Differential response of placental cells to high D-glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras-related protein RAB-7A.

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5-mM D-glucose (i.e. control) or 25-mM D-glucose (high glucose). Cell-conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K-g, pellet 10K-g, and pellet 100K-g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Viral shedding and viraemia of Andes virus during acute hantavirus infection: a prospective study.

BACKGROUND: Andes virus (ANDV) is a zoonotic Orthohantavirus leading to hantavirus cardiopulmonary syndrome. Although most transmissions occur through environmental exposure to rodent faeces and urine, rare person-to-person transmission has been documented, mainly for close contacts. This study investigates the presence and infectivity of ANDV in body fluids from confirmed cases and the duration of viraemia. METHODS: In this prospective study, 131 participants with confirmed ANDV infection were enrolled in Chile in a prospective study between 2008 and 2022. Clinical samples (buffy coat, plasma, gingival crevicular fluid [GCF], saliva, nasopharyngeal swabs [NPS], and urine) were collected weekly for 3 weeks together with clinical and epidemiological data. Samples were categorised as acute or convalescent (up to and after 16 days following onset of symptoms). Infectivity of positive fluids was assessed after the culture of samples on Vero E6 cells and use of flow cytometry assays to determine the production of ANDV nucleoprotein. FINDINGS: ANDV RNA was detected in 100% of buffy coats during acute phase, declining to 95% by day 17, and to 93% between days 23-29. ANDV RNA in GCF and saliva decreased from 30% and 12%, respectively, during the acute phase, to 12% and 11% during the convalescent phase. Successful infectivity assays of RT-qPCR-positive fluids, including GCF, saliva, NPS, and urine, were observed in 18 (42%) of 43 samples obtained during the acute phase of infection. After re-culture, the capacity to infect Vero E6 cells was maintained in 16 (89%) of 18 samples. Severity was associated with the presence of ANDV RNA in one or more fluids besides blood (odds ratio 2·58 [95% CI 1·42-5·18]). INTERPRETATION: ANDV infection is a systemic and viraemic infection, that affects various organs. The presence of infectious particles in body fluids contributes to our understanding of potential mechanisms for person-to-person transmission, supporting the development of preventive strategies. Detection of ANDV RNA in additional fluids at hospital admission is a predictor of disease severity. FUNDING: National Institutes of Health and Agencia de Investigación y Desarrollo. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Pork-cat syndrome as cause of anaphylactic reaction to well-cooked meat / Síndrome gato-porco como causa de reação anafilática à carne cozida

Pork-cat syndrome is a rare clinical syndrome that can cause lifethreatening reactions. Occuring in patients allergic to cat dander, it involves cross-reactivity between cat and pig serum albumin. Cat allergy usually precedes food allergies, suggesting primary sensitization to cat serum albumin. Since these proteins are thermolabile, the reaction tends to be more severe in undercooked meat. A 27-year-old woman with persistent moderate-to-severe rhinoconjunctivitis since childhood reported 2 immediate mucocutaneous reactions after eating small amounts of pork. Skin prick tests with commercial extracts showed sensitization to pork, and prick-to-prick tests confirmed sensitization to raw pork and raw beef. Specific IgE was positive for pork, and ISAC microarray also showed sensitization to Fel d 2. SDS-PAGE and IgE immunoblotting assays were performed with raw and cooked pork extract and detected in a 60 kDa band. In the immunoblotting-inhibition assays, cat serum albumin completely inhibited IgE binding to pork extract. The patient underwent 2 oral food challenges with well-cooked pork and beef, both causing an anaphylactic reaction. The patient's history and in-vivo and in-vitro tests led to a diagnosis of pork-cat syndrome with clinical cross-reactivity to another mammalian serum albumin. This case should stimulate oral food challenges with other well-cooked mammalian meats in patients with this syndrome to establish a tolerance threshold and avoid possible unexpected anaphylactic reactions.

A síndrome gato-porco é rara e ocorre em doentes alérgicos ao pêlo de gato, envolvendo reatividade cruzada entre as albuminas séricas (AS) de gato e de porco. Normalmente, a doença respiratória a pêlo de gato precede a alergia alimentar, sugerindo uma sensibilização primária à albumina sérica de gato. Uma vez que estas proteínas são termolábeis, as reações tendem a ser mais graves com carnes menos cozidas. Mulher de 27 anos com rinoconjuntivite persistente moderada a grave desde a infância que refere duas reações imediatas mucocutâneas após ingestão de pequenas quantidades de carne de porco. Os testes cutâneos por picada com extratos comerciais mostraram sensibilização à carne de porco e os testes prick-to-prick confirmaram sensibilização à carne de porco e de vaca cruas. A IgE específica (sIgE) foi positiva para carne de porco, e o ensaio ISAC mostrou sensibilização a Fel d 2. Foram realizados ensaios de immunoblotting SDS-PAGE IgE com extratos de carne de porco crua e cozidas que detectaram uma banda de 60 kDA. Nos ensaios de inibição por immunoblotting a albumina sérica de gato produziu uma inibição total da ligação da IgE ao extrato de carne de porco. A doente realizou duas provas de provocação oral com carne de porco e de vaca cozidas, ambas positivas com desenvolvimento de reação anafilática. A história clínica, os testes in-vivo e in-vitro levaram ao diagnóstico de síndrome gato-porco com reatividade cruzada clínica a outras albuminas séricas de mamíferos. A síndrome gato-porco é rara e pode causar reações fatais. Este caso frisa a importância da realização de provas de provocação oral com outras carnes de mamíferos bem cozidas em doentes com esta síndrome, de forma a estabelecer um limiar de tolerância e evitar possíveis reações anafiláticas inesperadas.

Detección del SARS-CoV-2 mediante RT-qPCR utilizando saliva en pacientes ambulatorios con estudio de COVID-19

Resumen Introducción: La pandemia de COVID-19 ha afectado a millones de personas en todo el mundo. La identificación de sujetos infectados ha sido importante para el control. Objetivo: Evaluar el rendimiento de una reacción de polimerasa en cadena (RPC) cuantitativa en tiempo real (en inglés: RT-qPCR) para SARS-CoV-2, utilizando saliva como matriz en comparación con un hisopado nasofaríngeo (HNF). Metodología: Se reclutaron adultos en atención ambulatoria, la mayoría sintomáticos. Fueron estudiadas 530 muestras pareadas de saliva e HNF con RT-qPCR. Resultados: Fueron positivas 59 muestras de HNF y 54 de saliva. La sensibilidad con saliva fue 91%, especificidad 100%, el valor predictor positivo (VPP) 100%, valor predictor negativo (VPN) 98%. El índice Kappa fue de 0,95 y LR-0,08. En promedio, el umbral de ciclo (en inglés cycle threshold-CT) de la saliva fue 3,99 puntos más alto que los de HNF (p < 0,0001) mostrando que la carga viral (CV) es menor en saliva. La carga viral en ambas disminuyó con el tiempo después del inicio de los síntomas. El muestreo de saliva fue preferido por los sujetos en lugar de HNF. Conclusión: Este estudio demuestra que la RPC para SARS-CoV-2 utilizando saliva, es adecuada para el diagnóstico de COVID-19 en adultos ambulatorios, especialmente en la etapa temprana de los síntomas.

Abstract Background: The COVID-19 pandemic has affected millions of people around the world. Part of control strategies is testing a large proportion of the population to identify and isolate the infected subjects. Aim: To evaluate the SARS-CoV-2 detection by the performance of a reverse transcription and quantitative polymerase chain reaction (RT-qPCR) against SARS-CoV-2, using saliva as a matrix compared to a nasopharyngeal swab (NPS) to simplify obtaining a diagnostic sample. Methods: Adults in outpatient care were recruited, 95% of them symptomatic. We studied 530 paired saliva and NPS samples by SARS-CoV-2 RT-qPCR. Results: Fifty-nine individuals tested positive in NPS and 54 in saliva samples. Sensitivity for saliva sample was 91%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value (NPV) 98%. The Kappa index was 0.95 and LR-0.08. On average, the cycle threshold (CT) of saliva was 3.99 points higher than those of NPS (p < 0.0001) showing that viral load (VL) is lower in saliva than in NPS. Viral load in both decreased over the time after onset of symptoms. Saliva sampling was preferred by subjects instead of NPS. Conclusion: This study demonstrates that SARS-CoV-2 RT-qPCR using saliva, even with lower VL, is suitable for the diagnosis of COVID-19 in outpatient adults, especially at early stage of symptoms.

Cribado de infecciones cervicales de transmisión sexual en mujeres embarazadas y su relación con la microbiota vaginal / Screening of cervical sexually transmitted infections in pregnant women and the relation with the vaginal microbiota

Resumen Introducción: La mujer embarazada está expuesta anumerosas infecciones de transmisión sexual (ITS), las que pueden producir aborto, enfermedad en el feto y/o en el recién nacido, además de alteraciones en el curso normal del embarazo. Objetivo: Realizar tamizaje de infección cervical asintomática en mujeres embarazadas y su relación con la microbiota. Pacientes y Métodos: Se enrolaron 85 mujeres embarazadas sin cervicitis clínica que consultaron en control de rutina de embarazo (47 pacientes) o que fueron derivadas a una unidad de ITS (38 pacientes). Se tomaron muestras de fondo de saco vaginal, que fueron analizadas por técnicas clásicas de microscopía y cultivo corriente y reacción de polimerasa en cadena para Neisseria gonorrhoeae, Trichomonas vaginalis y Chlamydia trachomatis. Resultados: Se encontró 12,9% de infección por C. trachomatis, 2,4% de T. vaginalis. En este estudio no se encontró N. gonorrhoeae. El 23,3% de pacientes con microbiota alterada (vaginosis bacteriana y microbiota intermedia) fue positiva para C. trachomatis. Conclusión: En este trabajo, encontramos una alta frecuencia de infección por C. trachomatis, que se relaciona en forma significativa con la presencia de microbiota alterada. Esta alta frecuencia debería promover estrategias preventivas en los controles de salud de la mujer embarazada.

Background: Pregnant woman is exposed to many sexual transmitted infections (STI). Many of these infections may produce diseases in the fetus and newborn, and also alteration in the normal course of the pregnancy. Aim: Screening of asymptomatic cervical infection in pregnant woman and its relationship with the vaginal microbiota. Patients and Methods: 85 pregnant women without clinical cervicitis who consult in the routine pregnant control (47 patients) and women derived from STI service (38 patients). The samples were obtained from the vaginal fund sac and were analyzed with optic microscopy, cultures and PCR of Neisseria gonorrhoeae, Trichomonas vaginalis and Chlamydia trachomatis. Results: 12,9% of the enrolled women were positive for C. trachomatis, 2,4% for T. vaginalis. In this study, we did not found N. gonorrhoeae. We observed 23,3% of patients with altered microbiota (bacterial vaginosis and intermediate microbiota) was positive for C. trachomatis. Conclusions: In this study, we found a high frequency of C. trachomatis infection, that correlates with the presence of altered microbiota. This high frequency would promote preventive strategies in the pregnant women routine controls.

Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae / Accuracy and diagnostic utility of IgM in Bartonella henselae infections

Introduction: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. Objective: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. Material and Methods: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). Results: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. Conclusions: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

Introducción: El diagnóstico de laboratorio de la enfermedad por arañazo de gato (EAG) se basa en la determinación de anticuerpos específicos anti-Bartonella henselae por distintas técnicas. El CDC de E.U.A. recomienda como estándar de oro la IgG mediante inmunofluorescencia (IF). Objetivo: Determinar la exactitud y utilidad diagnóstica de la IgM anti-B. henselae por IF para EAG. Material y Método: En suero de 108 pacientes a quienes se realizó IgG anti-B. henselae por sospecha de EAG, se determinó la presencia de IgM específica, en forma separada y ciega por dos profesionales de laboratorio ampliamente entrenados. Se calculó sensibilidad (S), especificidad (E), valores predictores positivo (VPP) y negativo (VPN) y likelihood ratio (LR) para una IgM positiva (LR+) y negativa (LR-). Resultados: En 37 pacientes con IgG anti-B. henselae positiva, la IgM fue positiva en 16 y negativa en 11; en 71 pacientes con IgG negativa, la IgM fue negativa en 69 y positiva en 2. Por consiguiente, la IgM presentó S 43%, E 97%, VPP 88%, VPN 77%, LR(+) 15 y LR(-) 0,58. Conclusiones: Los resultados sugieren que una IgM positiva apoya el diagnóstico de EAG, pero una negativa no permite descartarlo. Por tanto, la IgG debe seguir considerándose como el estándar de oro para el diagnóstico de infecciones por B. henselae.

Escala visual analógica, testes cutâneos ou imunoglobulinas específicas séricas: avaliação comparativa na imunoterapia específica com ácaros do pó doméstico / Visual analog scale, skin tests or serum specific immunoglobulins in the comparative evaluation of specific immunotherapy’s efficacy

Introdução: A eficácia imunológica da imunoterapia específica (ITE) é freqüentemente avaliada pela evolução de testes cutâneos, imunoglobulinas específicas ou por escalas visuais analógicas (VAS). Contudo, poucos trabalhos comparam diretamente a evolução destes parâmetros nos mesmos doentes. Objectivo: Comparar a evolução destes parâmetros, após um ano de ITE e sua correlação com o limiar de positividade alergênica na provocação nasal. Material e Métodos: 75 pacientes com rinite alérgica, dos quais 50 efetuaram ITE com extrato de D. pteronyssinus. Estes 50 doentes foram adicionalmente divididos em dois grupos: um que recebeu doses mais baixas do extrato alergênico (grupo 1) e outro que recebeu doses elevadas (grupo 2). Todosos doentes efetuaram provocação nasal especifica, auto-avaliação sintomática em VAS, testes cutâneos de puntura e doseamentos de IgE e IgG específicas séricas para D. pteronyssinus antes do início da ITE e após um ano. Resultados: No grupo controle não houve variações significativas em qualquer parâmetro. Nos doentes sob ITE observaram-se reduções das pápulas cutâneas e IgE específicas, e aumento de IgG específica, dos valores médios da VAS e dos limiares de provocação alergênica (p<0,05). Os testes cutâneos e os valores da VAS são os únicos parâmetros cuja evolução se correlaciona com a dos limiares alergênicos de provocação nasal. Conclusão: Os testes cutâneos permitem evidenciar mais precocemente melhorias imunológicas induzidas pela ITE, correlacionando-se com a evolução dos limiares alergênicos de provocação nasal, tal como a VAS. Contudo, esta última apresentou menor capacidade discriminativa na avaliação comparativa entre os dois grupos submetidos à ITE.

Desenvolvendo um sistema de informação geográfico para vigilância ambiental em saúde / Developing a geographical information system for environmental health surveillance

Os autores propõem o desenvolvimento de um sistema de informação que permita a tomada de decisão através da avaliação integral dos riscos ambientais a que uma população está exposta. Este sistema deverá ter a capacidade de integrar as informações disponíveis nos sistemas já desenvolvidos e que estão sendo utilizados pelos órgãos de controle ambiental de forma individualizada, cruzar as informações, visualizá-las cartograficamente e, através de indicadores, permitir a avaliação da situação de saúde da população, o desempenho da vigilância ambiental e a efetividade das intervenções aplicadas. Basicamente a estrutura do sistema deverá ser modular, composto de um módulo central cuja função será de coletar e sistematizar os dados e informações disponíveis nos módulos periféricos e processar as informações de acordo com a necessidade do usuário. Este sistema irá favorecer e facilitar a implantação do Sistema Nacional de Vigilância Ambiental em Saúde nos dois níveis de Gestão da Saúde, municipal e estadual. Este sistema deverá ser desenvolvido em ambiente Web, com acesso através da internet ou intranet.

Ativaçäo celular das reaçöes alérgicas / Cellular activation in allergic reactions

Sao analisados os mecanismos de ativaçao celular de monócito-macrófagos, mastócitos, basófilos, eosinófilos, neutrófilos e plaquetas nas reaçoes alérgicas.

Técnica quirúrgica para la cardiomioplastía dinámica / Procedure for dynamic cardiomyoplasty

Nuestro grupo ha realizado el procedimiento de Cardiomioplastía Dinámica en 13 pacientes con miocardiopatía dilatada. En 10 casos la operación fue llevada a cabo en 2 etapas. La primera para someter al músculo dorsal ancho a un período de entrenamiento con un generador de trenes de pulso y la segunda de Cardiomioplastía Dinámica para suturar el músculo entrenado alrededor del corazón. Los principales aspectos de ésta técnica quirúrgica son: 1) Incisión única en la línea axilar posterior izquierdo para disecar el músculo dorsal ancho que será introducido en el tórax a través de una ventana a nivel de la segunda costilla. Usando la misma incisión se accede al corazón por una toracotomía en el 5§ espacio intercostal. Este acceso único reduce significativamente el tiempo quirúrgico, la manipulación del paciente y las posibilidades de morbilidad por hemorragia o infecciones de herida. Además se logra una mejor exposición de las paredes lateral y posterior de los ventrículos. 2) El músculo es colocado en posición contraria a las agujas del reloj al rodear a los ventrículos. 3) La distancia entre los electrodos utilizados por nosotros es mayor que la señalada por otros autores. 4) No utilizamos sustitutos de pericardio y 5) El pericardio es parcialmente saturado. En éste trabajo detallamos la técnica quirúrgica empleada y enfatizamos sus beneficios en comparación con la utilizada por otros autores