 54) and GSH (183 ± 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2 -) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 μM ONOO- showed a O2 - production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 μM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 μM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants. (C) 2000 Elsevier Science Inc.Fil: Valdez, Laura Batriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Alvarez, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Lores Arnaiz, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Schöpfer, Francisco. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; ArgentinaFil: Carreras, Maria Cecilia. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Poderoso, Juan José. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Boveris, Alberto Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentin

Áreas de riesgo agroclimático para cuatro rotaciones de cultivos de la región oriental agropecuaria de La Pampa (Argentina) = Agroclimatic risk areas for four crop rotation schedules in the agricultural eastern region of La Pampa (Argentina)

Los riesgos climáticos son, en esencia, la probabilidad de ocurrencia de eventos de baja frecuencia que más tarde o más temprano terminan desarrollándose, guiados por su particular periodicidad. Estos riesgos se convierten en agroclimáticos cuando afectan a la producción agropecuaria. En el presente trabajo se calculó un índice de riesgo agroclimático (RAC) basado en el estudio de la climatología del agua del suelo simulando diferentes planteos rotacionales. El RAC toma valores entre 0 (riesgo mínimo) y 1 (riesgo máximo). Se utilizaron cuatro planteos rotacionales en secano: barbecho-trigo, alfalfa-trigo, barbecho-girasol y verdeo de invierno-girasol en veintiuna localidades de la región oriental agropecuaria de La Pampa. Los mapas muestran que en las cuatro rotaciones analizadas, durante todas las décadas que involucra cada planteo, se presentaron condiciones de sequía condicional o absoluta dentro de la época del año correspondiente al semestre cálido. La rotación barbecho-trigo presenta menor riesgo climático (RAC: 0.1 a 0.4) que alfalfatrigo (RAC: 0.1 a 0.6), barbecho-girasol (RAC: 0.4 a 0.5) y verdeo de invierno-girasol (RAC: 0.4 a 0.5). Los planteos rotacionales que involucran a un cultivo anual invernal (trigo) y los que consideran un cultivo anual estival (girasol) presentan mayor riesgo agroclimático de deficiencia hídrica en la zona centro-sur de la región agropecuaria pampeana que en la del nor-noreste.Climatic risks imply the probability of occurrence of extreme weather conditions, events that can threaten nature and human activities with a particular pattern of low frequency. They are termed agroclimatic risks when affecting farm production. The aim of this work was to assess an index of agroclimatic risk (ACR), based on the study of soil water climatology and simulation of different crop rotation schedules. The ACR index scoring was based on a scale of values running from 0 (minimum risk) to 1 (maximum risk). Four crop rotation schedules, under conditions of dry-land farming, were tested in 21 sites all over the Eastern region of La Pampa province: fallow land - wheat crop (FL-WC), lucerne pasture - wheat crop (LP-WC), fallow land - sunflower crop (FL-SC) and winter annual grass pasture - sunflower crop (WP-SC). Maps, generated with information involving the application for decades of FL-WC, LP-WC, FL-SC and WP-SC rotation schedules, showed the yearly occurrence of partial or absolute droughts during the warm semester. Rotation schedule FL-WC exhibited lower ACR values (0.1 to 0.4) than LP-WC (0.1 to 0.6), FL-SC (0.4 to 0.5) and WP-SC (0.4 to 0.5 ). In all cases, ACR index took higher values for the Centraland South-Eastern regions than the North-Eastern region of La Pampa province.EEA AnguilFil: Casagrande, Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Anguil; Argentina. Universidad Nacional de La Pampa. Facultad de Agronomía; ArgentinaFil: Vergara, Graciela Teresa. Universidad Nacional de La Pampa. Facultad de Agronomía; ArgentinaFil: Belmonte, María Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Anguil; ArgentinaFil: Fuentes, Maria Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Anguil; ArgentinaFil: Arnaiz, Juan Pablo. Universidad Nacional de La Pampa. Facultad de Agronomía; ArgentinaFil: Forte Lay, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Response to fluoxetine in children and adolescents: a weighted gene co-expression network analysis of peripheral blood

The inconclusive and non-replicated results of pharmacogenetic studies of antidepressant response could be related to the lack of acknowledgement of its mechanism of action. In this scenario, gene expression studies provide and interesting framework to reveal new candidate genes for pharmacogenetic studies or peripheral biomarkers of fluoxetine response. We propose a system biology approach to analyse changes in gene expression induced by eight weeks of treatment with fluoxetine in peripheral blood. 21 naïve child and adolescents participated in the present study. Our analysis include the identification of gene co-expression modules, using Weighted Gene Co-expression Network Analysis (WGCNA), followed by protein-protein interaction (PPi) network construction coupled with functional annotation. Our results revealed two modules of co-expression genes related to fluoxetine treatment. The constructed networks from these modules were enriched for biological processes related to cellular and metabolic processes, cell communication, immune system processes, cell death, response to stimulus and neurogenesis. Some of these processes, such as immune system, replicated previous findings in the literature, whereas, neurogenesis, a mechanism proposed to be involved in fluoxetine response, had been identified for first time using peripheral tissues. In conclusion, our study identifies several biological processes in relation to fluoxetine treatment in peripheral blood, offer new candidate genes for pharmacogenetic studies and valuable markers for peripheral moderator biomarkers discovery

Safety and vaccine-induced HIV-1 immune responses in healthy volunteers following a late MVA-B boost 4 years after the last immunization

Background: We have previously shown that an HIV vaccine regimen including three doses of HIV-modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B) was safe and elicited moderate and durable (1 year) T-cell and antibody responses in 75% and 95% of HIV-negative volunteers (n = 24), respectively (RISVAC02 study). Here, we describe the long-term durability of vaccine-induced responses and the safety and immunogenicity of an additional MVA-B boost. Methods: 13 volunteers from the RISVAC02 trial were recruited to receive a fourth dose of MVA-B 4 years after the last immunization. End-points were safety, cellular and humoral immune responses to HIV-1 and vector antigens assessed by ELISPOT, intracellular cytokine staining (ICS) and ELISA performed before and 2, 4 and 12 weeks after receiving the boost. Results: Volunteers reported 64 adverse events (AEs), although none was a vaccine-related serious AE. After 4 years from the 1st dose of the vaccine, only 2 volunteers maintained low HIV-specific T-cell responses. After the late MVA-B boost, a modest increase in IFN-γ T-cell responses, mainly directed against Env, was detected by ELISPOT in 5/13 (38%) volunteers. ICS confirmed similar results with 45% of volunteers showing that CD4+ T-cell responses were mainly directed against Env, whereas CD8+ T cell-responses were similarly distributed against Env, Gag and GPN. In terms of antibody responses, 23.1% of the vaccinees had detectable Env-specific binding antibodies 4 years after the last MVA-B immunization with a mean titer of 96.5. The late MVA-B boost significantly improved both the response rate (92.3%) and the magnitude of the systemic binding antibodies to gp120 (mean titer of 11460). HIV-1 neutralizing antibodies were also enhanced and detected in 77% of volunteers. Moreover, MVA vector-specific T cell and antibody responses were boosted in 80% and 100% of volunteers respectively. Conclusions: One boost of MVA-B four years after receiving 3 doses of the same vaccine was safe, induced moderate increases in HIV-specific T cell responses in 38% of volunteers but significantly boosted the binding and neutralizing antibody responses to HIV-1 and to the MVA vector

iHIVARNA phase IIa, a randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of iHIVARNA-01 in chronically HIV-infected patients under stable combined antiretroviral therapy

Background: HIV therapeutic vaccination aims to improve the immune responses against HIV in order to control viral replication without the need for combined antiretroviral therapy (cART). iHIVARNA-01 is a novel vaccine combining mRNA delivery and T-cell immunogen (HTI) based on conserved targets of effective antiviral T-cell responses. In addition, it holds adequate stimuli required for activating antigen presenting cells (APC)s and co-activating specific T-cells (TriMix), including human CD40L, constitutively active TLR4 (caTLR4) and CD70. We propose that in-vivo targeting of dendritic cells (DCs) by direct administration of a HIV mRNA encoding these immune modulating proteins might be an attractive alternative to target DCs in vitro. Methods/design: This is a phase-IIa, randomized, double-blinded, placebo-controlled, multicenter study in chronically HIV-1 infected patients under stable cART. One of the three study arms is randomly allocated to subjects. Three vaccinations with either HIVACAT T-cell immunogen (HTI)-TriMix (iHIVARNA-01), TriMix or water for injection (WFI) (weeks 0, 2 and 4) are administered by intranodal injection in the inguinal region. Two weeks after the last immunization (week 6) cART is stopped for 12 weeks. The two primary endpoints are: (1) safety and tolerability of intranodal iHIVARNA-01 vaccination compared with TriMix or WFI and (2) induced immunogenicity, i.e., increase in the frequency of HIV-specific T-cell responses between baseline, week 6 and 12 weeks after treatment interruption in iHIVARNA-01-treated patients as compared to the control groups, immunized with TriMix-mRNA or WFI measured by an IFNγ ELISPOT assay. Secondary endpoints include the evaluation of time to viral rebound, plasma viral load (pVL) at w18, the proportion of patients with control of viral load, induction of T-cell responses to new HIV epitopes, polyfunctionality of HIV-specific T-cells, CD8+ T-cell in-vitro HIV suppressive capacity, the effect on viral reservoir (measured by proviral DNA and cell-associated RNA), assessment of viral immune escape by mutation and mRNA expression profiles of host immune genes. Discussion: This trial aims to direct target DC in situ with mRNA encoding HTI and TriMix for co-stimulation. Intranodal injection circumvents laborious DC isolation and handling in the laboratory. The trial extends on the safety results of a phase-I dose-escalating trial. This candidate vaccine could complement or even replace cART for chronic HIV infection and could be applicable to improve the care and cost of HIV infection

Randomized placebo-controlled phase II trial of autologous mesenchymal stem cells in multiple sclerosis.

OBJECTIVE: Uncontrolled studies of mesenchymal stem cells (MSCs) in multiple sclerosis suggested some beneficial effect. In this randomized, double-blind, placebo-controlled, crossover phase II study we investigated their safety and efficacy in relapsing-remitting multiple sclerosis patients. Efficacy was evaluated in terms of cumulative number of gadolinium-enhancing lesions (GEL) on magnetic resonance imaging (MRI) at 6 months and at the end of the study. METHODS: Patients unresponsive to conventional therapy, defined by at least 1 relapse and/or GEL on MRI scan in past 12 months, disease duration 2 to 10 years and Expanded Disability Status Scale (EDSS) 3.0-6.5 were randomized to receive IV 1-2×10(6) bone-marrow-derived-MSCs/Kg or placebo. After 6 months, the treatment was reversed and patients were followed-up for another 6 months. Secondary endpoints were clinical outcomes (relapses and disability by EDSS and MS Functional Composite), and several brain MRI and optical coherence tomography measures. Immunological tests were explored to assess the immunomodulatory effects. RESULTS: At baseline 9 patients were randomized to receive MSCs (n = 5) or placebo (n = 4). One patient on placebo withdrew after having 3 relapses in the first 5 months. We did not identify any serious adverse events. At 6 months, patients treated with MSCs had a trend to lower mean cumulative number of GEL (3.1, 95% CI = 1.1-8.8 vs 12.3, 95% CI = 4.4-34.5, p = 0.064), and at the end of study to reduced mean GEL (-2.8±5.9 vs 3±5.4, p = 0.075). No significant treatment differences were detected in the secondary endpoints. We observed a non-significant decrease of the frequency of Th1 (CD4+ IFN-γ+) cells in blood of MSCs treated patients. CONCLUSION: Bone-marrow-MSCs are safe and may reduce inflammatory MRI parameters supporting their immunomodulatory properties. ClinicalTrials.gov NCT01228266

Immunological function restoration with Lopinavir/ritonavir vs Efavirenz containing regimens in HIV infected patients: a randomized clinical trial

CD4+ count increase has been reported to be different with lopinavir/r (LPV/r) and efavirenz (EFV)-containing regimens. The different effect of these two regimens on other immune function parameters and the relationship with the gain of CD4+ count have not been assessed in a randomized clinical trial. Fifty antiretroviral treatment (cART) naïve HIV-infected individuals were randomized to receive LPV/r or EFV both with tenofovir/emtricitabine for 48 weeks. A substudy of immunological function restoration was performed in 22 patients (LPV/r n=10 and EFV n=12). Activation, thymic function, apoptosis, senescence, exhaustion, Treg cells, interleukin (IL)-7-receptor/IL-7 system, thymic volume, and lymphoid tissue fibrosis were evaluated at baseline and at week 48. Both groups experienced a CD4+ count increase that was higher in the EFV group (ΔCD4+ 88 vs. 315 cells/μl LPV/r vs. EFV, respectively, p<0.001). Despite this difference in CD4+ gain, the change in other immune function parameters was similar in both treatment groups. Most of parameters evaluated tended to normalize after 48 weeks of cART. A significant decrease in levels of activation, senescence, exhaustion, and apoptosis on CD4+ and CD8+ T cells (p<0.001 for all) and a significant increase in markers of thymic function, IL-7 receptor, and in the levels of central memory CD4+ T cells and naive subsets of CD8+ T cells (p<0.001 for all) with respect to baseline values were observed without any difference between groups. These data indicate that the differences in CD4+ gain with different cART regimens are not immunologically meaningful and might explain the similar clinical efficacy of these regimens

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality