Avaliação da influência dos marcadores inflamatórios na mortalidade de pacientes com AVC isquêmico

À medida que a população envelhece e a expectativa de vida aumenta, a incidência global e a prevalência de AVC isquêmico tendem a aumentar significativamente. Nesse contexto, surge a necessidade de avaliar novos marcadores preditores de mortalidade, como a contagem absoluta de monócitos, relação linfócitos sobre monócitos, relação neutrófilos sobre linfócitos e níveis de proteína C reativa ultrassensível, que além de serem de fácil acesso e baixocusto, sugerem indicar desfecho no paciente com AVC agudo. O objetivo deste estudo foi avaliar a associação dos marcadores inflamatórios com a mortalidade de pacientes com AVC isquêmico. Métodos: trata-se de um estudo retrospectivo observacional a partir de prontuários eletrônicos e exames laboratoriais de pacientes com AVC isquêmico em uma unidade hospitalar de Cascavel/PR. Umaanálise estatística descritiva foi conduzida para determinar o perfil dospacientes segundo o desfecho e aplicado um modelo de regressãologística para verificar as variáveis associadas a mortalidade. Foramconsiderados significativos apenas os dados com p-valor 0,05.Resultados: Dos 65 pacientes que foram admitidos no estudo, 50receberam alta hospitalar e 15 foram a óbito no hospital. Entre osmarcadores inflamatórios, a relação de neutrófilos sobre linfócitos(OR 1,55; p-valor 0,01) mostrou-se significativamente associada amaior chance de óbito. Os pacientes que faleceram apresentaramníveis superiores de PCR ultrassensível, maior contagem absoluta demonócitos, relação linfócitos sobre monócitos diminuída, e relaçãoneutrófilos sobre linfócitos elevada. Conclusão: a relação de neutrófilos sobre linfócitos elevada pode estar significativamente associada ao desfecho desfavorável após um AVC isquêmico

Vagotomy diminishes obesity in cafeteria rats by decreasing cholinergic potentiation of insulin release

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORHerein, we investigated whether subdiaphragmatic vagotomy has benefits on obesity, body glucose homeostasis, and insulin secretion in cafeteria (CAF)-obese rats. Wistar rats were fed a standard or CAF diet for 12 weeks. Subsequently, CAF rats were randomly submitted to truncal vagotomy (CAF Vag) or sham operation (CAF Sham). CAF Sham rats were hyperphagic, obese, and presented metabolic disturbances, including hyperinsulinemia, glucose intolerance, insulin resistance, hyperglycemia, and hypertriglyceridemia. Twelve weeks after vagotomy, CAF Vag rats presented reductions in body weight and perigonadal fat stores. Vagotomy did not modify glucose tolerance but normalized fed glycemia, insulinemia, and insulin sensitivity. Isolated islets from CAF Sham rats secreted more insulin in response to the cholinergic agent, carbachol, and when intracellular cyclic adenine monophosphate (cAMP) is enhanced by forskolin or 3-isobutyl-1-methylxanthine. Vagotomy decreased glucose-induced insulin release due to a reduction in the cholinergic action on beta-cells. This effect also normalized islet secretion in response to cAMP. Therefore, vagotomy in rats fed on a CAF-style diet effectively decreases adiposity and restores insulin sensitivity. These effects were mainly associated with the lack of cholinergic action on the endocrine pancreas, which decreases insulinemia and may gradually reduce fat storage and improve insulin sensitivity.Herein, we investigated whether subdiaphragmatic vagotomy has benefits on obesity, body glucose homeostasis, and insulin secretion in cafeteria (CAF)-obese rats. Wistar rats were fed a standard or CAF diet for 12 weeks. Subsequently, CAF rats were randomly submitted to truncal vagotomy (CAF Vag) or sham operation (CAF Sham). CAF Sham rats were hyperphagic, obese, and presented metabolic disturbances, including hyperinsulinemia, glucose intolerance, insulin resistance, hyperglycemia, and hypertriglyceridemia. Twelve weeks after vagotomy, CAF Vag rats presented reductions in body weight and perigonadal fat stores. Vagotomy did not modify glucose tolerance but normalized fed glycemia, insulinemia, and insulin sensitivity. Isolated islets from CAF Sham rats secreted more insulin in response to the cholinergic agent, carbachol, and when intracellular cyclic adenine monophosphate (cAMP) is enhanced by forskolin or 3-isobutyl-1-methylxanthine. Vagotomy decreased glucose-induced insulin release due to a reduction in the cholinergic action on beta-cells. This effect also normalized islet secretion in response to cAMP. Therefore, vagotomy in rats fed on a CAF-style diet effectively decreases adiposity and restores insulin sensitivity. These effects were mainly associated with the lack of cholinergic action on the endocrine pancreas, which decreases insulinemia and may gradually reduce fat storage and improve insulin sensitivity724625633CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORsem informaçã

Influence of N-Glycosylation on the Morphogenesis and Growth of Paracoccidioides brasiliensis and on the Biological Activities of Yeast Proteins

The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology

Diabetes Mellitus: Um estudo epidemiológico retrospectivo e observacional

ABSTRACT: Diabetes mellitus is a chronic disease in which hyperglycemia occurs and causes several changes throughout the patients' lives. Given the changes in all areas due to Covid-19, the epidemiological study carried out showed a drop in the incidence of cases of Diabetes Mellitus diagnosed in the period from January 1 2015 to July 31 2021, which may be related to the direction of the outpatients clinics for the exclusive care of patients who have contracted covid-19.RESUMO: O diabetes mellitus é uma doença crônica em que ocorre hiperglicemia e acarreta em várias alterações ao longo da vida dos pacientes. Diante das mudanças em todos os âmbitos devido a Covid-19, o estudo epidemiológico realizado mostrou queda na incidência dos casos de Diabetes Mellitus diagnosticado no período de 01 janeiro de 2015 até 31 de julho de 2021, podendo ser relacionado ao direcionamento dos ambulatórios para o atendimento exclusivo de pacientes que contraíram a covid-19.&nbsp

Toxoplasma gondii chitinase induces macrophage activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 degrees C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathoge1012112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/10741-8]2013/10741-8SEM INFORMAÇÃOThis study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2013/10741-8). Additional financial help was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio ao Ensino, Pe

Recombinant paracoccin reproduces the biological properties of the native protein and induces protective Th1 immunity against Paracoccidioides brasiliensis infection.

BACKGROUND: Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. METHODOLOGY/PRINCIPAL FINDINGS: The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. CONCLUSIONS/SIGNIFICANCE: Our results showed that the recombinant protein reproduced the biological properties described for the native protein-including binding to laminin in a manner that is dependent on carbohydrate recognition-showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects

Electrophoretic profile of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> Induction time/response rPCN_exon4 detected in the lysates of <i>E. coli.</i> The time elapsed since IPTG induction is shown in hours. <b>Panel B:</b> The bound material to glutathione-sepharose was separated by 12% SDS-PAGE Coomassie blue staining revealed a single band (lane 1), which was recognized by specific antibodies against GST (lane 2) and PCNprep (lane 3). <b>Panel C:</b> rPCN_full was purified and evaluated for its ability to bind to <i>N</i>-acetylglucosamine. The bound material was separated by 10% SDS-PAGE under reducing conditions, and then stained with Coomassie blue. Lane 1, material before refolding; Lane 2, material after refolding (a single band was detected with an apparent molecular mass of 28 kDa). Molecular markers were a mixture of pre-stained proteins (Fermentas). <b>Panel D:</b> The rPCN_full band was recognized by an anti-paracoccin antibodies (lane 2) and not by antibodies from pre-immune sera.</p

Cloning strategies for cloning the paracoccin ORF for expression.

<p>The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from <i>P. brasiliensis</i> strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by <i>Bam</i>HI and <i>Eco</i>RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.</p

Anti-rPCN_full antibody reactivity on the yeast cell surface.

<p>Fluorescence labeling with anti-rPCN_full (<b>Panel A–D</b>) was evenly distributed over the yeast cell surface, with more intense labeling in some budding regions. Panel D is the merge of panels B and C.</p

Biological and enzymatic properties of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> production of TNF-α, <b>Panel B:</b> production of NO by induced murine macrophages following <i>in vitro</i> stimulation with PCNprep and rPCN_exon4 or PCNprep and rPCN_full. Cells were harvested from the peritoneal cavity of C57BL/6 mice and induced with thioglycollate. Adherent cells were incubated for 48 h with different recombinant proteins (0.25 mg/mL), medium (negative control), or LPS+IFNγ (positive control). The standard deviation was calculated based on tests performed in triplicate. The activity of the samples was compared to that of the medium alone. <b>Panel C:</b> The PCNprep, rPCN_exon4 and rPCN_full were assayed for NAGase activity. A colorimetric assay was performed in spectrophotometer set at λ = 405 nm. The standard deviation was calculated by analysis of experiments performed in triplicate. <b>Panel D:</b> Binding of rPCN_full to laminin. Different amounts of biotinylated recombinant protein were incubated with laminin (250 ng) coated in the microplate wells. The binding of the biotinylated protein was detected with a neutravidin-peroxidase conjugate and a chemiluminescent substrate. Luminescence readings are reported as relative luminescence units (RLU). <b>Panel E:</b> Inhibition of rPCN_full binding to laminin by sugars. Different concentrations of GlcNAc, d-glucose, and d-galactose were pre-incubated with the recombinant protein (100 ng), and the mixture was then added to the laminin-coated wells. The margin of error was calculated by analysis of triplicate experiments. Each sample with sugar was compared to a sample without sugar.</p