Detección de células secretoras de anticuerpos totales y específicas de rotavirus en adultos sanos

Antibodies (Ab) play a critical role in the immune response against rotavirus (RV). Protector Ab, are produced by antibody secreting cells (ASC). The capacity to detect ASC becomes fundamental in process like antiviral response analysis and vaccination testing. Here, we analyzed the frequency and isotype of total and RV-specific ASC by ELISPOT (functional assay) and flow cytometry (FC) in healthy adults using CD38and CD27 like ASC markers. In each million of peripherals blood mononuclear cells (PBMC), approximately 2,550 totals ASC were detected. IgA was the isotype preferentially expressed for circulating ASC followed by IgG and IgM with 63%, 29.4% and 7.6% of total ASC respectively. Paired analysis of immunomagnetic sorting, ELISPOT and FC shown that 85% and 90% of total ASC expressed CD38 and CD27 respectively. CD38 and CD27 ASC enrichment population were used to detect RV-specific ASC. None to 40 ASC per million of PBMC were detected by ELISPOT and FC and more interesting yet, a high correlation were found in the frequency of RV-specific ASC detected by both methods. This mixture assay can be used in the evaluation of new RV vaccines.Ya que los anticuerpos (Ac) son uno de los principales mecanismos de defensa contra la infección por rotavirus (RV), la capacidad de identificar a las células que secretan anticuerpos (CSA) totales y RV específicos es fundamental para procesos como el análisis de la respuesta inmune antiviral y la evaluación de nuevas vacunas. Aquí, se analiza por ELISPOT (ensayo funcional) y citometría de flujo (CF ensayo fenotípico) la frecuencia e isotipo de CSA totales y RV específicas en adultos sanos, usando células mononucleares de sangre periférica totales (CMSP) y a las moléculas CD38 y CD27 como marcadores para enriquecer en CSA. Por cada millón de CMSP, aproximadamente 2,550 células producían Ac totales. La IgA fue el isotipo más frecuente, seguido de la IgG e IgM, con el 63%, 29.4% y 7.6% respectivamente. El análisis combinado del ELISPOT y la CF mostró que el 85% de las CSA expresaron CD38 y el 90% de ellas expresaron CD27. Una alta y significativa correlación entre las CSA detectadas por el ensayo funcional y el fenotípico fue encontrada cuando estos ensayos se realizaron en las poblaciones purificadas con CD38 y CD27. Con el enriquecimiento de CSA usando al CD38 y CD27, se logró además detectar CSA RV específicas que se encuentran en circulación en tan baja frecuencia como 0 a 40 CSA-RV por millón de CMSP. Para las células RV específicas, también una buena correlación fue encontrada entre el ELISPOT y la CF. A pesar de su muy baja frecuencia, CSA antígeno específicas pueden ser detectadas en circulación de voluntarios sanos. Este acercamiento puede ser usado en la evaluación de vacunas, que para el caso particular del RV son necesarias mejorar

Detección y cuantificación de virus dengue 2 en lisado celular y plasma de niños por qPCR en tiempo real usando un estuche comercial y el equipo EcoTM System-Illumina

Methods  for  Dengue  virus  (DENV)  diagnosis  in  endemic areas  are  greatly  needed.  One of  them  is  the  real-time polymerase  chain  reaction  (qPCR)  that  also  enables  to quantitate  the  viral  genome.  Kits of  qPCR  for  DENV  are expensive and restrict their use to a small number of qPCR devices, which limits the application of the technique. Here, we evaluated the performance of a commercial kit of qPCR to DENV-2 detection  on  a  locally  available  qPCR  device (EcoTM System, Illumina), not cited by the kit manufacturer.VERO-76 cells lysate and plasma from children, both with confirmed ongoing DENV-2 infection, were evaluated. As specificity control, cell lysates and plasma  from  children infected  with  DENV-1,  and  uninfected  lysate,  were  also included. The reactions were simultaneously evaluated in an Applied Bio systems  7300  device.  The standard  curve generated  by  EcoTM  was  robust  (R2=  0.99)  with  low variability in the replicates (<10%). The reaction efficiency was high (88.8%) and signal was only obtained in lysates and plasma infected  with  DENV-2.  There was  a  strong positive  correlation  (R2=  1.0,  P=  0.0028)  between  the number of copies of viral RNA in the samples detected by both  qPCR  devices.  Thus, the use  of  the  evaluate  kit  for detection of DENV-2 here tested can be extended to EcoTM. With  this  work,  technological  capacity  for  DENV  study  in an endemic zone is greatly strengthened.Métodos para diagnóstico de dengue virus (DENV) en zonas endémicas son altamente necesarios. Uno de ellos es la reacción en cadena de polimerasa en tiempo real (qPCR), método que permite además la cuantificación del genoma viral. Los estuches comerciales de qPCR para DENV son costosos y restringen su uso a un número pequeño de dispositivos de qPCR, limitando la aplicación de la técnica. Aquí se evaluó el desempeño de un estuche comercial de qPCR para la detección de DENV-2 en un dispositivo de qPCR (EcoTM System, Illumina) localmente disponible, no listado por el fabricante del estuche. Lisado de células VERO-76 y plasma de niños, ambos con infección confirmada por DENV-2, fueron evaluados. Como controles de especificidad, lisado celular y plasma de niños infectados con DENV-1, además de lisado no infectado, fueron también incluidos. Las reacciones fueron además evaluadas simultáneamente en un equipo Applied Biosystem 7300, uno de los recomendados por el fabricante del estuche. La curva estándar generada por el EcoTM fue robusta (R2= 0.99), con baja variabilidad en las réplicas (<10%). La eficiencia de la reacción fue buena (88.8%) y sólo hubo amplificación en los lisados y plasma de niños infectados con DENV-2. Hubo una fuerte correlación positiva (R2= 1.0, P=0.0028) entre el número de copias de ARN viral en las muestras detectadas por los dos dispositivos de qPCR usados. Así, el uso del estuche para detección de DENV-2 aquí probado puede extenderse al EcoTM de forma segura. Este trabajo fortalece la capacidad tecnológica para el estudio de DENV en un área endémica

Temporal and spatial variations of CO2 diffuse volcanic degassing on Cuicocha Caldera Lake – Ecuador

Cuicocha Caldera is the youngest eruptive center of Cotacachi-Cuicocha Volcanic Complex, located at the north of Ecuador. The caldera contains a lake of 3.95 km2 surface, and a maximum depth of 148 m. Cuicocha Lake is characterized by the presence of CO2 gaseous diffuse emissions, perceptible as bubbling zones. Since 2011, CO2 diffuse flux measurements have been performed in this lake using the accumulation chamber method. The data obtained from twenty surveys were processed by means of the Graphical Statistical Approach and the Sequential Gaussian Simulation. The results reveal that Cuicocha lake has released a total estimated amount of ~400 kt of CO2 in the period between March 2011 and May 2019, with an average rate of 135 t/day. Furthermore, the spatial and temporal analysis of the data made possible the understanding of the processes occurring in the lake: 1) Lake stratification caused by the seasons seem to favor CO2 accumulation in the hipolimnion and its posterior releasing. Minimum total flux values of ~50 t/day have been estimated during “warm” stratified periods and maximum flux values of ~170 t/day have been recorded during “cold” overturn periods. Additionally, at least two anomalous degassing episodes were identified in 2012–2013, seemingly associated to changes in the volcanic activity also detected through seismicity. 2) Cuicocha CO2 degassing seems to be controlled by the existence of diffuse degassing structures at the lake bottom, which correspond to high permeability zones resulting from the intersection between ~NE-SW and ~WNW-ESE oriented structures. We propose a conceptual model to explain the systematic apparition of CO2 anomalies on specific areas of the lake surface.Fil: Sierra Vaca, Daniel Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Estudios Andinos "Don Pablo Groeber". Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Estudios Andinos "Don Pablo Groeber"; Argentina. Instituto Geofísico de la Escuela Politécnica Nacional; EcuadorFil: Hidalgo, Silvana. Instituto Geofísico de la Escuela Politécnica Nacional; EcuadorFil: Almeida, Marco. Instituto Geofísico de la Escuela Politécnica Nacional; EcuadorFil: Vigide, Nicolás Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Estudios Andinos "Don Pablo Groeber". Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Estudios Andinos "Don Pablo Groeber"; ArgentinaFil: Lamberti, María Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Estudios Andinos "Don Pablo Groeber". Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Estudios Andinos "Don Pablo Groeber"; ArgentinaFil: Proaño, Antonio. Instituto Geofísico de la Escuela Politécnica Nacional; EcuadorFil: Narváez, Diego F.. Instituto Geofísico de la Escuela Politécnica Nacional; Ecuado

PIH11 out-of-pocket health expenditures related to prenatal care: evidence from Colombia, 2018

To estimate the out-of-pocket expenditures (OOPE) and indirect costs related to prenatal check-ups and pregnancy complications in women seen in a maternity hospital in Cartagena-Colombia, 2018. Cost description study. A survey was constructed to estimate OOPE and indirect costs owing to prenatal check-ups. Women were asked about sociodemographic variables. The survey investigated how much pregnant women spend in the care of prenatal check-ups. Also, it explored OOPE linked to pregnancy complications. Productivity losses were quantified from the reduction of work time produced by the prenatal check-up. Absolute and relative frequencies, averages and interquartile ranges (IQR) were used to describe the population and estimate the OOPE and indirect costs in pregnant women. The latter were calculated from the percentile method. We performed a bootstrapping to generate an empirical estimate of the complete sample distribution. Costs were reported in Colombian pesos (COP), 2018. Fifty-six pregnant women were surveyed, with an average age of 25.9 years (±6.2). 96.4% of the respondents had completed at least primary school studies, 7.3% were married. All women surveyed had OOPE in at least one cost-item. Transportation was the item with the highest frequency of expenses. The mean of OOPE for women who attend their prenatal check-up were COP$71,736 (IQR COP$53,400-$92,715). Twenty-five women reported some complication related to their pregnancy status. The mean OOPE associated with a pregnancy complication was COP$57,539 (IQR COP$28,686-$100,124). Women reported a time average of 4.2 hours (range 1-12) for attending the prenatal check-up, and their companions spent 4.5 (range 1-13). On average a woman had productivity losses of COP$13,541 (IQR COP$8,138- $16,276) and her companion COP$14,475 (range $8,130-$19,531). Expenses produced by prenatal care could totalize 9.2% (6.8-11.9) of the monthly income of a poor household, which unfortunately makes prenatal care an important source of economic burden, impacting poor population in Cartagena

Patrón de citoquinas en niños con dengue hemorrágico en Neiva, Colombia

Cytokines play a critical role in the pathogenesis of dengue hemorrhagic fever and have been used as markers of severity disease. In this study we measured serum levels of five cytokines in dengue infected children and correlate their levels with shock and complicated forms such as myocarditis, hepatitis or bleeding. Methods: 30 patients who met WHO criteria for dengue hemorrhagic fever (DHF) were enrolled and classified into two groups: group one without shock (grade I and II) and group two with shock (grade III and IV). Serum levels of TNFá, IL-6, IL-10, IL-4 and IFNã were measured by ELISA at first day of defervescence and were compared with serum levels of 28 healthy children. Results: from 30 patients, 9 were assigned to group number one (median age 67 months) and 21 to group two (median age 42 months). Statistical differences were found between dengue infected patients and controls: controls IL-6 (5.2 pg/ml), group 1 (485 pg/ml) (p=0.002) and group 2 (190 pg/ml) (p=0.001); TNFá, control group (70 pg/ml), group 1 (586.7 pg/ml) (p=0.021) and group 2 (320.7 pg/ml) (p<0.001) and for IFNã, control group (12.3 pg/ml), group 2 (27.5 pg/ml) (p=0.019). However, we could not find correlation between cytokines and shock or complicated forms of illness. IL-4 and IL-10 did not show differences between any tested groups. Conclusion: IL-6, TNFá and IFNã are elevated in children with dengue hemorrhagic fever, but there was not correlation with severe forms of shock. Las citoquinas juegan un papel crítico en la patogénesis de la fiebre dengue hemorrágico (FDH) y han sido usadas como marcadores de severidad. En este estudio se midieron los niveles de cinco citoquinas en niños infectados con dengue y se correlacionaron con el choque y las formas complicadas tales como miocarditis, hepatitis o sangrado. Método: 30 pacientes que cumplían los criterios de la OMS para FDH fueron incluidos y clasificados en dos grupos: grupo 1, sin choque (grado I y II) y grupo 2, con choque (grado III y IV). Niveles séricos de TNFá, IL-6, IL-10. IL-4 e IFN ã fueron medidos por ELISA en el primer día de la defervescencia y comparados con los respectivos niveles de 28 niños sanos. Resultados: de los 30 pacientes, 9 fueron clasificados en el grupo número 1 (media de edad de 67 meses) y 21 en el grupo 2 (media de la edad 42 meses). Diferencias estadísticamente significativas fueron encontradas entre niños infectados con dengue y controles sanos: sanos IL-6 (5,2 pg/ml), grupo 1 (485 pg/ml) (p = 0,002) y grupo 2 (190 pg/ml) (p = 0,001); TNFá, grupo control (70 pg/ml), grupo 1 (586,7 pg/ml) (p = 0,021) y grupo 2 (320,7 pg/ml) (p < 0,001) y para IFNã, grupo control (12,3 pg/ml), grupo 2 (27,5 pg/ml) (p = 0,019). Sin embargo, no se encontró correlación entre las citoquinas y el choque o las otras formas evaluadas. IL-4 e IL-10 no fueron diferentes en ninguno de los grupos analizados. Conclusión: IL-6, TNFá e IFNã están elevadas en niños con FDH, pero no hubo correlación con las formas severas de choque.

Concentraciones plasmáticas de linfopoyetina estromal tímica en niños afectados por virus dengue

Thymic stromal lymphopoietin (TSLP) is a recently described cytokine involved in the physiopathogenesis of allergic and parasitic diseases. After production by epithelial cells in response to different stimuli like bacterial and viral components, the TSLP modulate the function of dendritic cells to generate a Th2-lymphocyte response. In dengue, an endemic viral febrile disease in Colombia, a predominant Th2-cytokine pattern has been described in children suffering forms severe and potentially lethal dengue. Here, it was determined by ELISA, the plasma concentrations of TSLP in children undergoing the acute and convalescent stage, in comparison to healthy and atopic children's levels as controls groups. Results show no significant differences in plasma concentrations of TSLP between healthy and acute-phase dengue-infected children and just atopic children showed significant higher levels of TSLP. However, a significant decrease in plasmatic TSLP was noted between the acute and convalescent phase of the dengue infection. When comparing the TSLP levels to the number of platelets, a known marker of dengue severity, there was no correlation. These findings support the hypothesis that plasma TSLP could not be directly involved in the pathogenesis of dengue infection in children and other local T lymphocyte polarization and systemic factors should be explored. La linfopoyetina estromal tímica (TSLP) es un citoquina recientemente descrita que juega un papel clave en la fisiopatología de enfermedades alérgicas y parasitarias. La TSLP, luego de ser producida por el epitelio en respuesta a diferentes estímulos como componentes bacterianos y virales, condiciona a células dendríticas locales a generar una respuesta de linfocitos Th2. En el dengue, una enfermedad viral febril endémica en Colombia, un predominante perfil de citoquinas Th2 ha sido descrito en los niños que presentan formas severas y potencialmente letales de la enfermedad. Aquí se determinaron por ELISA, las concentraciones plasmáticas de TSLP en niños con dengue en fase aguda y convaleciente de la enfermedad, comparándolos con los niveles presentes en niños sanos y atópicos como controles. Los resultados indican que no hay diferencias significativas en las concentraciones plasmáticas de TSLP entre los niños sanos y los niños infectados con dengue en fase aguda. Sin embargo, una disminución significativa fue encontrada en los niños infectados entre la etapa aguda y convaleciente de la infección. Cuando se compararon los niveles de TSLP con el número de plaquetas, un conocido marcador de severidad en el dengue, no hubo correlación. Estos hallazgos apoyan la hipótesis de que el TSLP plasmático podría no estar implicado directamente en la fisiopatología de la infección por virus dengue. Otros factores de polarización de linfocitos T locales y sistémicos deberían ser evaluados

A genome-wide association study follow-up suggests a possible role for PPARG in systemic sclerosis susceptibility

Introduction: A recent genome-wide association study (GWAS) comprising a French cohort of systemic sclerosis (SSc) reported several non-HLA single-nucleotide polymorphisms (SNPs) showing a nominal association in the discovery phase. We aimed to identify previously overlooked susceptibility variants by using a follow-up strategy.<p></p> Methods: Sixty-six non-HLA SNPs showing a P value <10-4 in the discovery phase of the French SSc GWAS were analyzed in the first step of this study, performing a meta-analysis that combined data from the two published SSc GWASs. A total of 2,921 SSc patients and 6,963 healthy controls were included in this first phase. Two SNPs, PPARG rs310746 and CHRNA9 rs6832151, were selected for genotyping in the replication cohort (1,068 SSc patients and 6,762 healthy controls) based on the results of the first step. Genotyping was performed by using TaqMan SNP genotyping assays. Results: We observed nominal associations for both PPARG rs310746 (PMH = 1.90 × 10-6, OR, 1.28) and CHRNA9 rs6832151 (PMH = 4.30 × 10-6, OR, 1.17) genetic variants with SSc in the first step of our study. In the replication phase, we observed a trend of association for PPARG rs310746 (P value = 0.066; OR, 1.17). The combined overall Mantel-Haenszel meta-analysis of all the cohorts included in the present study revealed that PPARG rs310746 remained associated with SSc with a nominal non-genome-wide significant P value (PMH = 5.00 × 10-7; OR, 1.25). No evidence of association was observed for CHRNA9 rs6832151 either in the replication phase or in the overall pooled analysis.<p></p> Conclusion: Our results suggest a role of PPARG gene in the development of SSc

IgA vasculitis: influence of CD40, BLK and BANK1 gene polymorphisms

CD40, BLK and BANK1 genes involved in the development and signaling of B-cells are identified as susceptibility loci for numerous inflammatory diseases. Accordingly, we assessed the potential influence of CD40, BLK and BANK1 on the pathogenesis of immunoglobulin-A vasculitis (IgAV), predominantly a B-lymphocyte inflammatory condition. Three genetic variants within CD40 (rs1883832, rs1535045, rs4813003) and BLK (rs2254546, rs2736340, rs2618476) as well as two BANK1 polymorphisms (rs10516487, rs3733197), previously associated with inflammatory diseases, were genotyped in 382 Caucasian patients with IgAV and 955 sex- and ethnically matched healthy controls. No statistically significant differences were observed in the genotype and allele frequencies of CD40, BLK and BANK1 when IgAV patients and healthy controls were compared. Similar results were found when CD40, BLK and BANK1 genotypes or alleles frequencies were compared between patients with IgAV stratified according to the age at disease onset or to the presence/absence of gastrointestinal or renal manifestations. Moreover, no CD40, BLK and BANK1 haplotype differences were disclosed between patients with IgAV and healthy controls and between patients with IgAV stratified according to the clinical characteristics mentioned above. Our findings indicate that CD40, BLK and BANK1 do not contribute to the genetic background of IgAV.Funding: This study was supported by European Union FEDER funds and “Fondo de Investigaciones Sanitarias” (grants PI18/00042 and PI21/00042) from “Instituto de Salud Carlos III” (ISCIII, Health Ministry, Spain). D.P.-P. is a recipient of a Río Hortega program fellowship from the ISCIII, co-funded by the European Social Fund (ESF, “Investing in your future”) (grant number CM20/00006). F.G. is supported by funds of the RICORS Program from ISCIII, co-funded by the European Union (grant number RD21/0002/0025). V.P.-C. is supported by funds of PI18/00042. S.R.-M. is supported by funds of the RETICS Program (RD16/0012/0009) (ISCIII, co-funded by the European Regional Development Fund (ERDF)). O.G. is a staff member of Xunta de Galicia (Servizo Galego de Saude (SERGAS)) through a research-staff stabilization contract (ISCIII/SERGAS) and his work is funded by ISCIII and the European Union FEDER fund (grant numbers RD16/0012/0014 (RIER) and PI17/00409). He is a beneficiary of project funds from the Research Executive Agency (REA) of the European Union in the framework of MSCA-RISE Action of the H2020 Program, project 734899—Olive-Net. R.L.-M. is a recipient of a Miguel Servet type II program fellowship from the ISCIII, co-funded by ESF (“Investing in your future”) (grant number CPII21/00004). Acknowledgments: We are indebted to the patients and healthy controls for their essential collaboration on this study. We also thank the National DNA Bank Repository (Salamanca) for supplying part of the control samples

IgA Vasculitis: Influence of CD40, BLK and BANK1 Gene Polymorphisms

CD40, BLK and BANK1 genes involved in the development and signaling of B-cells are identified as susceptibility loci for numerous inflammatory diseases. Accordingly, we assessed the potential influence of CD40, BLK and BANK1 on the pathogenesis of immunoglobulin-A vasculitis (IgAV), predominantly a B-lymphocyte inflammatory condition. Three genetic variants within CD40 (rs1883832, rs1535045, rs4813003) and BLK (rs2254546, rs2736340, rs2618476) as well as two BANK1 polymorphisms (rs10516487, rs3733197), previously associated with inflammatory diseases, were genotyped in 382 Caucasian patients with IgAV and 955 sex- and ethnically matched healthy controls. No statistically significant differences were observed in the genotype and allele frequencies of CD40, BLK and BANK1 when IgAV patients and healthy controls were compared. Similar results were found when CD40, BLK and BANK1 genotypes or alleles frequencies were compared between patients with IgAV stratified according to the age at disease onset or to the presence/absence of gastrointestinal or renal manifestations. Moreover, no CD40, BLK and BANK1 haplotype differences were disclosed between patients with IgAV and healthy controls and between patients with IgAV stratified according to the clinical characteristics mentioned above. Our findings indicate that CD40, BLK and BANK1 do not contribute to the genetic background of IgAV

Prevalence of Anxiety and Depression among Outpatients with Type 2 Diabetes in the Mexican Population

Depression and anxiety are common in diabetic patients; however, in recent years the frequency of these symptoms has markedly increased worldwide. Therefore, it is necessary to establish the frequency and factors associated with depression and anxiety, since they can be responsible for premature morbidity, mortality, risk of developing comorbidities, complications, suffering of patients, as well as escalation of costs. We studied the frequency of depression and anxiety in Mexican outpatients with type 2 diabetes and identified the risk factors for depression and anxiety.We performed a study in 820 patients with type 2 diabetes. The prevalence of depression and anxiety was estimated using the Hamilton Depression Rating Scale and the Hamilton Anxiety Rating Scale, respectively. We calculated the proportions for depression and anxiety and, after adjusting for confounding variables, we performed multivariate analysis using multiple logistic regressions to evaluate the combined effect of the various factors associated with anxiety and depression among persons with type 2 diabetes. The rates for depression and anxiety were 48.27% (95% CI: 44.48–52.06) and 55.10% (95% CI: 51.44–58.93), respectively. Occupation and complications in diabetes were the factors associated with anxiety, whereas glucose level and complications in diabetes were associated with depression. Complications in diabetes was a factor common to depression and anxiety (p<0.0001; OR 1.79, 95% CI 1.29–2.4).Our findings demonstrate that a large proportion of diabetic patients present depression and/or anxiety. We also identified a significant association between complications in diabetes with depression and anxiety. Interventions are necessary to hinder the appearance of complications in diabetes and in consequence prevent depression and anxiety