ATP-P2X7R pathway activation limits the Tfh cell compartment during pediatric RSV infection.

Background: Follicular helper T cells (Tfh) are pivotal in B cell responses. Activation of the purinergic receptor P2X7 on Tfh cells regulates their activity. We investigated the ATP-P2X7R axis in circulating Tfh (cTfh) cells during Respiratory Syncytial Virus (RSV) infection. Methods: We analyzed two cohorts: children with RSV infection (moderate, n=30; severe, n=21) and healthy children (n=23). We utilized ELISA to quantify the levels of PreF RSV protein-specific IgG antibodies, IL-21 cytokine, and soluble P2X7R (sP2X7R) in both plasma and nasopharyngeal aspirates (NPA). Additionally, luminometry was employed to determine ATP levels in plasma, NPA and supernatant culture. The frequency of cTfh cells, P2X7R expression, and plasmablasts were assessed by flow cytometry. To evaluate apoptosis, proliferation, and IL-21 production by cTfh cells, we cultured PBMCs in the presence of Bz-ATP and/or P2X7R antagonist (KN-62) and a flow cytometry analysis was performed. Results: In children with severe RSV disease, we observed diminished titers of neutralizing anti-PreF IgG antibodies. Additionally, severe infections, compared to moderate cases, were associated with fewer cTfh cells and reduced plasma levels of IL-21. Our investigation revealed dysregulation in the ATP-P2X7R pathway during RSV infection. This was characterized by elevated ATP levels in both plasma and NPA samples, increased expression of P2X7R on cTfh cells, lower levels of sP2X7R, and heightened ATP release from PBMCs upon stimulation, particularly evident in severe cases. Importantly, ATP exposure decreased cTfh proliferative response and IL-21 production, while promoting their apoptosis. The P2X7R antagonist KN-62 mitigated these effects. Furthermore, disease severity positively correlated with ATP levels in plasma and NPA samples and inversely correlated with cTfh frequency. Conclusion: Our findings indicate that activation of the ATP-P2X7R pathway during RSV infection may contribute to limiting the cTfh cell compartment by promoting cell death and dysfunction, ultimately leading to increased disease severity.

Extracellular ATP and Imbalance of CD4+ T Cell Compartment in Pediatric COVID-19.

Severe COVID-19 in children is rare, but the reasons underlying are unclear. Profound alterations in T cell responses have been well characterized in the course of adult severe COVID-19, but little is known about the T cell function in children with COVID-19. Here, we made three major observations in a cohort of symptomatic children with acute COVID-19: 1) a reduced frequency of circulating FoxP3+ regulatory T cells, 2) the prevalence of a TH17 polarizing microenvironment characterized by high plasma levels of IL-6, IL-23, and IL17A, and an increased frequency of CD4+ T cells expressing ROR-γt, the master regulator of TH17 development, and 3) high plasma levels of ATP together with an increased expression of the P2X7 receptor. Moreover, that plasma levels of ATP displayed an inverse correlation with the frequency of regulatory T cells but a positive correlation with the frequency of CD4+ T cells positive for the expression of ROR-γt. Collectively, our data indicate an imbalance in CD4+ T cell profiles during pediatric COVID-19 that might favor the course of inflammatory processes. This finding also suggests a possible role for the extracellular ATP in the acquisition of an inflammatory signature by the T cell compartment offering a novel understanding of the involved mechanisms.

Efecto del empleo de prescripciones informatizadas en la seguridad de pacientes pediátricos hospitalizados / Effect of electronic prescriptions on the safety of hospitalized pediatric patients

Introducción. Los errores en prescripción médica (EPM) son la causa más frecuente de errores prevenibles. El empleo de sistemas de prescripción informatizada (PI) contribuiría a disminuir el error y a mejorar la calidad de atención. Objetivos. Evaluar el efecto de la PI en la prevalencia de EPM y eventos adversos (EA) relacionados en pacientes pediátricos hospitalizados. Evaluar la adherencia, aceptabilidad y apropiabilidad de la herramienta por parte de los usuarios. Método. Diseño híbrido, descriptivo y cuasiexperimental tipo antes-después. Se incluyeron prescripciones médicas de pacientes hospitalizados, calculando la prevalencia de EPM y EA relacionados, en los períodos pre-y pos implementación de la PI en un hospital pediátrico (HP) y en uno general (HG) que se tomó como control. Se evaluó la adherencia mediante la proporción de las PI sobre las totales registradas en el período posimplementación. Se evaluó la aceptabilidad y apropiabilidad de la implementación por encuesta a los usuarios. Resultados. Al comparar la prevalencia de EPM pre- y posimplementación en el HP, se observó una disminución estadísticamente significativa en los dos hospitales: HP 29,1 versus 19,9 EPM/100 prescripciones (OR: 1,65; IC95 %: 1,34-2,02; p < 0,01). En el HG 24,9 versus 13,6 EPM/100 prescripciones (OR: 2,1; IC95 %: 1,5-2,8; p < 0,01). La tasa de adherencia global a la PI fue del 83 %. La implementación presentó aceptabilidad y apropiabilidad satisfactoria. Conclusión. La prevalencia de EPM se redujo un 30 % posimplementación. La adherencia global a la herramienta fue satisfactoria

Introduction. Prescription errors are the most common cause of preventable errors. Electronic prescription (EP) systems may help to reduce errors and improve the quality of care. Objectives. To assess the effect of EP on the prevalence of prescription errors and related adverse events (AE) among hospitalized pediatric patients. To assess EP adherence, acceptability, and suitability among users. Method. Hybrid, descriptive, and quasi-experimental, before-and-after design. Prescriptions made to hospitalized patients were included, estimating the prevalence of prescription errors and related AE in the pre- and post- EP implementation periods at a children's hospital (CH) and a general hospital (GH) used as control. Adherence was assessed based on the proportion of EP among all prescriptions registered in the post-implementation period. The acceptability and suitability of EP implementation was assessed via a user survey. Results. The prevalence of prescription errors pre- and post-EP implementation at the CH was compared and a statistically significant reduction was observed in both hospitals: CH: 29.1 versus 19.9 prescription errors/100 prescriptions (OR: 1.65; 95% CI: 1.34-2.02;p < 0.01). GH: 24.9 versus 13.6 prescription errors/100 prescriptions (OR: 2.1; 95% CI: 1.5-2.8; p < 0.01). The rate of overall adherence to EP was 83%. The implementation of EP was adequately acceptable and suitable. Conclusion. The prevalence of prescription errors reduced 30% after the implementation of EP. The overall adherence to EP was adequate.

Pacientes asistidos en el Departamento de Medicina de un hospital pediátrico en el inicio de la pandemia COVID-19 en Buenos Aires, Argentina / Patients assisted at the Department of Medicine of a pediatric hospital at the beginning of the COVID-19 pandemic in Buenos Aires, Argentina

Introducción. A pesar de la magnitud de la pandemia por COVID-19, la información sobre su desarrollo en pediatría es aún limitada. Se describen las características de pacientes hospitalizados por COVID-19 en un hospital pediátrico durante los primeros tres meses de la pandemia.Método. Estudio descriptivo, que incluyó a todos los pacientes hospitalizados por COVID-19, desde el 1/4/2020 al 30/6/2020.Resultados. Se hospitalizaron 191 pacientes por COVID-19; la edad mediana fue 7,7 años; el 89 % presentaban antecedente de contacto. El 35,6 % se consideraron asintomáticos; el 61,2 %, leves, y el 3,2 %, moderados (no se observaron pacientes graves). Ninguno recibió tratamiento específico para la enfermedad. Los síntomas más frecuentes fueron fiebre, odinofagia y tos. La duración de la hospitalización tuvo una mediana de 6 días.Conclusión. Se reportaron 191 casos de niños y adolescentes hospitalizados por COVID-19. La mayoría fueron asintomáticos o presentaron enfermedad leve.

Introduction. Despite the magnitude of the COVID-19 pandemic, the information about its development in pediatrics is still limited. This report describes the characteristics of patients admitted to a children's hospital due to COVID-19 during the first three months of the pandemic.Method. Descriptive study including all patients hospitalized due to COVID-19 between 4/1/2020 and 6/30/2020.Results. A total of 191 patients were hospitalized due to COVID-19; their median age was 7.7 years; 89 % had a history of close contact. Of them, 35.6 % were considered asymptomatic; 61.2 %, mild cases; and 3.2 %, moderate cases (no severe cases). None of them received a specific treatment for the disease. The most common symptoms were fever, sore throat, and cough. The median length of stay was 6 days.Conclusion. A total of 191 cases of children and adolescents admitted due to COVID-19 are reported. Most were asymptomatic or presented with a mild disease

Epithelial cells activate plasmacytoid dendritic cells improving their anti-HIV activity.

Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.

Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-alpha, IL-6, IL-12p70 and interferon-gamma while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE(2) we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-alpha and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia(b) and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.

Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor-alpha.

We have previously reported that human neutrophils pretreated with tumour necrosis factor-alpha (TNF-alpha) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12-myristate 13-acetate (PMA), C5a, fMLP, or granulocyte-macrophage colony-stimulating factor (GM-CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF-alpha is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac-1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down-regulation in expression of the anti-apoptotic protein, Mcl-1. Interestingly, we also found that the anti-apoptotic cytokine interleukin-1 (IL-1) improves the ability of TNF-alpha to promote apoptosis, supporting the notion than TNF-alpha, acting together with IL-1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation.

Human platelets produce granulocyte-macrophage colony-stimulating factor and delay eosinophil apoptosis.

An association between eosinophils and platelets has been described in several diseases, most notably asthma. Although the mechanisms through which platelets influence eosinophil behavior are not well defined, platelets seem to contribute to the selective accumulation of eosinophils at sites of allergic inflammation by virtue of their ability to produce eosinophil chemotactic factors. We report here for the first time that platelets delay apoptosis, thus enhancing eosinophil survival. A marked inhibition of spontaneous apoptosis was observed using eosinophil:platelet ratios of 1:50, 1:25, 1:10, and 1:5. Moreover, promotion of eosinophil apoptosis by either pronase or dexamethasone was also inhibited greatly in the presence of platelets. The antiapoptotic effect mediated by platelets was dependent on the release of soluble products and was significantly inhibited by neutralizing antibodies directed to GM-CSF. Studies performed by flow cytometry, directed to analyze the cellular source of this cytokine, demonstrated that intracytoplasmic GM-CSF is present in resting platelets. Moreover, GM-CSF was found in platelet supernatants, at concentrations able to prevent eosinophil apoptosis. Our findings support a novel mechanism through which platelets may contribute to eosinophil accumulation at allergic inflammatory sites.

Control of dendritic cell differentiation by angiotensin II.

Here we analyze the role of the angiotensinergic system in the differentiation of dendritic cells (DC). We found that human monocytes produce angiotensin II (AII) and express AT1 and AT2 receptors for AII. DC differentiated from human monocytes in the presence of AT1 receptor antagonists losartan or candesartan show very low levels of CD1a expression and poor endocytic and allostimulatory activities. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in the development of nonadherent cells with CD1a expression and endocytic and allostimulatory activities higher than control DC. Similar contrasting effects were observed in mouse DC obtained from bone marrow cultures supplemented with granulocyte-monocyte colony-stimulating factor. DC differentiated in the presence of the AT1 receptor antagonist losartan express lower levels of CD11c, CD40, and Ia and display a lower ability to endocyte horseradish peroxidase (HRP) and to induce antibody responses in vivo, compared with controls. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in cells with high levels of CD11c, CD40, and Ia, as well as high ability to endocyte HRP and to induce antibody responses in vivo. Our results support the notion that the differentiation of DC is regulated by AII.

Stimulation of neutrophil apoptosis by immobilized IgA.

In the current study, we analyzed whether immunoglobulin A (IgA) is able to modulate neutrophil apoptosis. We found that culture of neutrophils on immobilized plasma IgA (iIgAp) or secretory IgA (iIgAs) induced a marked increase in apoptotic rates. By contrast, soluble IgAp, IgAs, or aggregated IgAp exerted no effect. Promotion of apoptosis by iIgA was almost completely prevented by blocking antibodies directed to CD18 or CD11b and was shown to be dependent on the activation of the respiratory burst as suggested by the ability of catalase to prevent apoptosis stimulation; the effect of azide, an heme enzyme inhibitor that significantly increased promotion of apoptosis by iIgA; and the inability of iIgA to stimulate apoptosis of neutrophils isolated from chronic granulomatous disease patients. Stimulation of neutrophil apoptosis by IgA might contribute to the control of inflammatory processes in certain autoimmune diseases such as IgA nephropathy in which tissue deposits of IgA or IgA containing immune complexes are found.

Síndrome urémico hemolítico asociado a infección por Shigella flexneri / Haemolytic Uraemic Syndrome (Martin Bell): cytogenetical study vs molecular biology techniques

Niña de 9 años de edad de deficiente medio socio-económico con mal estado nutricional.Inició su enfermedad con dolor abdominal,vómitos y diarrea.Tratada en un centro periférico con TMS presentó episodio convulsivo por lo que la derivaron a nuestro hospital.Ingresó gravemente enferma con letargo,palidez e ictericia,hemesturia macroscópica con cilindruria,proteinuria e isostenuria y cuadro de púrpura con trombocitopenia,esquizocitos e insuficiencia renal.Se plantearon como diagnósticos diferenciales:síndrome urémico hemolítico,púrpura trobótica trombocitopénica,leptospirosis,fiebre hemorrágica argentina,lupus erimatoso sistémico y sepsis.Serología para leptospirosis y fiebre hemorrágica argentina,FAN,anti ADN,factor reumatoideo,reacción de Widal y anticuerpos anti-veroxitina 1 y verotoxina 2 fueron todos negativos en tanto el coprocultivo desarrolló Shigella flexneri.Se efectuó plasmaférisis y transfusión de sedimiento globular.A partir del quinto día comenzó a recuperarse normalizanso su cuadro hematológico y humoral.La biopsis renal efectuada a los 17 días de evolución inmediatamente antes del alta mostró lesiones compatibles con síndrome urémico hemolítico en regresión.La edad,el estado nutritivo y las condiciones socio-ambientales son diferentes a lo observado en el síndrome urémico hemolítico endemoepidémico de nuestro país,generalmente asociado a infección por gérmenes productores de verotoxina.No se estudiaron citotoxinas en materia fecal pero anticuerpos anti-veroxina resultaron negativos,El resultado de coprocultivo y la bipsia renal muestran el síndrome urémico hemolítico como asociado a infección por Shigella flexneri,lo cual no ha sido referido hasta la fecha en nuestro país