Evaluation of the Gam-COVID-Vac and vaccine-induced neutralizing response against SARS-CoV-2 lineage P.1 variant in an Argentinean cohort.

We evaluated humoral immune-response elicited by Sputnik-V by measuring anti-Spike (S) IgG antibodies (Abs) and neutralizing antibodies (NAb) prior to, 14 and 42 days after-vaccination. The safety and disease rates among vaccinated individuals were also evaluated. Since SARS-CoV-2 lineage P.1 is rapidly spreading in Argentina, virus-neutralizing activity of Sputnik-V-elicited and infection-elicited NAb faced to P.1 were also assessed. A total of 285 participants were recruited; all reported good tolerance, without any severe adverse event. Nine COVID-19 cases were confirmed in fully vaccinated individuals and viable P.1 variant was successfully isolated from one of them. At day 42, 99.65% of the individuals had anti-S IgG; however, 23.15% had not detectable NAbs. Significantly higher neutralization potency against WT compared to P.1 (p < 0·001) was observed. Some samples failed to neutralize P.1, mainly among vaccinated-naїve subjects; however, no significant differences were observed among previously infected-vaccinated individuals. Our results corroborated that Sputnik-V is safe and induces an efficient humoral immune response, although not all immunized subjects develop Nabs. Herein, we show for the first time, evidence of infectious SARS-CoV-2 shedding from Sputnik-V fully vaccinated individuals, by the isolation of viable virus from the nasopharyngeal swab of one participant of our study, 139 days after receiving the second dose. Thereby, we provide evidence indicating that the vaccine might avoid severe forms of COVID-19 but does not prevent infection nor prevents transmission from a fully vaccinated individual.

The extent of infectious SARS-CoV-2 shedding in an Argentinean cohort.

BACKGROUND: To analyze the infectious extent of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in different settings where prevention strategies are critical to limit infection spread, we evaluated SARS-COV-2 viability to guide public health policies regarding isolation criteria and infection control. METHODS: We attempted viral isolation in 82 nasopharyngeal swabs from 72 patients with confirmed SARS-COV-2 infection. Study population was divided into four groups: (i) Patients during the first week of symptoms; (ii) Patients with prolonged positive PCR; (iii) Healthcare workers from a hospital participating of an outbreak investigation, with SARS-COV-2 infection confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and (iv) Recipients of convalescent immune plasma (CIP).Vero Cl76 cell-line (ATCC CRL-587) was used in assays for virus isolation. Plasma samples of CIP recipients were also tested with plaque-reduction neutralization test. RESULTS: We obtained infectious SARS-COV-2 isolates from 15/84 nasopharyngeal swabs. The virus could not be isolated from upper respiratory tract samples collected 10-day after onset of symptoms (AOS) in patients with mild-moderate disease. CONCLUSION: The knowledge of the extent of SARS-CoV-2 infectivity AOS is relevant for effective prevention measures. This allows to discuss criteria for end isolation despite persistence of positive PCR and improve timing for hospital discharge with consequent availability of critical beds.

Impacto de la implementación de un sistema documental en el aseguramiento de la calidad en un laboratorio de análisis clínicos de un hospital público / Impact of the implementation of a documentary system on quality assurance in a clinical analysis laboratory of a public hospital

El objetivo de este trabajo fue establecer el impacto de la implementación de un sistema de gestión de calidad (SGC) en el desempeño del laboratorio de análisis clínicos del Hospital Materno Provincial. Se diseñaron diez indicadores de calidad (IC), que se midieron pre y posimplementación del sistema documental. Se encontró para el indicador solicitud médica incorrecta (SMI) una disminución de 11,2% a 6% cuando se implementó la gestión documental. Para el indicador omisión del diagnóstico (OD), el porcentaje bajó de 41,6% a 27,9% luego de la intervención. Se encontró un 5% de errores en ingreso al sistema informático del laboratorio (EI-SIL) en situación basal y 3,9% posimplementación, mientras que el indicador muestras mal remitidas (MMR) disminuyó de 3,1% a 1,9%. El 58% de los analitos disminuyó el índice de error total (IET) y el 80% aumentó el valor de sigma luego de la intervención. El 61% de los analitos disminuyó el valor de incertidumbre, mejorando de esta manera el desempeño de los métodos analíticos. El porcentaje de valores críticos (VC) comunicados al médico terapeuta antes de los 60 minutos aumentó del 20 al 54% después de la implementación de la gestión documental, mientras que el indicador reimpresión de informes (RI) disminuyó de 5,2 a 1,8%. El tiempo de respuesta (TAT) disminuyó de 164 a 125 minutos. La implementación de un SGC bajo los requisitos de normas internacionales mejoró el conocimiento y funcionamiento de los procesos del laboratorio clínico, evidenciado por la disminución de los errores en las etapas preanalítica, analítica y posanalítica.

The purpose of this study was to analyze the impact of implementing a quality management system (QMS) in the performance of a clinical laboratory of Hospital Materno Provincial. Ten quality indexes (QI) have been designed; they were measured before and after the implementation of the document management system (DMS). For the "incorrect medical application" index, there was a reduction from 11.2% to 6% when the DMS was implemented. For the "diagnostic omission" index, the percentage decreased from 41.6% to 27.9% after the implementation. Five per cent of mistakes were found in the admission of the laboratory computer system in the baseline state and 3.9% of the mistakes were found after implementation of the system. Meanwhile, the "incorrect sent samples" index decreased from 3.1% to 1.9%. Fifty eight per cent of the analytes decreased the total error index and 80% of them increased the sigma value after the intervention. Sixty one per cent of the analytes decreased the uncertainty value, thus improving the performance of analytical methods. The percentage of critical values communicated to the physician before the 60 minutes increased from 20% to 54% after the implementation of the DMS, while the "reprint of reports" index decreased from 5.2% to 1.8%. The turn around time (TAT) decreased from 164 to 125 minutes. The implementation of a QMS, under the requirements of international standards, improved the knowledge and the functioning of different processes of the clinical laboratory. This has been evidenced by the decrease of mistakes in the pre-analytical, analytical and post-analytical phases.

O objetivo deste trabalho foi estabelecer o impacto da implementação de um sistema de gestão de qualidade (SGC) no desempenho do laboratório de análises clínicas do Hospital Materno Provincial. Foram desenhados dez indicadores de qualidade (IQ), que foram medidos antes e depois da implementação do sistema documental. Foi achada uma diminuição de 11,2% para 6% no indicador solicitação médica incorreta, (SMI) quando se aplica a gestão documental. Para o indicador omissão do diagnóstico, a porcentagem baixou de 41,6% para 27,9% após a intervenção. Foram encontrados 5% de erros em entrada ao sistema informático do laboratório (EI-SIL) em situação basal e 3,9% pós-implantação, enquanto que o indicador amostras mal encaminhadas (AME) diminuiu de 3,1% para 1,9%. cincuenta e ocho por ciento dos analitos diminuíram o índice de erro total (IET) e 80% aumentou o valor de sigma após a intervenção. Sesenta e uno por ciento dos analitos diminuiu o valor de dúvidas, melhorando desta maneira o desempenho dos métodos analíticos. A porcentagem de valores críticos (VC) comunicados ao médico terapeuta antes dos 60 minutos aumentou de 20 para 54% depois da implementação da gestão documental, enquanto que o indicador reimpressão de relatórios diminuiu de 5,2 para 1,8%. O tempo de resposta diminuiu de 164 para 125 minutos. A implementação de um SGC sob os requisitos de normas internacionais melhorou o conhecimento e funcionamento dos processos do laboratório clínico, evidenciado pela diminuição dos erros nas etapas pré-analítica, analítica e pós-analítica.

Laboratory Accreditation in Argentina.

Laboratory accreditation is an essential element in the healthcare system since it contributes substantially to decision-making, in the prevention, diagnosis, treatment and follow-up of the health status of the patients, as well as in the organization and management of public healthcare. Therefore, the clinical biochemistry professional works continuously to provide reliable results and contributes to the optimization of operational logistics and integration of a laboratory into the health system. ISO 15189 accreditation, ensures compliance of the laboratory to minimize instances of error through the planning, prevention, implementation, evaluation and improvement of its procedures, which provides skill areas that involve both training undergraduate and graduate professionals in clinical biochemistry.

Standardized flow cytometry assay for identification of human monocytic heterogeneity and LRP1 expression in monocyte subpopulations: decreased expression of this receptor in nonclassical monocytes.

In this article, we present a flow cytometry assay by which human blood monocyte subpopulations-classical (CD14(++) CD16(-)), intermediate (CD14(++) CD16(+)), and nonclassical (CD14(+) CD16(++)) monocytes-can be determined. Monocytic cells were selected from CD45(+) leukocyte subsets by differential staining of the low-density lipoprotein receptor-related protein 1 (LRP1), which allows reducing the spill-over of natural killer cells and granulocytes into the CD16(+) monocyte gate. Percentages of monocyte subpopulations established by this procedure were significantly comparable with those obtained by a well-standardized flow cytometry assay based on the HLA-DR monocyte-gating strategy. We also demonstrated that LRP1 is differentially expressed at cell surface of monocyte subpopulations, being significantly lower in nonclassical monocytes than in classical and intermediate monocytes. Cell surface expression of LRP1 accounts for only 20% of the total cellular content in each monocyte subpopulation. Finally, we established the within-individual biological variation (bCV%) of circulating monocyte subpopulations in healthy donors, obtaining values of 21%, 20%, and 17% for nonclassical, intermediate, and classical monocytes, respectively. Similar values of bCV% for LRP1 measured in each monocyte subpopulation were also obtained, suggesting that its variability is mainly influenced by the intrinsic biological variation of circulating monocytes. Thus, we conclude that LRP1 can be used as a third pan-monocytic marker together with CD14 and CD16 to properly identify monocyte subpopulations. The combined determination of monocyte subpopulations and LRP1 monocytic expression may be relevant for clinical studies of inflammatory processes, with special interest in atherosclerosis and cardiovascular disease.

Eficacia del cordón umbilical humano como sustrato para detección de anticuerpos anti endomisio / Efficiency of human umbilical cord as substrate or detection of antiemdomysium antibodies / Aeficácia do cordão umbilical humano como substrato para a detenção de anticorpos anti-endomísio

Los anticuerpos anti endomisio IgA (EMA) están dirigidos hacia antígenos del tejido conectivo que rodea a las fibras del músculo liso. El objetivo de este trabajo fue evaluar la eficacia del cordón umbilical humano (CUH) como sustrato para detectar EMA mediante inmunofluorescencia indirecta y compararlo con una de las metodologías disponibles comercialmente, la cual utiliza como sustrato esófago de mono. Se obtuvieron 100 sueros de pacientes con diagnóstico de enfermedad celíaca y 50 sueros de pacientes clínicamente sanos con biopsia de mucosa intestinal normal, los cuales realizaron su consulta y atención en el Hospital Privado Centro Médico de Córdoba, en un periodo de tiempo comprendido entre los años 2006 y 2009. Los resultados obtenidos mostraron una "muy buena" concordancia entre ambos métodos. Se estimó para el método que utiliza CUH una sensibilidad y especificidad de 98% (93-99%) y 100% (93-100%) respectivamente con una eficacia del 99%. De acuerdo con lo anterior se concluye que utilizar CUH como sustrato para evaluar la presencia de EMA es confiable y efectivo para detectar pacientes con enfermedad celíaca no tratada.

The antiendomysium antibodies (EMA) are directed toward antigens of connective tissue that surrounds the smooth muscle fibers. The aim of this study was to evaluate the efficiency of human umbilical cord (HUC) as substrate to detect EMA by indirect immunofluorescence and to compare it with one of the commercially available methodologies which use monkey esophagus as substrate. Serum samples obtained from 100 patients with celiac disease diagnosis and 50 healthy controls with normal intestinal mucosa were evaluated. Patients were treated at the Hospital Privado Centro Médico de Córdoba over a period of time between 2006 and 2009. The results showed an "almost perfect" concordance between both methods. The calculated sensitivity and specificity for HUC was 98% (93-99%) and 100% (93-100%) respectively, with an efficiency of 99%. This results indicate that the use of HUC as substrate to evaluate the presence of EMA is reliable and effective for the detection of patients with untreated celiac disease.

Os anticorpos anti-endomísio IgA (EMA) são direcionados contra os antígenos do tecido conectivo que cercam as fibras do músculo liso. O objetivo deste trabalho foi avaliar a eficácia do cordão umbilical humano (CUH) como substrato para detectar EMA através da imunofluorescência indireta e compará-lo com uma das metodologias disponíveis comercialmente, a qual utiliza como substrato esôfago de macaco. Foram obtidos 100 soros de pacientes com diagnóstico de doença celíaca e 50 soros de pacientes clinicamente saudáveis com biópsia de mucosa intestinal normal, os quais realizaram sua consulta e atendimento no Hospital Privado Centro Médico de Córdoba, em um período de tempo compreendido entre os anos 2006 e 2009. Os resultados obtidos mostraram uma "ótima" concordância entre ambos os métodos. Foi calculada para o método que utiliza CUH uma sensibilidade e especificidade de 98% (93-99%) e 100% (93-100%) respectivamente com uma eficácia de 99%. De acordo com o acima exposto, se conclui que utilizar CUH como substrato para avaliar a presença de EMA é confiável e eficaz para detectar pacientes com doenças celíacas não tratadas.

Eficacia del cordón umbilical humano como sustrato para detección de anticuerpos anti endomisio / Efficiency of human umbilical cord as substrate or detection of antiemdomysium antibodies / Aeficácia do cordÒo umbilical humano como substrato para a detenþÒo de anticorpos anti-endomísio

Los anticuerpos anti endomisio IgA (EMA) están dirigidos hacia antígenos del tejido conectivo que rodea a las fibras del músculo liso. El objetivo de este trabajo fue evaluar la eficacia del cordón umbilical humano (CUH) como sustrato para detectar EMA mediante inmunofluorescencia indirecta y compararlo con una de las metodologías disponibles comercialmente, la cual utiliza como sustrato esófago de mono. Se obtuvieron 100 sueros de pacientes con diagnóstico de enfermedad celíaca y 50 sueros de pacientes clínicamente sanos con biopsia de mucosa intestinal normal, los cuales realizaron su consulta y atención en el Hospital Privado Centro Médico de Córdoba, en un periodo de tiempo comprendido entre los años 2006 y 2009. Los resultados obtenidos mostraron una "muy buena" concordancia entre ambos métodos. Se estimó para el método que utiliza CUH una sensibilidad y especificidad de 98% (93-99%) y 100% (93-100%) respectivamente con una eficacia del 99%. De acuerdo con lo anterior se concluye que utilizar CUH como sustrato para evaluar la presencia de EMA es confiable y efectivo para detectar pacientes con enfermedad celíaca no tratada.(AU)

The antiendomysium antibodies (EMA) are directed toward antigens of connective tissue that surrounds the smooth muscle fibers. The aim of this study was to evaluate the efficiency of human umbilical cord (HUC) as substrate to detect EMA by indirect immunofluorescence and to compare it with one of the commercially available methodologies which use monkey esophagus as substrate. Serum samples obtained from 100 patients with celiac disease diagnosis and 50 healthy controls with normal intestinal mucosa were evaluated. Patients were treated at the Hospital Privado Centro Médico de Córdoba over a period of time between 2006 and 2009. The results showed an "almost perfect" concordance between both methods. The calculated sensitivity and specificity for HUC was 98% (93-99%) and 100% (93-100%) respectively, with an efficiency of 99%. This results indicate that the use of HUC as substrate to evaluate the presence of EMA is reliable and effective for the detection of patients with untreated celiac disease.(AU)

Os anticorpos anti-endomísio IgA (EMA) sÒo direcionados contra os antígenos do tecido conectivo que cercam as fibras do músculo liso. O objetivo deste trabalho foi avaliar a eficácia do cordÒo umbilical humano (CUH) como substrato para detectar EMA através da imunofluorescÛncia indireta e compará-lo com uma das metodologias disponíveis comercialmente, a qual utiliza como substrato es¶fago de macaco. Foram obtidos 100 soros de pacientes com diagnóstico de doenþa celíaca e 50 soros de pacientes clinicamente saudáveis com biópsia de mucosa intestinal normal, os quais realizaram sua consulta e atendimento no Hospital Privado Centro Médico de Córdoba, em um período de tempo compreendido entre os anos 2006 e 2009. Os resultados obtidos mostraram uma "ótima" concordÔncia entre ambos os métodos. Foi calculada para o método que utiliza CUH uma sensibilidade e especificidade de 98% (93-99%) e 100% (93-100%) respectivamente com uma eficácia de 99%. De acordo com o acima exposto, se conclui que utilizar CUH como substrato para avaliar a presenþa de EMA é confiável e eficaz para detectar pacientes com doenþas celíacas nÒo tratadas.(AU)

Verificación de métodos en un laboratorio acreditado y planificación del control de calidad interno / Method verification in a certified laboratory and internal quality control planification / Verificação de métodos num laboratório credenciado e planejamento do controle de qualidade interno

En el proceso de verificación de métodos se obtienen datos del desempeño del sistema de medición en las condiciones de trabajo del laboratorio; luego esto es cotejado con las especificaciones brindadas por el fabricante de reactivos y con los requerimientos de calidad disponibles de distintas fuentes. Los objetivos del presente trabajo fueron aplicar protocolos de evaluación de métodos publicados en guías internacionales (CLSI, Clinical Laboratory Standard Institute) para verificar el correcto rendimiento de las metodologías evaluadas y diseñar e implementar una estrategia de control de calidad interno (CCI) que permita evaluar la estabilidad del sistema de medición en el tiempo. Se evaluaron glucosa, creatinina y láctico deshidrogenasa (LDH); sobre las metodologías para la valoración de los mismos se realizaron los ensayos correspondientes a la verificación de precisión y veracidad, linealidad, límite de detección, comparación de equipos y establecimiento de valores de referencia de acuerdo con lo establecido en las guías CLSI EP15-A2, EP6-A, EP17-A, EP9-A2, C28-A2, respectivamente. En todos los ensayos realizados se cumplieron con las especificaciones estipuladas por el fabricante para cada analito, como así también con los requerimientos de calidad elegidos para el error total permitido. Además, se determinó el punto operativo y se especificaron las reglas de CCI adecuadas para el seguimiento del desempeño de estas metodologías. Con los resultados obtenidos se construyó una matriz de calidad para hacer el seguimiento mensual de los parámetros evaluados. Aplicando procedimientos de verificación de métodos se demostró la aceptabilidad de los parámetros analíticos evaluados. La verificación de los métodos permite diseñar y aplicar una estrategia de CCI para evaluar la estabilidad analítica de los sistemas de medición en el tiempo, dentro de un marco de seguridad analítica exigido para métodos acreditados.

The process of method verification (MV) generates data about the performance of a measuring system in current laboratory working conditions; later, the information obtained is compared to the specifications issued by the assay manufacturer and International Regulating Organizations. The objectives of this study were: 1) to apply evaluation protocols established by international guidelines (CLSI, Clinical Laboratory Standards Institute) to verify the correct performance of the methodologies under analysis; 2) to design and implement a strategy of internal quality control (IQC) that allows evaluating the stability of the measuring system in time. Glucose, creatinine and lactate deshidrogenase were subject to analysis. Verification of precision, trueness, linearity detection limit, instrument comparison and reference values were performed under the provisions of CLSI EP-15A2, EP6A, EP17-A, EP9-A2 and C28-A2 guidelines, respectively. For every assay, the procedures indicated by the manufacturer were respected, as well as the quality requirements chosen for total allowed error. An operative point was obtained and adequate ICQ rules for monitoring the performance of those methodologies were established. With the results obtained, a quality matrix was built for a monthly follow-up of the evaluated parameters. The acceptability of the evaluated analytical parameters was proved by the application of MV procedures. Furthermore, the MV enabled to design and implementation of an ICQ strategy to test the analytical stability of the measuring systems, in the analytical safety environment required for accredited methods.

No processo de verificação de métodos são obtidos dados do desempenho do sistema de medição nas condições de trabalho do laboratório; depois isto é comparado com as especificações oferecidas pelo fabricante de reagentes de laboratório e com os requerimentos de qualidade disponíveis em diversas fontes. Os objetivos do presente trabalho foram aplicar protocolos de avaliação de métodos publicados em guias internacionais (CLSI, Clínical Laboratory Standard Institute) para verificar o correto rendimento das metodologias avaliadas e desenhar e implementar uma estratégia de controle de qualidade interno (CCI) que permita avaliar a estabilidade do sistema de medição no tempo. Foram avaliadas glicose, creatinina e desidrogenase lática (LDH); sobre as metodologias para a avaliação dos mesmos foram realizados os ensaios correspondentes à verificação de precisão e veracidade, linearidade, limite de detecção, comparação de equipamentos e estabelecimento de valores de referência conforme o estabelecido nos guias CLSI EP15-A2, EP6-A, EP17-A, EP9-A2, C28-A2, respectivamente. Em todos os ensaios realizados foram cumpridas as especificações estabelecidas pelo fabricante para cada analito, bem como com os requerimentos de qualidade selecionados para o erro total permitido. Além disso, foi possível determinar o ponto operacional e especificar as regras do CCI adequadas para o acompanhamento do desempenho destas metodologias. Com os resultados obtidos se construiu uma matriz de qualidade para fazer o acompanhamento mensal dos parâmetros avaliados. Aplicando procedimentos de verificação de métodos foi demonstrada a aceitabilidade dos parâmetros analíticos avaliados. A verificação dos métodos permite desenhar e aplicar uma estratégia de CCI para avaliar a estabilidade analítica dos sistemas de medição no tempo, dentro de um quadro de segurança analítica exigido para métodos com credenciamento.

Mycetoma of the scalp due to Microsporum canis: hystopathologic, mycologic, and immunogenetic features in a 6-year-old girl.

Dermatophytic mycetoma is an extremely rare subcutaneous mycosis. Here, we report the case of a 6-year-old girl with clinical, histologic, and mycologic findings consistent with a mycetoma of the scalp caused by Microsporum canis. To our knowledge, this is the first report showing the immunologic and immunogenetic features of a patient with a recalcitrant dermatophytic mycetoma.

Establecimiento de intervalos de referencia en subpoblaciones linfocitarias por citometría de flujo / Reference values in lymphocyte subpopulations using flow cytometry

La determinación de Subpoblaciones Linfocitarias Humanas (SLH) por Citometría de Flujo, Linfocitos CD3+ (LT), LTCD4+, LTCD8+ y la relación LTCD4+/LTCD8+, permiten monitorear pacientes infectados con virus de la inmunodeficiencia humana (VIH), los cuales son comparados con Valores de Referencia (VR). A su vez los VR informados por la bibliografía son variables debido a la falta de validación analítica y partición estadística de los datos de VR, entre otros. En este trabajo, utilizando un ensayo validado (ISO15189), se establecieron VR para SLH con criterios estadísticos de partición en individuos con serología negativa para VIH (397 mujeres (F) y 279 varones (M), edad: 17-83 años). Las SLH fueron medidas en un Citómetro de Flujo (Coulter Epics XL-MCL). Los datos fueron procesados empleando como criterio estadístico de partición el algoritmo de Martin Gellerstedt (AMG). Del análisis estadístico y del AMG se determinó la partición de los datos en 6 subpoblaciones definidas por edad (intervalo de años) y género (F y M): grupos I y IV: 17-35 (F y M); grupos II y V: 36-50 (F y M); y grupos III y VI: >50 años (F y M). Para los 6 grupos se definieron VR de linfocitos totales, LT, LTCD4+, LTCD8+ y LTCD4+/LTCD8+. Para cada límite inferior y superior de VR se estableció el intervalo de confianza al 90%. En conclusión, este estudio permitió establecer VR para SLH en un ensayo validado empleando un criterio estadístico de partición, lo cual permitiría incrementar la confiabilidad de los resultados y uniformar a nivel internacional los VR en SLH en diferentes poblaciones.

The determination of Human Lymphocyte Subpopulations (HLS) including Lymphocytes CD3+ (LT), LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio by flow cytometry, makes it possible to monitor patients infected who HIV, which are compared against reference values (RV). However, RV reported by the international bibliography are variable due to the absence of two main factors: analytical validation procedures and partitioning statistical criteria. This study, using a validated method (ISO15189), RV were established for HLS in individuals with negative serology for HIV (397 females (F) and 279 males (M), age: 17-83 years) applying partitioning statistical criteria. The values of HLS were obtained by flow cytometry (Coulter Epics XL-MCL). The data were processed using partitioning statistical criteria based on Martin Gellerstedt's algorithm (MGA). From the statistical analysis and MGA the partition of the data was determined in 6 subpopulations defined by age (interval of years) and gender (F and M): groups I and IV: 17-35 (F and M); groups II and V: 36-50 (F and M); and groups III and VI: >50 years (F and M). Hence, RV of total lymphocytes, LT, LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio were defined. For each limit (lower and upper) of reference values, the 90% confidence interval was determined. In conclusion, this study allowed establishing RV for HLS in a validated method using a partitioning statistical criterion, which would allow increasing the assurance results and establishing an international criterion for reference values in HLS in different populations.

A dopamine mechanism is implied in the acquisition and expression of amphetamine and stress-induced effects observed in the lymphocyte subpopulations.

Drugs of abuse and stress are associated with changes in circulating cell populations and reductions in cell-mediated immune responses. The main goal of this study was to determine the influence of repeated and acute d-amphetamine treatments on the foot-shock stress-induced effects on the peripheral lymphocyte subpopulations, and the involvement of a dopamine mechanism in the development and expression of this phenomenon. Wistar rats received an acute (5 mg/kg/day i.p.) or a repeated (2 mg/kg/day i.p. during 9 days) amphetamine treatment, and were exposed to a foot-shock stress (1 mA, 3 s) 4 days after the last amphetamine injection. Another group was administered with haloperidol (1 mg/kg/day i.p.) 15 min previous to each daily amphetamine injection or previous to the foot-shock stress session. Then, blood cells stained with monoclonal antibodies against CD3-FITC, CD8-PE and CD4-Cy-Chrome, and against CD161a-FITC, CD3-PE, and CD45RA-Cy-Crhome, were analyzed by multiparameter flow cytometry. The exposure to a foot-shock stress induced a decrease in the absolute number of peripheral lymphocytes, as well as in CD4+ and CD8+ T-cells and B-cells in acute and repeatedly amphetamine-treated rats, whereas the NK-cell population remained unchanged. Haloperidol administration previous to each drug administration or the foot-shock stress session reversed these effects. This study provides strong evidence that dopamine can play a more general role in the influence of amphetamine on the stress-induced effects on the lymphocyte subsets.

Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

Statistical criteria to establish optimal antibody dilution in flow cytometry analysis.

BACKGROUND: In direct techniques of flow cytometry, the optimal antibody dilution or titer point is established from the plateau area of the antibody titration curve. However, the plateau area is defined without any statistical criteria, which may lead to an incorrect selection of antibody dilution. Herein, we report statistical criteria to establish the optimal antibody dilution for CD14, CD8, CD4, and CD3 analysis by flow cytometry in peripheral whole blood. METHODS: The unpaired t-test (two-tail P value) was used as statistical criteria to analyze the titration curve of the following monoclonal antibody panels: CD14-FITC, CD8-FITC, CD4-RD1, and CD3-PC5. RESULTS: Using the unpaired t-test (two-tail P value), the plateau area from the antibody titration curve was fitted when two consecutive antibody volumes showed mean peak of channel fluorescence (MPCF) values not significantly different. When the antibody was used at volume corresponding to that of the antibody titration point, the flow cytometry analysis of whole blood samples with different density of cell antigens can be correctly discriminated. CONCLUSION: This statistical criteria allows the fitting of the plateau area of MPCF versus antibody volume and consequently, to define the optimal antibody dilution.

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.

Amphetamine triggers an increase in met-enkephalin simultaneously in brain areas and immune cells.

We analyzed effects of amphetamine on proenkephalin-derived peptides in brain areas and immune cells in rats. Acute, as well as a repeated amphetamine treatment, decreased the concanavalin-A-induced lymphocyte proliferation, concomitantly with an increase of free met-enkephalin in nucleus accumbens, prefrontal cortex, spleen, thymus and splenic macrophages. Proenkephalin protein increased in prefrontal cortex, thymus (32 kDa isoform), nucleus accumbens and spleen (44 kDa isoform), while proenkephalin mRNA levels decreased in brain stem. The influence of met-ENK in key brain areas for sensitization and in immune organs is consistent with the idea that changes on met-ENK could underlie amphetamine's effects on brain and IS.

[Function of flow cytometry on the dosage of antibodies against double stranded DNA in systemic lupus erythematosus]. / Función de la citometría de flujo en el dosaje de anticuerpos anti-DNA de doble cadena en el lupus eritematoso sistémico.

BACKGROUND: Among the diverse number of antibodies observed in systemic lupus erythematosus, antibodies against double stranded DNA (anti-dsDNA) represent important serologic markers for the disease diagnosis and the follow-up of the disease activity. OBJECTIVE: To evaluate the role of a new quantitative methodology to detect antibodies against double stranded DNA in systemic lupus erythematosus and its association with the disease activity. MATERIAL AND METHODS: The performance of the indirect immunofluorescence flow cytometry with Crithidia luciliae as substrate was compared with the Crithidia luciliae indirect immunofluorescence assay and the ELISA technique in order to detect antibodies against double stranded DNA in 54 sera from 47 patient with systemic lupus erythematosus and 100 sera from normal controls. RESULTS: The new method showed a sensitivity of 78% and a specificity of 81% when the Crithidia luciliae indirect immunofluorescence assay was the gold standard. Compared with the ELISA technique, the flow cytometry showed a sensitivity of 78% and a specificity of 86%. No correlation was found among antibodies against double stranded DNA values detected with flow cytometry and the MEX-SLEDAI activity scores. However, the flow cytometry showed a sensitivity of 70% and a specificity of 42% to distinguish patients with systemic lupus erythematosus with and without activity (MEX-SLEDAI score > or = 5). The Rho intra-observer coefficient was 0.61 (p < 0.0001). CONCLUSIONS: In spite of the fact that this new method might represent an interesting advance for antibodies against double stranded DNA quantitative testing, a clear superiority does not emerge when it was compared with more traditional assays. Difficulties related with its reproducibility might represent a limitation in the routine use of this new method.

Inmunodeficiencia común variable: hallazgos recientes sobre anormalidades celulares / Common variable immunodeficiency: recent findings in cellular abnormalities

La Inmunodeficiencia Común Variable (IDCV), también llamada hipogammaglobulinemia adquirida o disgammaglobulinemia, es un grupo heterogéneo de desórdenes que afecta a los linfocitos (L)T y a los LB. Constituye uno de los principales síndromes de deficiencia de anticuerpos, presenta una pérdida o disminución de la respuesta inmune humoral a antígenos específicos, particularmente polisacáridos capsulares bacterianos. Estos pacientes tienen una alta susceptibilidad a infecciones del tracto respiratorio y la IDCV tiene una estrecha relación con patologías tales como deficiencias de IgA y subclases de IgG. El objetivo de esta revisión es actualizar los conocimientos de este grupo de desórdenes inmunológicos, con especial énfasis en los nuevos criterios utilizados para el diagnóstico y seguimiento

Inmunodeficiencia común variable: hallazgos recientes sobre anormalidades celulares / Common variable immunodeficiency: recent findings in cellular abnormalities

La Inmunodeficiencia Común Variable (IDCV), también llamada hipogammaglobulinemia adquirida o disgammaglobulinemia, es un grupo heterogéneo de desórdenes que afecta a los linfocitos (L)T y a los LB. Constituye uno de los principales síndromes de deficiencia de anticuerpos, presenta una pérdida o disminución de la respuesta inmune humoral a antígenos específicos, particularmente polisacáridos capsulares bacterianos. Estos pacientes tienen una alta susceptibilidad a infecciones del tracto respiratorio y la IDCV tiene una estrecha relación con patologías tales como deficiencias de IgA y subclases de IgG. El objetivo de esta revisión es actualizar los conocimientos de este grupo de desórdenes inmunológicos, con especial énfasis en los nuevos criterios utilizados para el diagnóstico y seguimiento (AU)

[Expression of leukocytic adhesion molecules in a patient with common variable immunodeficiency]. / Expresión de las moléculas de adhesión leucocitaria en un paciente con inmunodeficiencia variable común.

BACKGROUND: Common variable immunodeficiency is one of the main antibodies' deficiency syndromes. OBJECTIVE: To present the immunological study of a 29-year-old patient with common variable immunodeficiency who assisted to a check-up after being four years without treatment with gammaglobuline. MATERIAL AND METHODS: We studied a sample of peripheral blood and saliva of a patient with common variable immunodeficiency and that of a healthy patient (control). Assessment of immunoglobulin G, immunoglobulin A and immunoglobuin M was performed by a simple radial immunodiffusion test, and immunoglobulin E by immunoassay. Immunophenotypic study of leukocytic subpopulations was done by citometry by using the following panel of monoclonal antibodies: CD3 (Leu-4), CD4 (Leu-3a), CD8 (Leu-2a), CD19 (Leu-12), CD14 (Leu-M3), CD11a (LFA-I), CD49d (VLA-4), CD54 (ICAM-1), CD31 (PECAM). RESULTS: It was found a significant reduction in most of the serum and secretory immunoglobulins, levels of unusual expression of integrines CD11a and CD31 in lymphocytes T related to the low percentage of activated lymphocytes T/memory.