Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and na\uefve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM 12 plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies

Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with Covid-19 pneumonia.

We provide an in-depth investigation of the T cell compartment and functionality, cytokine production and plasma levels in a total of 39 patients affected by Covid-19 pneumonia. At admission, patients were lymphopenic; for all, SARS-CoV-2 was detected in a nasopharyngeal swab specimen by real-time RT-PCR, and pneumonia was subsequently confirmed by X-rays. Detailed 18-parameter flow cytometry coupled with unsupervised data analysis revealed that patients showed similar percentages of CD4+ and CD8+ T cells, but a decreased absolute number in both populations. For CD4+ T lymphocytes, we found a significant decrease in the number of na\uefve, central and effector memory cells and an increased percentage of terminally differentiated cells, regulatory T cells, and of those that were activated or that were expressing PD1 and CD57 markers. Studies on chemokine receptors and lineage-specifying transcription factors revealed that, among CD4+ T cells, patients displayed a lower percentage of cells expressing CCR6 or CXCR3, and of those co-expressing CCR6 and CD161, but higher percentages of 62 CXCR4+ or CCR4+ cells. No differences were noted in the expression of T-bet or GATA-3. Analyses of patients' CD8+ T cells showed decreased numbers of na\uefve and central memory and increased amounts of activated cells, accompanied by increased percentages of activated cells and of lymphocytes expressing CD57, PD1, or both. CD8+ T cells expressed lower percentages of CCR6+, CXCR3+ or T-bet+ cells and of CXCR3+,T-bet+ or CCR6+,CD161+ lymphocytes. We also found higher percentages of cells expressing CCR4+, CXCR4 or GATA-3. Analyses of lymphocyte proliferation revealed that terminally differentiated CD4+ and CD8+ T cell from patients had a lower proliferative index than controls, whereas cellular bioenergetics, measured by the quantification of mitochondrial oxygen consumption and extracellular acidification rate, was similar in CD4+ T cells from both groups. We measured plasma level of 31 cytokines linked to inflammation, including T helper (TH)type-1 and TH2 cytokines, chemokines, galectins, pro- and anti-inflammatory mediators, finding that most were dramatically increased in Covid-19 patients, confirming the presence of a massive cytokine storm. Analysis of the production of different cytokines after stimulation by anti-CD3/CD28 monoclonal antibodies revealed that patients not only had a high capacity to produce tumour necrosis factor (TNF)-\u3b1, interferon (IFN)-\u3b3 and interleukin (IL)-2, but also showed a significant skewing of CD4+ T cells towards the TH17 phenotype. A therapeutic approach now exists based on the administration of drugs that block IL-6pathway, and seems to improve the disease. IL-17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid-19. We show here that a skewing of activated T cells towards the TH17 functional phenotype exists in Covid-19 patients. We therefore suggest that blocking the IL-17 pathway by biological drugs that are already used to treat different pathologies could provide a novel, additional strategy to improve the health of patients infected by SARS-CoV-2

Effects of cytokine blocking agents on hospital mortality in patients admitted to ICU with acute respiratory distress syndrome by SARS-CoV-2 infection: Retrospective cohort study

Background: The use of cytokine-blocking agents has been proposed to modulate the inflammatory response in patients with COVID-19. Tocilizumab and anakinra were included in the local protocol as an optional treatment in critically ill patients with acute respiratory distress syndrome (ARDS) by SARS-CoV-2 infection. This cohort study evaluated the effects of therapy with cytokine blocking agents on in-hospital mortality in COVID-19 patients requiring mechanical ventilation and admitted to intensive care unit. Methods: The association between therapy with tocilizumab or anakinra and in-hospital mortality was assessed in consecutive adult COVID-19 patients admitted to our ICU with moderate to severe ARDS. The association was evaluated by comparing patients who received to those who did not receive tocilizumab or anakinra and by using different multivariable Cox models adjusted for variables related to poor outcome, for the propensity to be treated with tocilizumab or anakinra and after patient matching. Results: Sixty-six patients who received immunotherapy (49 tocilizumab, 17 anakinra) and 28 patients who did not receive immunotherapy were included. The in-hospital crude mortality was 30,3% in treated patients and 50% in non-treated (OR 0.77, 95% CI 0.56-1.05, p=0.069). The adjusted Cox model showed an association between therapy with immunotherapy and in-hospital mortality (HR 0.40, 95% CI 0.19-0.83, p=0.015). This protective effect was further confirmed in the analysis adjusted for propensity score, in the propensity-matched cohort and in the cohort of patients with invasive mechanical ventilation within 2 hours after ICU admission. Conclusions: Although important limitations, our study showed that cytokine-blocking agents seem to be safe and to improve survival in COVID-19 patients admitted to ICU with ARDS and the need for mechanical ventilation

Radical Anions of Fullerene Bisadducts: A Multifrequency CW-EPR study.

The radical anions of three C60 bisadducts (from now on termed CIS1, CIS2, and CIS3) have been studied in liquid solution and glassy matrix by X-band and high frequency CW-EPR spectroscopy. The three adducts CIS1, CIS2, and CIS3 are characterized by a pyrrolidinic ring and an isoxazolinic ring in position cis1-O, cis2-C, and cis3-C, respectively; the two rings are connected by a methylenic chain. In the X-band spectra of CIS1, one species has been observed showing hyperfine coupling constants with the pyrrolidinic nitrogen and nuclei, whereas in the X-band spectra of CIS2 and CIS3 more species are apparent, some with and other without hyperfine coupling with the pyrrolidinic . High field spectra at 110 and 220 GHz for all the bisadducts have been obtained; in all the cases, more species are evident with small differences in the g-factors. The occurrence of more species are discussed and put in relation with different bisadduct conformations. Furthermore, on the basis of the mechanism proposed for the hcc in previously studied fulleropyrrolidine (FP) monoanions, the presence or absence of hcc for CIS1 and the different conformations of CIS2 and CIS3 is discussed. The temperature dependence of the EPR linewidths and the g-factors of all the species have been determined and discussed in term of the different structural stiffness and symmetry of the three bisadducts

Influência da suplementação de vitaminas A, D e E na função imune de bezerros alimentados com dieta á base de feno capim tifton (Cynodon spp.)

Resumo: As vitaminas são compostos orgânicos necessários em poucas quantidades no organismo, todavia indispensáveis para as funções metabólicas. Elas se inserem em inúmeras reações metabólicas, fisiológicas e imunes das células, necessárias para a manutenção da saúde animal, além de atuarem como imunoestimulante. Embora a dieta rica em folhagens verdes frescas forneça quantidades suficientes de vitaminas A, D e E a suplementação intensiva com alimentos conservados na forma de feno ou silagem pode reduzir em até 50 % dos teores destas vitaminas no alimento. Diante disso, a proposta do trabalho foi verificar se a administração parenteral de vitaminas A, D e E age como imunoestimulante em garrotes estabulados e alimentados exclusivamente com feno de tifton. Para tanto 14 bovinos foram divididos em dois grupos homogêneos, sendo o grupo S, suplementado com vitamina A, D e E em dose única de 30 mL por via intramuscular; e o grupo C, sem suplementação. Ambos os grupos foram alojados em baias parcialmente privadas de sol, e alimentados com feno por um período de três meses. A avaliação imune foi realizada por hemogramas e ensaio de função leucocitária (metabolismo oxidativo e fagocitose) nos momentos antes do tratamento, três e dez dias após os tratamentos. Tendo em vista que a suplementação com polivitamínicos A, D e E aumentou a porcentagem da atividade de células granulocítica e a intensidade da atividade de células mononucleares, além de intensificar o efeito antioxidante prolongando a sobrevida de hemácias e neutrófilos, conclui-se que esta suplementação promoveu efeito benéfico na resposta imune de bezerros da Raça Holandesa, apesar dos efeitos deletérios da alimentação exclusiva com feno e da privação parcial da incidência solar direta

Carotid Flow Velocity/diameter Ratio Is A Predictor Of Cardiovascular Events In Hypertensive Patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objective:The aim of this study was to evaluate the performance of common carotid artery diameter, flow velocity and flow velocity/artery diameter ratio as predictors of major adverse cardiovascular events (MACEs) in a sample of hypertensive patients.Methods:A cohort of 403 hypertensive patients was followed up for a median of 1260 (714) days, and 27 suffered MACEs. At baseline, participants were evaluated by clinical, laboratory, echocardiographic and carotid ultrasound analysis.Results:Patients with peak-systolic flow velocity (sFV) less than the median value and systolic artery diameter (sAD) greater than the median value presented the worst clinical outcome compared to those with isolated sFV less than the median value or sAD greater than the median value, suggesting an additive effect of these two variables. Further, Kaplan-Meier analysis demonstrated worse outcome for individuals with sFV/sAD ratio less than 85.7/s (optimal cut-off point obtained by receiver-operating characteristic analysis) compared to those with higher sFV/sAD values (log-rank test: P<0.0001). In stepwise multivariable Cox-regression analyses, sFV/sAD was significantly associated with MACEs (P<0.001), whereas carotid intima-media thickness and clinically defined high cardiovascular risk did not. Furthermore, area under the receiver-operating characteristic curve for sFV/sAD was higher than that for Framingham risk score (0.77 versus 0.64; P=0.045), whereas adding sFV/sAD to the Framingham risk factors resulted in increased net reclassification improvement (P=0.041) and integrated discrimination improvement (P<0.001).Conclusions:Lower carotid sFV/sAD was associated with MACEs in hypertensive patients independent of cardiovascular prediction models and carotid intima-media thickness. These findings suggest that this index may be a promising approach to identify hypertensive subjects at increased risk for future cardiovascular events.331020542060Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq [476909/2012-0, 304245/2013-5

Influence of the C242T Polymorphism of the p22-phox Gene (CYBA) on the Interaction between Urinary Sodium Excretion and Blood Pressure in an Urban Brazilian Population

<div><p>Background</p><p>Reactive oxygen species are implicated in the physiopathogenesis of salt-induced hypertension and the C242T polymorphism of the p22-phox gene has been associated with higher superoxide production. This study investigated the impact of this polymorphism on the relationship between urinary sodium excretion (USE) and blood pressure levels in an urban Brazilian population.</p><p>Methods</p><p>We cross-sectionally evaluated 1,298 subjects from the city of Vitoria-ES, located in the Southeast region of Brazil, by clinical history, physical examination, anthropometry, analysis of laboratory parameters, USE measurement and p22-phox C242T polymorphism genotyping.</p><p>Results</p><p>No significant differences in studied parameters were detected between the studied genotype groups (CC vs. CT+TT). Systolic blood pressure exhibited significant correlation with USE only in T allele carriers (r = 0.166; p<0.001), while diastolic blood pressure and hypertension status correlated with USE in both genotypes albeit more weakly in subjects with CC genotype (r = 0.098; p = 0.021 and r = 0.105; p = 0.013, respectively) than in T carriers (r = 0.236; p<0.001 and r = 0.213; p<0.001, respectively). Regression analyses adjusted for confounding factors showed that USE remained independently associated with systolic (p<0.001) and diastolic blood pressure (p<0.001) and hypertension status (p = 0.004) only in T allele carriers. Finally, higher diastolic and systolic blood pressure levels were detected in T allele carriers than in CC genotype individuals in the highest tertile of USE.</p><p>Conclusions</p><p>The p22-phox 242T allele is associated with higher blood pressure levels among subjects with higher USE in an urban Brazilian population.</p></div

Features of studied subjects according to the tertiles of urinary sodium excretion and p22-phox C242T genotypes.

<p><b>Legend.</b> HDL – high-density-lipoprotein; LDL – low-density-lipoprotein;</p>*<p>p<0.05 in comparison with CC genotype.</p

Linear regression analysis for systolic and diastolic blood pressure.

<p><b>Legend.</b> SBP – systolic blood pressure; DBP – diastolic blood pressure; HDL – high-density-lipoprotein; LDL – low-density-lipoprotein.</p

Logistic regression analysis for hypertension status.

<p><b>Legend.</b> Model 1 also included triglycerides >102 mg/dL, creatinine >1.0 mg/dL and uric acid >4.6 mg/dL, while model 2 also included diabetes mellitus, triglycerides >99 mg/dL, creatinine >1.0 mg/dL, uric acid >4.6 mg/dL and urinary potassium >19.4 mEq/12 h. Only variables that exhibited significant association and urinary sodium excretion are presented in the table.</p