Desafíos para la recolección de sangre y el análisis bioquímico en un gran estudio multicéntrico con adolescentes en las escuelas : lecciones del ERICA en Brasil

O Estudo de Riscos de Cardiovasculares em Adolescentes (ERICA) é um estudo pioneiro que tem como objetivo avaliar a prevalência de fatores de risco cardiovascular, incluindo componentes da síndrome metabólica, entre adolescentes brasileiros. Este artigo tem como objetivo descrever os aspectos metodológicos relacionados com a coleta de sangue, assim como informar os resultados da preparação, transporte, armazenamento e exames no ERICA. Os exames foram realizados em um único laboratório e as amostras de sangue foram coletadas de forma padronizada. A logística envolveu o transporte aéreo das amostra até o laboratório de referência, com a temperatura controlada desde a coleta do sangue. O soro foi armazenado em biorrepositores locais em quatro centros e se serão utilizados em análises futuras. Durante o estudo foram realizados 284.247 exames e a taxa de participação foi de 56,2%, representando 40.732 adolescentes. Do total, 92,6% das amostras chegaram ao laboratório de referência mantendo a temperatura entre 0-10°C. Não foram identificadas alterações clínicas significativas nos resultados devido a mudanças de temperatura. O controle de qualidade externo registrou resultados satisfatórios em 98,7% das avaliações. Foram criados quatro biorrepositores com amostras de 7.785 adolescentes. Assim, podemos considerar que a logística adotada no ERICA foi bastante exitosa e sua descrição, tal como as dificuldades experimentadas no Brasil, podem informar e facilitar o planejamento de futuros estudos, especialmente nos países em desenvolvimento.The Study of Cardiovascular Risk in Adolescents (ERICA) is a pioneering study that aimed to assess the prevalence of cardiovascular risk factors, including metabolic syndrome components in Brazilian adolescents. This study aims to describe the methodological aspects related to blood collection as well as to report pertaining results of the preparation, transport, storage, and exams in ERICA. Exams in ERICA were performed in a single laboratory and blood samples were collected in schools in a standardized manner. Logistics involved air transportation of samples to the reference laboratory with controlled temperature since sample collection. The serum was stored in local biorepositories in four centers to be used in future analyses. During the study, 284,247 exams were performed and rate of participation in exams was 56.2%, thus involving 40,732 adolescents. From the total, 92.6% of the samples reached the reference laboratory maintaining the temperature between 0-10°C. No clinical significant changes in results due to temperature changes were identified. External quality control recorded satisfactory results in 98.7% of the evaluations. Four biorepositories with samples of 7,785 adolescents were created. Thus, we can consider that the logistics adopted in ERICA was fairly successful and description of this as well as the difficulties experienced in Brazil can inform and facilitate the planning of future studies, especially in developing countries.El Estudio de Riesgos Cardiovasculares en Adolescentes (ERICA) es un estudio pionero que tiene como objetivo evaluar la prevalencia de factores de riesgo cardiovascular, incluyendo componentes del síndrome metabólico en adolescentes brasileños. Este estudio tiene como objetivo describir los aspectos metodológicos relacionados con la recolección de sangre, así como informar sobre los resultados de la preparación, transporte, almacenamiento y exámenes en el ERICA. Los exámenes en ERICA se realizaron en un solo laboratorio y se recogieron muestras de sangre en las escuelas de manera estandarizada. La logística involucró el transporte aéreo de muestras al laboratorio de referencia con temperatura controlada desde la recolección de muestras. El suero fue almacenado en biorepositories locales en cuatro centros que se utilizarán en análises futuros. Durante el estudio se realizaron 284.247 exámenes y la tasa de participación fue de 56,2%, lo que involucró a 40.732 adolescentes. Del total, el 92,6% de las muestras alcanzaron el laboratorio de referencia manteniendo la temperatura entre 0-10°C. No se identificaron cambios clínicos significativos en los resultados debido a cambios de temperatura. El control de calidad externo registró resultados satisfactorios en el 98,7% de las evaluaciones. Se crearon cuatro biorrepositores con muestras de 7.785 adolescentes. Así, podemos considerar que la logística adoptada en el ERICA fue bastante exitosa y su descripción así como las dificultades experimentadas en Brasil pueden informar y facilitar la planificación de futuros estudios, especialmente en los países en desarrollo

Desafíos para la recolección de sangre y el análisis bioquímico en un gran estudio multicéntrico con adolescentes en las escuelas: lecciones del ERICA en Brasil

The Study of Cardiovascular Risk in Adolescents (ERICA) is a pioneering study that aimed to assess the prevalence of cardiovascular risk factors, including metabolic syndrome components in Brazilian adolescents. This study aims to describe the methodological aspects related to blood collection as well as to report pertaining results of the preparation, transport, storage, and exams in ERICA. Exams in ERICA were performed in a single laboratory and blood samples were collected in schools in a standardized manner. Logistics involved air transportation of samples to the reference laboratory with controlled temperature since sample collection. The serum was stored in local biorepositories in four centers to be used in future analyses. During the study, 284,247 exams were performed and rate of participation in exams was 56.2%, thus involving 40,732 adolescents. From the total, 92.6% of the samples reached the reference laboratory maintaining the temperature between 0-10°C. No clinical significant changes in results due to temperature changes were identified. External quality control recorded satisfactory results in 98.7% of the evaluations. Four biorepositories with samples of 7,785 adolescents were created. Thus, we can consider that the logistics adopted in ERICA was fairly successful and description of this as well as the difficulties experienced in Brazil can inform and facilitate the planning of future studies, especially in developing countries.O Estudo de Riscos de Cardiovasculares em Adolescentes (ERICA) é um estudo pioneiro que tem como objetivo avaliar a prevalência de fatores de risco cardiovascular, incluindo componentes da síndrome metabólica, entre adolescentes brasileiros. Este artigo tem como objetivo descrever os aspectos metodológicos relacionados com a coleta de sangue, assim como informar os resultados da preparação, transporte, armazenamento e exames no ERICA. Os exames foram realizados em um único laboratório e as amostras de sangue foram coletadas de forma padronizada. A logística envolveu o transporte aéreo das amostra até o laboratório de referência, com a temperatura controlada desde a coleta do sangue. O soro foi armazenado em biorrepositores locais em quatro centros e se serão utilizados em análises futuras. Durante o estudo foram realizados 284.247 exames e a taxa de participação foi de 56,2%, representando 40.732 adolescentes. Do total, 92,6% das amostras chegaram ao laboratório de referência mantendo a temperatura entre 0-10°C. Não foram identificadas alterações clínicas significativas nos resultados devido a mudanças de temperatura. O controle de qualidade externo registrou resultados satisfatórios em 98,7% das avaliações. Foram criados quatro biorrepositores com amostras de 7.785 adolescentes. Assim, podemos considerar que a logística adotada no ERICA foi bastante exitosa e sua descrição, tal como as dificuldades experimentadas no Brasil, podem informar e facilitar o planejamento de futuros estudos, especialmente nos países em desenvolvimento.El Estudio de Riesgos Cardiovasculares en Adolescentes (ERICA) es un estudio pionero que tiene como objetivo evaluar la prevalencia de factores de riesgo cardiovascular, incluyendo componentes del síndrome metabólico en adolescentes brasileños. Este estudio tiene como objetivo describir los aspectos metodológicos relacionados con la recolección de sangre, así como informar sobre los resultados de la preparación, transporte, almacenamiento y exámenes en el ERICA. Los exámenes en ERICA se realizaron en un solo laboratorio y se recogieron muestras de sangre en las escuelas de manera estandarizada. La logística involucró el transporte aéreo de muestras al laboratorio de referencia con temperatura controlada desde la recolección de muestras. El suero fue almacenado en biorepositories locales en cuatro centros que se utilizarán en análises futuros. Durante el estudio se realizaron 284.247 exámenes y la tasa de participación fue de 56,2%, lo que involucró a 40.732 adolescentes. Del total, el 92,6% de las muestras alcanzaron el laboratorio de referencia manteniendo la temperatura entre 0-10°C. No se identificaron cambios clínicos significativos en los resultados debido a cambios de temperatura. El control de calidad externo registró resultados satisfactorios en el 98,7% de las evaluaciones. Se crearon cuatro biorrepositores con muestras de 7.785 adolescentes. Así, podemos considerar que la logística adoptada en el ERICA fue bastante exitosa y su descripción así como las dificultades experimentadas en Brasil pueden informar y facilitar la planificación de futuros estudios, especialmente en los países en desarrollo

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 μM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 μM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 μM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 μM) is optimal for bioimaging, whereas a high concentration (200 μM CdTe) could be toxic to cells. Taken together, our data indicate that 2 μM QD can be used for the successful long-term study of the parasite-vector interaction in real time

Mouse Chow Composition Influences Immune Responses and Food Allergy Development in a Mouse Model

Our diet is known to substantially influence the immune response not only by support of mucosal barriers but also via direct impact on immune cells. Thus, it was of great interest to compare the immunological effect of two mouse chows with substantial differences regarding micro-, macronutrient, lipid and vitamin content on the food allergic response in our previously established mouse model. As the two mouse chows of interest, we used a soy containing feed with lower fatty acid (FA) amount (soy-containing feed) and compared it to a soy free mouse chow (soy-free feed) in an established protocol of oral immunizations with Ovalbumin (OVA) under gastric acid suppression. In the animals receiving soy-containing feed, OVA-specific IgE, IgG1, IgG2a antibody levels were significantly elevated and food allergy was evidenced by a drop of body temperature after oral immunizations. In contrast, mice on soy-free diet had significantly higher levels of IL-10 and were protected from food allergy development. In conclusion, soy-containing feed was auxiliary during sensitizations, while soy-free feed supported oral tolerance development and food allergy prevention

Gastric Enzyme Supplementation Inhibits Food Allergy in a BALB/c Mouse Model

Impaired gastric digestion due to suppressed gastric acidity enhances the risk for food allergy development. In the current study, we aimed to evaluate the impact of a supported gastric digestion via application of a pharmaceutical gastric enzyme solution (GES) on food allergy development and allergic reactions in a BALB/c mouse model. The ability of the GES to restore hypoacidic conditions was tested in mice treated with gastric acid suppression medication. To evaluate the impact on allergic symptoms, mice were orally sensitized with ovalbumin (OVA) under gastric acid suppression and subjected to oral challenges with or without GES. The immune response was evaluated by measurement of antibody titers, cytokine levels, mucosal allergy effector cell influx and regulatory T-cell counts. Clinical response was objectified by core body temperature measurements after oral OVA challenge. Supplementation of GES transiently restored physiological pH levels in the stomach after pharmaceutical gastric acid suppression. During oral sensitization, supplementation of gastric enzymes significantly reduced systemic IgE, IgG1 and IgG2a levels and allergic symptoms. In food allergic mice, clinical symptoms were reduced by co-administration of the gastric enzyme solution. Support of gastric digestion efficiently prevents food allergy induction and alleviates clinical symptoms in our food allergy model

Nutrients / Immune Effects of the Nitrated Food Allergen Beta-Lactoglobulin in an Experimental Food Allergy Model

Food proteins may get nitrated by various exogenous or endogenous mechanisms. As individuals might get recurrently exposed to nitrated proteins via daily diet, we aimed to investigate the effect of repeatedly ingested nitrated food proteins on the subsequent immune response in non-allergic and allergic mice using the milk allergen beta-lactoglobulin (BLG) as model food protein in a mouse model. Evaluating the presence of nitrated proteins in food, we could detect 3-nitrotyrosine (3-NT) in extracts of different foods and in stomach content extracts of non-allergic mice under physiological conditions. Chemically nitrated BLG (BLGn) exhibited enhanced susceptibility to degradation in simulated gastric fluid experiments compared to untreated BLG (BLGu). Gavage of BLGn to non-allergic animals increased interferon- and interleukin-10 release of stimulated spleen cells and led to the formation of BLG-specific serum IgA. Allergic mice receiving three oral gavages of BLGn had higher levels of mouse mast cell protease-1 (mMCP-1) compared to allergic mice receiving BLGu. Regardless of the preceding immune status, non-allergic or allergic, repeatedly ingested nitrated food proteins seem to considerably influence the subsequent immune response.(VLID)458847

Plasma Levels of the Bioactive Sphingolipid Metabolite S1P in Adult Cystic Fibrosis Patients: Potential Target for Immunonutrition?

Recent research has linked sphingolipid (SL) metabolism with cystic fibrosis transmembrane conductance regulator (CFTR) activity, affecting bioactive lipid mediator sphingosine-1-phosphate (S1P). We hypothesize that loss of CFTR function in cystic fibrosis (CF) patients influenced plasma S1P levels. Total and unbound plasma S1P levels were measured in 20 lung-transplanted adult CF patients and 20 healthy controls by mass spectrometry and enzyme-linked immunosorbent assay (ELISA). S1P levels were correlated with CFTR genotype, routine laboratory parameters, lung function and pathogen colonization, and clinical symptoms. Compared to controls, CF patients showed lower unbound plasma S1P, whereas total S1P levels did not differ. A positive correlation of total and unbound S1P levels was found in healthy controls, but not in CF patients. Higher unbound S1P levels were measured in ΔF508-homozygous compared to ΔF508-heterozygous CF patients (p = 0.038), accompanied by higher levels of HDL in ΔF508-heterozygous patients. Gastrointestinal symptoms were more common in ΔF508 heterozygotes compared to ΔF508 homozygotes. This is the first clinical study linking plasma S1P levels with CFTR function and clinical presentation in adult CF patients. Given the emerging role of immunonutrition in CF, our study might pave the way for using S1P as a novel biomarker and nutritional target in CF

Tumor-Extrinsic Axl Expression Shapes an Inflammatory Microenvironment Independent of Tumor Cell Promoting Axl Signaling in Hepatocellular Carcinoma

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl−/− mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl−/− tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment

Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFβ stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT