Characterization of Dengue Virus 4 Cases in Paraguay, 2019-2020.

In 2019-2020, dengue virus (DENV) type 4 emerged to cause the largest DENV outbreak in Paraguay's history. This study sought to characterize dengue relative to other acute illness cases and use phylogenetic analysis to understand the outbreak's origin. Individuals with an acute illness (≤7 days) were enrolled and tested for DENV nonstructural protein 1 (NS1) and viral RNA by real-time RT-PCR. Near-complete genome sequences were obtained from 62 DENV-4 positive samples. From January 2019 to March 2020, 799 participants were enrolled: 253 dengue (14 severe dengue, 5.5%) and 546 other acute illness cases. DENV-4 was detected in 238 dengue cases (94.1%). NS1 detection by rapid test was 52.5% sensitive (53/101) and 96.5% specific (387/401) for dengue compared to rRT-PCR. DENV-4 sequences were grouped into two clades within genotype II. No clustering was observed based on dengue severity, location, or date. Sequences obtained here were most closely related to 2018 DENV-4 sequences from Paraguay, followed by a 2013 sequence from southern Brazil. DENV-4 can result in large outbreaks, including severe cases, and is poorly detected with available rapid diagnostics. Outbreak strains seem to have been circulating in Paraguay and Brazil prior to 2018, highlighting the importance of sustained DENV genomic surveillance.

Serum biomarkers and anti-flavivirus antibodies at presentation as indicators of severe dengue.

BACKGROUND: Dengue is the most common vector-borne viral disease worldwide. Most cases are mild, but some evolve into severe dengue (SD), with high lethality. Therefore, it is important to identify biomarkers of severe disease to improve outcomes and judiciously utilize resources. METHODS/PRINCIPAL FINDINGS: One hundred forty-five confirmed dengue cases (median age, 42; range <1-91 years), enrolled from February 2018 to March 2020, were selected from an ongoing study of suspected arboviral infections in metropolitan Asunción, Paraguay. Cases included dengue virus types 1, 2, and 4, and severity was categorized according to the 2009 World Health Organization guidelines. Testing for anti-dengue virus IgM and IgG and serum biomarkers (lipopolysaccharide binding protein and chymase) was performed on acute-phase sera in plate-based ELISAs; in addition, a multiplex ELISA platform was used to measure anti-dengue virus and anti-Zika virus IgM and IgG. Complete blood counts and chemistries were performed at the discretion of the care team. Age, gender, and pre-existing comorbidities were associated with SD vs. dengue with/without warning signs in logistic regression with odds ratios (ORs) of 1.07 (per year; 95% confidence interval, 1.03, 1.11), 0.20 (female; 0.05,0.77), and 2.09 (presence; 1.26, 3.48) respectively. In binary logistic regression, for every unit increase in anti-DENV IgG in the multiplex platform, odds of SD increased by 2.54 (1.19-5.42). Platelet count, lymphocyte percent, and elevated chymase were associated with SD in a combined logistic regression model with ORs of 0.99 (1,000/µL; 0.98,0.999), 0.92 (%; 0.86,0.98), and 1.17 (mg/mL; 1.03,1.33) respectively. CONCLUSIONS: Multiple, readily available factors were associated with SD in this population. These findings will aid in the early detection of potentially severe dengue cases and inform the development of new prognostics for use in acute-phase and serial samples from dengue cases.

Etiological Roles of p75NTR in a Mouse Model of Wet Age-Related Macular Degeneration.

Choroidal neovascularization (CNV) is a pathological angiogenesis of the choroidal plexus of the retina and is a key feature in the wet form of age-related macular degeneration. Mononuclear phagocytic cells (MPCs) are known to accumulate in the subretinal space, generating a chronic inflammatory state that promotes the growth of the choroidal neovasculature. However, how the MPCs are recruited and activated to promote CNV pathology is not fully understood. Using genetic and pharmacological tools in a mouse model of laser-induced CNV, we demonstrate a role for the p75 neurotrophin receptor (p75NTR) in the recruitment of MPCs, in glial activation, and in vascular alterations. After laser injury, expression of p75NTR is increased in activated Muller glial cells near the CNV area in the retina and the retinal pigmented epithelium (RPE)-choroid. In p75NTR knockout mice (p75NTR KO) with CNV, there is significantly reduced recruitment of MPCs, reduced glial activation, reduced CNV area, and the retinal function is preserved, as compared to wild type mice with CNV. Notably, a single intravitreal injection of a pharmacological p75NTR antagonist in wild type mice with CNV phenocopied the results of the p75NTR KO mice. Our results demonstrate that p75NTR is etiological in the development of CNV.

LRP1 mediates the IGF-1-induced GLUT1 expression on the cell surface and glucose uptake in Müller glial cells.

Insulin-like Growth Factor-1 (IGF-1) is involved in the normal development and survival of retinal cells. Low-density lipoprotein Receptor-related Protein-1 (LRP1) plays a key role on the regulation of several membrane proteins, such as the IGF-1 receptor (IGF-1R). In brain astrocytes, LRP1 interact with IGF-1R and the glucose transporter type 1 (GLUT1), regulating the glucose uptake in these cells. Although GLUT1 is expressed in retinal Müller Glial Cells (MGCs), its regulation is not clear yet. Here, we investigated whether IGF-1 modulates GLUT1 traffic to plasma membrane (PM) and glucose uptake, as well as the involvement of LRP1 in this process in the human Müller glial-derived cell line (MIO-M1). We found that IGF-1 produced GLUT1 translocation to the PM, in a time-dependent manner involving the intracellular signaling activation of MAPK/ERK and PI3K/Akt pathways, and generated a significant glucose uptake. Moreover, we found a molecular association between LRP1 and GLUT1, which was significantly reduced by IGF-1. Finally, cells treated with specific siRNA for LRP1 showed an impaired GLUT1 expression on PM and decreased glucose uptake induced by IGF-1. We conclude that IGF-1 regulates glucose homeostasis in MGCs involving the expression of LRP1.

Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor.

Hemin is an erythropoietic inductor capable of inducing autophagy in erythroid-like cell lines. Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor involved in a wide range of cellular processes, such as proliferation, differentiation, and metabolism. Our aim was to evaluate whether LRP1 is responsible for hemin activity in K562 cells, with the results demonstrating a three-fold increase in LRP1 gene expression levels (P-values <0.001) when assessed by quantitative real-time RT-PCR (qRT-PCR). Moreover, a 70% higher protein amount was observed compared with control condition (P-values <0.01) by Western blot (WB). Time kinetic assays demonstrated a peak in light chain 3 (LC3) II (LC3II) levels after 8 h of hemin stimulation and the localization of LRP1 in the autophagosome structures. Silencing LRP1 by siRNA decreased drastically the hemin-induced autophagy activity by almost 80% compared with control cells (P-values <0.01). Confocal localization and biochemical analysis indicated a significant redistribution of LRP1 from early endosomes and recycling compartments to late endosomes and autophagolysosomes, where the receptor is degraded. We conclude that LRP1 is responsible for hemin-induced autophagy activity in the erythroblastic cell line and that hemin-LRP1 complex activation promotes a self-regulation of the receptor. Our results suggest that hemin, via the LRP1 receptor, favors erythroid maturation by inducing an autophagic response, making it a possible therapeutic candidate to help in the treatment of hematological disorders.

Fasciola hepatica Kunitz type molecule decreases dendritic cell activation and their ability to induce inflammatory responses.

The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.

Differential effects of insulin on peripheral diabetes-related changes in mitochondrial bioenergetics: involvement of advanced glycosylated end products.

Large scale clinical trials have demonstrated that an intensive antihyperglycemic treatment in diabetes mellitus (DM) in individuals reduces the incidence of micro- and macrovascular complications, e.g. nephropathy, retinopathy, DM-accelerated atherosclerosis, myocardial infarction, or limb amputations. Here, we investigated the effect of short- and long-term insulin administration on mitochondrial function in peripheral tissues of streptozotocin (STZ)-induced hyperglycemic rats. In addition, the in vitro effect of methylglyoxal (MG), advanced glycation end products (AGEs) and human diabetic plasma on mitochondrial activity was investigated in skeletal muscle and liver mitochondria and in rat skin primary fibroblasts. Hyperglycemic STZ rats showed tissue-specific patterns of energy deficiency, evidenced by reduced activities of complexes I, II and/or IV after 30 days of hyperglycemia in heart, skeletal muscle and liver; moreover, cardiac tissue was found to be the most sensitive to the diabetic condition, since energy metabolism was impaired after 10 days of the hyperglycemia. Insulin-induced tight glycemic control was effective in protecting against the hyperglycemia-induced inhibition of mitochondrial enzyme activities. Furthermore, the long-term hormone replacement (30 days) also increased these activities in kidney from STZ-treated animals, where the hyperglycemic state did not modify the electron transport activity. Results from in vitro experiments indicate that mitochondrial impairment could result from oxidative stress-induced accumulation of MG and/or AGEs. Further investigations demonstrated that human plasma AGE accumulation elicits reduced mitochondrial function in skin fibroblast. These data suggest that persistent hyperglycemia results in tissue-specific patterns of energy deficiency and that early and continuous insulin therapy is necessary to maintain proper mitochondrial metabolism.

Regulaçäo como instrumento das políticas públicas do Estado contemporâneo: Agência Nacional de Saúde Suplementar - ANS / Regulation as public policy tools of contemporary State: National Supplementary Agency - ANS

Identifica as relaçöes das políticas públicas de saúde com a assistência médica suplementar no Brasil, mostrando os aspectos relevantes da criaçäo e do desenho institucional da Agência Nacional de Saúde Suplementar -ANS, bem como suas relaçöes estabelecidas com as operadoras, os consumidores e os prestadores de serviços de saúde. Discutem-se também a repercussäo e o impacto das açöes da ANS junto aos diferentes atores: públicos, privados e outros segmentos sociais envolvidos. A primeira etapa baseou-se em pesquisa bibliográfica, que fundamentou o marco referencial teórico e permitiu a seleçäo de conteúdos históricos de influência, como a regulaçäo e a conformaçäo da assistência médica suplementar no Brasil. A segunda teve como abordagem metodológica uma pesquisa qualitativa, que abrangeu 12 entrevistas realizadas, através de formulários semi-estruturados, com representantes de instituiçöes que têm uma vinculaçäo significativa com o tema. As informaçöes obtidas por meio das dez questöes formuladas foram agrupadas em quatro blocos temáticos, sendo que a análise elaborada considerou o papel social dos entrevistados de tal sorte que expusesse o entendimento central sobre o assunto. A atual conformaçäo da assistência médica brasileira é resultado de medidas implementadas pelas políticas públicas setoriais desde a década de 30, quando o Estado optou pelo investimento e pela compra de serviços de saúde do setor privado, financiando seu desenvolvimento e ampliaçäo. A criaçäo da ANS foi considerada, pelo diversos atores sociais, uma medida positiva e que deu credibilidade aos atos normativos. Para os entrevistados, o recorte regulatório baseado na padronizaçäo do produto focalizou os interesses dos consumidores e teve como resultado quase que imediato a eliminaçäo do mercado de empresas sem solidez financeira. Embora este mercado encontre-se em acomodaçäo, foi apontada a tendência de uma competiçäo entre as diversas modalidades existentes. Por fim, detectou-se que há um vazio normativo entre as relaçöes estabelecidas entre operadoras, prestadores de serviços e o Sistema Unico de Saúde que requer revisäo, pois este fato influencia o resultado final da assistência médica

Projeto urgência emergência: sistema regional referência hospitalar / Emergencie Project: regional system hospital reference

Uma das grandes dificuldades por que passa o Sistema Único de Saúde na questão assistencial, tem a ver com o crescente desinteresse dos Hospitais Filantrópicos de perfil regional em atender pacientes na Urgência-Emergência. Independente dos motivos que levaram a esta situação, a posição desses hospitais sobrecarrega as Unidades de Emergência dos Hospitais Universitários, com casos que muito provavelmente poderiam ser resolvidos no nível de referência local ou micro-regional. A partir desse diagnóstico, a Secretaria de Estado da Saúde propõe criar um Sistema organizado e regulado, para atender e garantir o acesso ao tratamento adequado, aos cidadãos que sofrem agravos agudos a sua saúde, sejam por causas externas, doenças agudas ou complicações de doenças crônicas pré-existentes (AU).