Reduced T Cell and Antibody Responses to Inactivated Coronavirus Vaccine Among Individuals Above 55 Years Old.

CoronaVac is an inactivated SARS-CoV-2 vaccine that has been rolled out in several low and middle-income countries including Brazil, where it was the mainstay of the first wave of immunization of healthcare workers and the elderly population. We aimed to assess the T cell and antibody responses of vaccinated individuals as compared to convalescent patients. We detected IgG against SARS-CoV-2 antigens, neutralizing antibodies against the reference Wuhan SARS-CoV-2 strain and used SARS-CoV-2 peptides to detect IFN-g and IL-2 specific T cell responses in a group of CoronaVac vaccinated individuals (N = 101) and convalescent (N = 72) individuals. The frequency among vaccinated individuals, of whom 96% displayed T cell and/or antibody responses to SARS-CoV-2, is comparable to 98.5% responses of convalescent individuals. We observed that among vaccinated individuals, men and individuals 55 years or older developed significantly lower anti-RBD, anti-NP and neutralization titers against the Wuhan strain and antigen-induced IL-2 production by T cells. Neutralizing antibody responses for Gamma variant were even lower than for the Wuhan strain. Even though some studies indicated CoronaVac helped reduce mortality among elderly people, considering the appearance of novel variants of concern, CoronaVac vaccinated individuals above 55 years old are likely to benefit from a heterologous third dose/booster vaccine to increase immune response and likely protection.

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape.

Introduction: Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information. Methods: We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. Results: We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity. Discussion: This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Astronium fraxinifolium Schott Exerts Leishmanicidal Activity by Providing a Classically Polarized Profile in Infected Macrophages.

BACKGROUND: Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary. PURPOSE: The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages. METHODS: For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-ß by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique. RESULTS: The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-ß and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA. CONCLUSION: Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.

Antioxidant, anti-inflammatory and healing potential of ethyl acetate fraction of Bauhinia ungulata L. (Fabaceae) on in vitro and in vivo wound model.

The present work aimed to investigate the antioxidant, anti-inflammatory and wound healing potential of ethyl acetate fraction from Bauhinia ungulata L. (FABU) on in vitro and in vivo models. Wound healing assay using human lung adenocarcinoma A549 cell line was employed to evaluate the ability of FABU in modulating cell migration. In addition, a surgical wound model in C57BL/6 mice was used to study the healing potential of FABU incorporated into gel carbomer 940 (Carbopol®). Evaluation of lipid peroxidation, inflammatory and anti-inflammatory mediator gene expression, rate of wound closure, and histological analysis were done. FABU significantly reduced the gap area in in vitro wound healing assay, 24 h after treatment. In the animal model, FABU at 0.5% topically applied once-daily for 5 days to the surgical wounds significantly reduced the lesion area. Moreover, it significantly decreased the levels of lipid peroxidation in the lesions and decreased the relative gene expression levels of IL-1ß and TNF-α in the injured region. In conclusion, our study suggests that Bauhinia ungulata can effectively promote the wound healing, probably by regulating the inflammatory environment during the early stages of the process.

Nitro-imidazole-based ruthenium complexes with antioxidant and anti-inflammatory activities.

Inflammation is a physiological process triggered in response to tissue damage, and involves events related to cell recruitment, cytokines release and reactive oxygen species (ROS) production. Failing to control the process duration lead to chronification and may be associated with the development of various pathologies, including autoimmune diseases and cancer. Considering the pharmacological potential of metal-based compounds, two new ruthenium complexes were synthesized: cis-[Ru(NO2)(bpy)2(5NIM)]PF6 (1) and cis-[RuCl(bpy)2(MTZ)]PF6 (2), where bpy = 2,2'-bipyridine, 5NIM = 5-nitroimidazole and MTZ = metronidazole. Both products were characterized by spectroscopic techniques, followed by Density Functional Theory (DFT) calculations in order to support experimental findings. Afterwards, their in vitro cytotoxic, antioxidant and anti-inflammatory activities were investigated. Compounds 1 and 2 presented expressive in vitro antioxidant activity, reducing lipid peroxidation and decreasing intracellular ROS levels with comparable effectiveness to the standard steroidal drug dexamethasone or α-tocopherol. These complexes showed no noticeable cytotoxicity on the tested cancer cell lines. Bactericidal assay against metronidazole-resistant Helicobacter pylori, a microorganism able to disrupt oxidative balance, unraveled compound 1 moderate activity over that strain. Besides this, it was able to inhibit interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) production as well as interleukin-1ß (IL-1ß) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. This latter activity is remarkable, which has not been reported for other ruthenium-based complexes. Altogether, these results suggest cis-[Ru(NO2)(bpy)2(5NIM)]PF6 complex has potential pharmacological application as an anti-inflammatory agent that deserve further biological investigation.

Polymorphism of IL10, IL4, CTLA4, and DAO Genes in Cross-Reactive Nonsteroidal Anti-inflammatory Drug Hypersensitivity.

Our aim was to evaluate genetic polymorphism of molecules involved in immunoregulatory/allergic processes in patients who presented with cutaneous hypersensitivity caused by chemically unrelated nonsteroidal anti-inflammatory drugs. Polymorphisms at IL10 (-1082 G>A), IL4 (-589 C>T), CTLA4 (+49A>G), and DAO (+8956 C>G) genes were studied in 55 cases and 97 controls by the polymerase chain reaction-restriction fragment length polymorphism technique. With regard to the polymorphism at IL10 -1082, higher frequencies of the AG genotype (57% vs 39%) and G allele carriers (70% vs 48%) were found among the patients, indicating a risk effect (odds ratio [OR] = 2.56 and P = .01 for AG genotype and OR = 2.52; P = .01 for AG/GG). For the CTLA4 +49 A/G single-nucleotide polymorphism (SNP), AG genotype (31.0%) (P = .02) and G carrier (54.0%) (P = .05) frequencies were found to be significantly lower in the patient group compared with the control group (51.0% and 69.0%, respectively). The SNP DAO +8956 C>G was associated with a strong protective effect, with OR values of 0.83 for CG and 0.11 for GG genotype (P = .04 for the codominant model), suggesting an allele dose effect. The combination of IL10 and DAO SNPs in a multivariate model did not alter the OR values, suggesting independent effects for both SNPs. The results are striking. In conclusion, these results suggest that polymorphisms in regulatory targets of the immune response and in DAO gene could modulate an individual's susceptibility to nonsteroidal anti-inflammatory drug hypersensitivity reactions. Further studies will be necessary to complement our results.

The Effect of a Selective Inhibitor of Phosphodiesterase-9 on Oxidative Stress, Inflammation and Cytotoxicity in Neutrophils from Patients with Sickle Cell Anaemia.

The aim of the study was to investigate the possible anti-inflammatory and antioxidant effects of BAY 73-6691 on neutrophils from SCA patients. This study included 35 patients with a molecular diagnosis of SCA, whose neutrophils were isolated and treated with BAY 73-6691 at the concentrations 100, 10, 1.0 and 0.1 µg/mL. LDH release and MTT assays were performed to verify cell viability. To evaluate oxidative stress, the following parameters were determined by spectrophotometric assays: NO and malondialdehyde (MDA) levels and activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx). As inflammatory markers, myeloperoxidase (MPO) levels were evaluated by colorimetric assay and TNF-α by enzyme immunoassay. The results showed that neutrophils from SCA patients not treated with hydroxyurea (HU) had significantly lower NO levels and catalase and SOD activity, as well as significantly higher MDA, MPO and TNF-α levels when compared with neutrophils from SCA patients treated with HU and neutrophils from control group. Treatment of SCA neutrophils with BAY 73-6691 resulted in 94%, 200% and 168% increase in NOx levels, SOD and catalase activity, respectively. In addition, there was a reduction of approximately 46% and 45% in TNF-α and MPO levels, respectively. In SCAHU neutrophils, there was a 30% and 44% increase in NOx levels and SOD activity, respectively, and a 28% and 37% decrease in TNF-α and MPO levels, respectively. However, these effects were observed at cytotoxic doses only. The results of this study are original and demonstrate that inhibition of phosphodiesterase-9 in neutrophils from SCA patients with BAY 73-6691 was able to increase the NO bioavailability and attenuate oxidative stress and inflammation in neutrophils from patients not treated with HU.

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p<0.001) in LDH activity and an increase in the percentage of viable cells by the MTT (p<0.001). However, the SSHU group presented significantly higher DI values (49.57±6.0 U/A) when compared to the AA group (7.43 ± 0,94U/A) and the SS group (22.73 ±5.58 U/A) (p<0.0001), especially when treated for longer periods (>20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils.

A hidroxiuréia (HU) constitui o avanço mais importante no tratamento da anemia falciforme (AF) por prevenir complicações e aumentar a qualidade de vida dos pacientes. Entretanto, alguns aspectos do tratamento com HU permanecem obscuros, incluindo a sua ação e potencial toxicidade em outras células sanguíneas, tais como neutrófilos. Este estudo utilizou a mensuração da lactato desidrogenase (LDH) e do metil tiazoltetrazólio (MTT) e o ensaio do cometa para investigar a citotoxicidade e índice de dano (ID) ao DNA em neutrófilos de pacientes com AF em uso do medicamento. Nos ensaios de LDH e MTT, observou-se além de ausência de toxicidade, uma ação citoprotetora no grupo de pacientes tratados, Grupo SSHU (n=21, 11 mulheres e 10 homens, com idades entre 19-63 anos), quando comparados aos pacientes sem tratamento, Grupo SS (n=20, 13 mulheres e 07 homens, 18-69 anos), e grupo de indivíduos saudáveis (AA) usado como controle (n=52, 28 mulheres e 24 homens, 19-60 anos), com redução significativa (p<0,001) na atividade de LDH e aumento no percentual de células viáveis pelo MTT (p<0,001). Entretanto, o grupo SSHU apresentou valores de ID significativamente elevados (49,57±6,0 U/A), quando comparados ao grupo AA (7,43 ± 0,94U/A) e grupo SS (22,73 ±5,58 U/A) (p<0,0001), especialmente quando tratados por períodos mais longos (>20 meses), demonstrando que apesar dos efeitos citoprotetores quanto à viabilidade celular, o uso da HU pode induzir lesão ao DNA de neutrófilos.