Randomized clinical trial of BCG vaccine in patients with convalescent COVID-19: Clinical evolution, adverse events, and humoral immune response.

BACKGROUND: The Bacillus Calmette-Guérin (BCG) vaccine may confer cross-protection against viral diseases in adults. This study evaluated BCG vaccine cross-protection in adults with convalescent coronavirus disease 2019 (COVID-19). METHOD: This was a multicenter, prospective, randomized, placebo-controlled, double-blind phase III study (ClinicalTrials.gov: NCT04369794). SETTING: University Community Health Center and Municipal Outpatient Center in South America. PATIENTS: a total of 378 adult patients with convalescent COVID-19 were included. INTERVENTION: single intradermal BCG vaccine (n = 183) and placebo (n = 195). MEASUREMENTS: the primary outcome was clinical evolution. Other outcomes included adverse events and humoral immune responses for up to 6 months. RESULTS: A significantly higher proportion of BCG patients with anosmia and ageusia recovered at the 6-week follow-up visit than placebo (anosmia: 83.1% vs. 68.7% healed, p = 0.043, number needed to treat [NNT] = 6.9; ageusia: 81.2% vs. 63.4% healed, p = 0.032, NNT = 5.6). BCG also prevented the appearance of ageusia in the following weeks: seven in 113 (6.2%) BCG recipients versus 19 in 126 (15.1%) placebos, p = 0.036, NNT = 11.2. BCG did not induce any severe or systemic adverse effects. The most common and expected adverse effects were local vaccine lesions, erythema (n = 152; 86.4%), and papules (n = 111; 63.1%). Anti-severe acute respiratory syndrome coronavirus 2 humoral response measured by N protein immunoglobulin G titer and seroneutralization by interacting with the angiotensin-converting enzyme 2 receptor suggest that the serum of BCG-injected patients may neutralize the virus at lower specificity; however, the results were not statistically significant. CONCLUSION: BCG vaccine is safe and offers cross-protection against COVID-19 with potential humoral response modulation. LIMITATIONS: No severely ill patients were included.

Mitochondrial heat shock protein mortalin as potential target for therapies based on oxidative stress.

BACKGROUND: Treatments based on production of reactive oxygen species for bladder cancer such as photodynamic therapy (PDT) have been marginalized due to low specificity and the existence of resistance mainly associated with the up-regulation of Heat Shock Proteins (HSPs). To overcome these barriers, the establishment of strategies combining PDTs with HSP inhibitors may be promising and the identification of HSPs involved with oxidative stress from bladder tumors in animal models represents a key step in this direction. MATERIALS: Thus, the present study aims to identify cytosolic and mitochondrial HSPs up expressed in murine bladder tumors and in the urothelial carcinoma cell line MB49 by qRT-PCR screening, and to analyze the importance of the activity of the HSPs associated with oxidative stress protection in the survival of the MB49 using strategy of inhibition in vitro. RESULTS: Results showed that both tumor tissues and MB49 cells in culture had significant overexpression of the mitochondrial HSPA9 (mortalin) and HSP60 mRNAs, while the cytosolic HSP90 was overexpressed only in the tumor. The effect of mortalin in the MB49 cells survival under oxidative stress was evaluated in vitro in presence of the specific inhibitor MKT-077 and H2O2. The findings showed that MB49 viability was permanently reduced by the MKT-077 in a dose-dependent manner by inducing apoptosis or necrosis, mainly under oxidative stress conditions. CONCLUSION: Results suggest that mortalin is preferentially expressed in the MB49 cancer model and plays a key role in tumoral survival, especially under oxidative stress, making this HSP a potential target for an alternative treatment combining PDT with HSP inhibitors.

Tissue microarray: physical and chemical parameters involved in the construction of recipient blocks / Microarranjo de tecidos: parâmetros físicos e químicos envolvidos na construção dos blocos receptores

ABSTRACT Introduction: Tissue microarray (TMA) is considered an innovative method in several fields, with a great diversity of applications and advantages over traditional histomorphometric techniques. The most important advantage that TMA offers is the simultaneous evaluation of a large number of specimens from a limited source of material. However, TMA exhibits a high rate of non-viable samples in the final stages of the process, which compromise their use in analyzes that can not be repeated. Objective: Considering this disadvantage, the objective of this study was to optimize the methodology to maximize the viability of the samples, as well as to increase the efficiency of the technique. Material and methods: For this purpose, several variables involved in the construction of the recipient blocks, including paraffin composition, diameter, spacing distance, localization and type of the tissue samples in the block were tested in order to establish correlations between the quality of the values and the parameters studied. Results: The results showed that the blocks built with polymer-enriched paraffin, subjected to the fusion protocol at 37ºC, associated to a tempering, and constructed with one millimeter diameter samples and 1000 µm spacing between tissues, produced slides whith superior features. Conclusion: The data obtained from the physical and chemical adjustments of the TMA recipient blocks provided vital information that, when applied in TMA research projects, may reduce the losses associated with the method.

RESUMO Introdução: O microarranjo tecidual (MAT) é considerado um método inovador em vários campos, com uma vasta diversidade de aplicações e vantagens em relação às técnicas histomorfométricas clássicas. A vantagem mais importante que o MAT oferece é a avaliação simultânea de um grande número de espécimes de uma fonte limitada de material. Contudo, ele apresenta uma taxa elevada de amostras não viáveis nos estádios finais do processo, o que compromete sua utilização em análises que não podem ser repetidas. Objetivos: Considerando essa desvantagem, o objetivo deste estudo foi otimizar a metodologia para maximizar a viabilidade das amostras, bem como aumentar a eficiência da técnica. Material e métodos: Para tanto, foram testadas várias variáveis envolvidas na construção dos blocos receptores, como composição da parafina, diâmetro, distância de espaçamento, localização e tipo das amostras de tecido no bloco, a fim de estabelecer correlações entre a qualidade dos valores e os parâmetros estudados. Resultados: Os resultados mostraram que os blocos construídos com parafina enriquecida em polímero, submetidos ao protocolo de fusão a 37ºC, acoplados a ciclos de aquecimento e resfriamento e construídos com amostras de um milímetro de diâmetro e espaçamento entre os tecidos de 1000 µm, produziram lâminas com características superiores. Conclusão: Os dados obtidos dos ajustes físicos e químicos dos blocos de receptores de MAT forneceram informações vitais que, quando aplicadas em projetos de pesquisa de MAT, podem reduzir as perdas associadas ao método.

Th1/Th2 cytokines' expression and production by propolis-treated mice.

AIM OF THE STUDY: Propolis is a natural product extensively used in food and beverages to improve health and to prevent diseases, showing immunomodulatory properties. The goal of this work was to evaluate the effect of propolis administration over a short-term to mice on Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines' expression and production. MATERIALS AND METHODS: Propolis was administered for 3 days to mice by gavage, spleens were removed and RNA was extracted to assess cytokines' expression by real-time PCR. Supernatants of spleen cell cultures were used for cytokines determination by ELISA. RESULTS: Propolis administration to mice did not affect IL-2, IL-4 and IL-10 expression and production, while IFN-gamma production was inhibited in the splenocyte cultures stimulated or not by Con A. CONCLUSIONS: Since IFN-gamma is a pro-inflammatory cytokine, our data suggest that propolis administration over a short-term to mice may be associated with anti-inflammatory effects in vivo, and further assays could check propolis efficiency in inflammatory diseases.

Propolis effect on Th1/Th2 cytokines production by acutely stressed mice.

AIM OF THE STUDY: Propolis has gained special attention due to its biological properties, however, little is known about its immunomodulatory effects in stress conditions. The purpose of this study was to investigate propolis effect on Th1/Th2 cytokines production by spleen cells of acutely stressed mice. Serum corticosterone concentration was determined as a stress indicator. MATERIALS AND METHODS: Male BALB/c mice were submitted to restraint stress and treated with propolis (200mg/kg) for 3 days. Supernatants of splenocytes cultures were assessed for Th1/Th2 cytokines determination. RESULTS: Regarding Th1 cytokines production, no alterations were seen in IL-2 production; however, IFN-gamma production was inhibited in stressed mice, even when treated with propolis. As to Th2 cytokines, IL-4 was inhibited in stressed mice, but normal levels were seen when these animals were treated with propolis. No significant differences were found in IL-10 production between the experimental groups. Stressed groups (treated or not with propolis) showed higher corticosterone concentrations in comparison to control group. CONCLUSIONS: Data suggest that propolis treatment was not able to counteract the stress-induced immunosuppressive effect on IFN-gamma production; however, propolis showed an immunorestorative role, increasing IL-4 production in stressed mice, favoring humoral immune response during stress. Since the exact mechanisms of this natural product on immune system are still unclear, further studies are still required for a better comprehension of propolis use as a therapeutic alternative against the stress-induced negative effects that could lead to the development of various diseases.

Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice.

INTRODUCTION: Propolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1beta and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells. METHODS: BALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator. RESULTS: Stressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1beta and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1beta and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress. CONCLUSION: Propolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions.

Th1/Th2 cytokine production by clove-treated mice.

Although clove possesses several biological and therapeutic properties, its immunomodulatory action has not been fully investigated. The goal of this work was to investigate the effect of administration of the water extract of clove over a short-term to BALB/c mice on Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokine production. After treatment, spleen cells were aseptically removed and cells were stimulated with concanavalin A. Supernatants of cell cultures were used for cytokine determination by ELISA. The chemical composition of the extract was also carried out, revealing that eugenol(4-allyl-2-methoxyphenol) was the major component in our sample. Although the anti-inflammatory action of clove has been mentioned, our data showed that clove administration to mice did not influence the Th1/Th2 cytokine balance. Further studies dealing with cytokine expression and production will provide a better understanding of clove's immunomodulatory and anti-inflammatory actions, using different extract concentrations and different intake periods.