Titulación de hemolisinas en concentrados plaquetarios obtenidos a partir de buffy coat de donantes voluntarios de sangre de grupo O Rh D positivo / Hemolysin titration in platelet concentrates obtained from buffy coat of voluntary blood donors of group O Rh D positive

Los posibles efectos adversos que se producen en transfusiones incompatibles ABO son un riesgo latente en el uso de concentrados de plaquetas grupo O, por lo que la titulación de hemolisinas anti-A/B constituye una de las estrategias para su prevención. El objetivo de este estudio fue determinar la frecuencia de títulos de hemolisinas de isotipos IgG e IgM anti-A/B en donantes de sangre. Se trató de un estudio descriptivo, transversal y aleatorio simple con un tamaño muestral de 308 muestras. Se aplicó la metodología en tubo, gel salino y anti-inmunoglobulina IgG y, mediante soluciones seriadas, se evidenció el título. Adicionalmente, se realizó una encuesta sobre los posibles factores de riesgo para el aumento de estos títulos. Se aplicó estadística descriptiva mediante el uso del software informático SPSS versión 22.0 y la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado y, para establecer la concordancia de las lecturas visuales de las tarjetas de gel, se aplicó el índice kappa. Se determinó la existencia de hemolisinas de isotipo IgG e IgM anti-A/B de títulos superiores a 1/64. Existió una relación estadísticamente significativa entre embarazos y títulos de IgG anti-A/B >1/128 y el aumento de hemolisinas de isotipo IgM y la ingesta de probióticos. Los resultados demostraron la necesidad de implementar la titulación de hemolisinas previo a la transfusión de concentrados plaquetarios no isogrupo, por lo que se recomienda una investigación de riesgo-beneficio y el seguimiento de pacientes con transfusiones de concentrados plaquetarios incompatibles ABO.

The possible adverse effects that occur in incompatible ABO transfusions are a latent risk in the use of group O platelet concentrates, so the titration of anti-A/B hemolysins is one of the strategies for its prevention. The objective of this study was to determine the frequency of hemolysins titers IgG and IgM anti-A/B isotypes in blood donours. It was a simple randomized descriptive cross-sectional study with a sample size of 308 samples. The methodology was applied in tube, saline gel and anti-IgG anti-immunoglobulin and by means of serial solutions the title was verified. Additionally, a survey was conducted on the possible risk factors for the increase in securities. Descriptive statistics were used through the application of the SPSS version 22.0 software and the relationship between independent variables through the Chi-square statistical analysis and the kappa index was applied to match the visual readings of the gel cards. The existence of IgG and IgM anti-A/B isotype hemolysins of titers greater than 1/64 was determined. There was a statistically significant relationship between pregnancies and anti-A/B IgG titres>1/128; and the increase in IgM isotype hemolysins and probiotic intake. The results demonstrate the need to implement hemolysin titration prior to transfusion of non-isogroup platelet concentrates, so a risk-benefit investigation and follow-up of patients with transfusions of ABO incompatible platelet concentrates is recommended.

Os possíveis efeitos adversos que ocorrem em transfusões incompatíveis ABO são um risco latente no uso de concentrados de plaquetas do grupo O, portanto a titulação de hemolisinas anti-A/B é uma das estratégias para sua prevenção. O objetivo deste estudo foi determinar a frequência de títulos de hemolisinas de isotipos IgG e IgM anti-A/B em doadores de sangue. Trata-se de um estudo descritivo transversal aleatório simples, com tamanho de amostra de 308 amostras. A metodologia foi aplicada em tubo, gel salino e anti-imunoglobulina IgG e utilizando soluções em série, o título foi verificado. Além disso, foi realizada uma pesquisa sobre os possíveis fatores de risco para o aumento destes títulos. A estatística descritiva foi utilizada através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes por meio da análise estatística do qui-quadrado e, para estabelecer a concordância com as leituras visuais dos cartões de gel, o índice kappa foi aplicado. Foi determinada a existência de hemolisinas de isotipo IgG e IgM anti-A/B de títulos maiores que 1/64. Existiu uma relação estatisticamente significante entre gestações e títulos de IgG anti-A/B>1/128; e o aumento de hemolisinas do isotipo IgM e a ingestão de probióticos. Os resultados demonstram a necessidade de implementar a titulação da hemolisina antes da transfusão de concentrados de plaquetas não isogrupo, por isso, recomenda-se uma investigação de risco-benefício e acompanhamento de pacientes com transfusões de concentrados de plaquetas incompatíveis com ABO.

The evaluation of the Myxovirus Resistance 1 protein in serum and saliva to monitor disease activation in primary Sjögren's syndrome

OBJECTIVE: Primary Sjögren's syndrome (pSjS) is a chronic autoimmune disease that causes dry eye and mouth. No laboratory parameters to monitor the activation of this disease have been identified. Therefore, any possible relationships between salivary and blood myxovirus resistance 1 (MX1) and pSjS must be prospectively studied. METHODS: Thirty female patients with pSjS, 30 women with rheumatoid arthritis (RA) without secondary Sjögren's syndrome (SjS) and 28 healthy control women were enrolled in this investigation. Analyses of MX1 by the enzyme-linked immunosorbent assay (ELISA) method, SS-A (Ro) and SS-B (La) tests by the strip immunoblot method, anti-nuclear antibody (ANA) tests by immunofluorescence and the measurement of serum rheumatoid factor (RF), C3, C4, immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were performed. RESULTS: The serum level of MX1 in patients without Raynaud phenomenon was higher than in those with Raynaud phenomenon (p:0.029, p<0.05, statistically significant). There was a statistically significant positive association between hemoglobin levels and MX1 serum levels. No statistically significant association was found among the other parameters. Low MX1 levels were shown to be associated with both a low disease activity score based on the European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI) and hydroxychloroquine use in all patients. CONCLUSION: MX1 levels have a considerable impact on the assessment of the disease activity in SjS. We believe that more-comprehensive studies should be performed on patients with pSjS who do not use hydroxychloroquine to prove this relationship and that MX1 levels should be used as a routine marker for the assessment of pSjS disease activity. Further studies are needed to create awareness of the role that MX1 has in the diagnosis of pSjS, which may help to uncover novel pathways for new therapeutic modalities.

Toxoplasmosis congénita: Diagnóstico serológico, RPC, aislamiento y caracterización molecular de Toxoplasma gondii / Congenital toxoplasmosis: Serology, PCR, parasite isolation and molecular characterization of Toxoplasma gondii

Resumen Introducción El diagnóstico de toxoplasmosis congénita (TC) en el recién nacido es muy importante porque debe recibir tratamiento siempre, sintomático o no, para evitar o aminorar las secuelas de la enfermedad. Objetivo Evaluación comparativa de los métodos disponibles en la institución para el diagnóstico de TC. Materiales y Métodos Se evaluaron métodos diagnósticos en 67 niños cuyas madres cursaron toxoplasmosis aguda durante el embarazo. Se utilizó la técnica de Sabin Feldman para IgG al nacimiento y durante el seguimiento serológico hasta el año de vida. Para determinar IgM, IgA e IgE se utilizó la técnica immunosorbent agglutination assay (ISAGA). El diagnóstico directo se realizó por reacción de polimerasa en cadena (RPC), aislamiento y caracterización molecular del parásito. Resultados La sensibilidad (S) de ISAGA IgM fue 87%, ISAGA IgA 91% y la especificidad (E) fue 100% para ambas; cuando se realizaron en conjunto, la S aumentó a 98%. La detección de IgE contribuyó al diagnóstico cuando se la detectó sólo en la sangre del neonato y no en sangre materna. Se aisló el parásito en cuatro casos de TC, uno fue genotipo II y los otros tres, genotipos "atípicos". La S del aislamiento fue 80% y la E 100%. Conclusión Los métodos serológicos utilizados mostraron una buena eficacia diagnóstica. Un caso fue detectado sólo por el aislamiento y la caracterización molecular tiene gran valor epidemiológico.

Background. Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child's serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were "atypical" genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.

Cerebrospinal fluid data compilation and knowledge-based interpretation of bacterial, viral, parasitic, oncological, chronic inflammatory and demyelinating diseases. Diagnostic patterns not to be missed in neurology and psychiatry / Liquido cefalorraquidiano: compilação dos dados e interpretação avançada de doenças bacterianas, virais, parasitarias, oncológicas, inflamatórias cronicas e desmielinizantes. Padrões diagnósticos que não devem faltar em neurologia e psiquiatria

ABSTRACT The analysis of intrathecal IgG, IgA and IgM synthesis in cerebrospinal fluid (CSF) and evaluation in combined quotient diagrams provides disease-related patterns. The compilation with complementary parameters (barrier function, i.e., CSF flow rate, cytology, lactate, antibodies) in a cumulative CSF data report allows a knowledge-based interpretation and provides analytical and medical plausibility for the quality assessment in CSF laboratories. The diagnostic relevance is described for neurological and psychiatric diseases, for which CSF analysis can’t be replaced by other diagnostic methods without loss of information. Dominance of intrathecal IgM, IgA or three class immune responses give a systematic approach for Facial nerve palsy, Neurotrypanosomiasis, Opportunistic diseases, lymphoma, Neurotuberculosis, Adrenoleucodystrophy or tumor metastases. Particular applications consider the diagnostic power of the polyspecific antibody response (MRZ-antibodies) in multiple sclerosis, a CSF-related systematic view on differential diagnostic of psychiatric diseases and the dynamics of brain- derived compared to blood-derived molecules in CSF for localization of paracytes.

RESUMO A análise da síntese intratecal de IgG, IgA e IgM no liquido cefalorraquidiano (LCR) e a avaliação destas em diagramas com quocientes sugere padrões de diversas doenças. Estes dados, juntamente com outros parâmetros como a função de barreira, o fluxo liquórico, a citologia, o lactato e a pesquisa de anticorpos, integrados em uma ficha de paciente, permite uma interpretação baseada em conhecimento e permite também uma aferição da qualidade em laboratórios de LCR. A relevância diagnóstica é descrita para doenças neurológicas e psiquiátricas pois a análise do LCR não pode ser substituída por outros metódos diagnósticos sem perda de informação para o diagnóstico do paciente. O aumento da síntese intratecal de IgM, IgA ou das 3 classes de imunoglobulinas sugerem um diagnóstico sistemático de paralisia facial periférica, neurotripanosomiase, doenças oportunísticas, linfoma, neurotuberculose, adrenoleucodistrofia ou metástases de tumores cerebrais. A resposta poliespecífica de anticorpos contra sarampo, rubéola e varicela zoster (MRZ reação) é sugestiva de esclerose múltipla. Uma visão sistemática considera o diagnóstico diferencial de doenças psiquiátricas e doenças chrônicas. A dinâmica de moléculas derivadas do cérebro comparadas com aqueles derivadas do sangue é importante para a localização de parasitos em doenças parasitárias do sistema nervoso.

Síndrome de Job asociado a tuberculosis miliar

El síndrome de hiper IgE también denominado síndrome de Job, es una inmunodeficiencia primaria poco frecuente, cuyo modo de herencia puede ser autosómico recesivo o dominante. Se caracteriza por altos niveles de IgE, eosinofilia, abscesos cutáneos, eccema, candidiasis mucocutánea crónica e infecciones pulmonares recidivantes que contribuyen al desarrollo de neumatoceles y bronquiectasias. El germen más frecuentemente aislado es el Staphylococcus aureus. En la actualidad, ante la mayor supervivencia de los pacientes se han comunicado infecciones oportunistas y linfomas. Existen escasas publicaciones de pacientes con enfermedad por Mycobacterium tuberculosis asociada a síndrome de hiper IgE, por lo que consideramos relevante comunicar el caso de un paciente con antecedentes de una tuberculosis pulmonar, que presentó una tuberculosis miliar con grave compromiso respiratorio, con buena respuesta al tratamiento estándar con drogas de primera línea.

The hyper Immunoglobulin E syndrome, also known as Job´s syndrome, is a rare primary immunodeficiency, its mechanisms of inheritance maybe recessive or dominant autosomal. It is characterized by high levels of IgE, eosinophilia, skin abscesses, eczema, chronic mucocutaneous candidiasis and recurrent pulmonary infections all of which contribute to the development of pneumatoceles and bronchiectasis. The most frequently isolated bacteria is Staphylococcus aureus. Currently, despite the highest survival of patients, lymphomas and other opportunistic infections have been reported. There are few reports of patients with Mycobacterium tuberculosis infection associated with hyper IgE syndrome. Therefore it is relevant that we report a case history of a patient with pulmonary tuberculosis, presenting miliary tuberculosis and severe respiratory compromise, who responded positively to standard anti-tuberculous treatment with first line drugs.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Increased IgE serum levels are unrelated to allergic and parasitic diseases in patients with juvenile systemic lupus erythematosus

OBJECTIVE: The aim of this study was to assess the IgE serum levels in juvenile systemic lupus erythematosus patients and to evaluate possible associations with clinical and laboratory features, disease activity and tissue damage. METHODS: The IgE serum concentrations in 69 consecutive juvenile systemic lupus erythematosus patients were determined by nephelometry. IgG, IgM and IgA concentrations were measured by immunoturbidimetry. All patients were negative for intestinal parasites. Statistical analysis methods included the Mann-Whitney, chi-square and Fisher's exact tests, as well as the Spearman rank correlation coefficient. RESULTS: Increased IgE concentrations above 100 IU/mL were observed in 31/69 (45%) juvenile systemic lupus erythematosus patients. The mean IgE concentration was 442.0 ± 163.4 IU/ml (range 3.5-9936.0 IU/ml). Fifteen of the 69 patients had atopic disease, nine patients had severe sepsis and 56 patients presented with nephritis. The mean IgE level in 54 juvenile systemic lupus erythematosus patients without atopic manifestations was 271.6 ± 699.5 IU/ml, and only nine of the 31 (29%) patients with high IgE levels had atopic disease. The IgE levels did not statistically differ with respect to the presence of atopic disease, severe sepsis, nephritis, disease activity, or tissue damage. Interestingly, IgE concentrations were inversely correlated with C4 levels (r = -0.25, p = 0.03) and with the SLICC/ACR-DI score (r = -0.34, p = 0.005). The IgE concentration was also found to be directly correlated with IgA levels (r = 0.52, p = 0.03). CONCLUSIONS: The present study demonstrated for the first time that juvenile systemic lupus erythematosus patients have increased IgE serum levels. This increase in IgE levels was not related to allergic or parasitic diseases. Our results are in line with the hypothesis that high IgE levels can be considered a marker of immune dysregulation.

Comparación de adenosina deaminasa y detección de anticuerpos anti-antígeno A60 para el diagnóstico de meningitis tuberculosa / A comparative study for adenosine deaminase and anti-antigen A-60 antibodies detection for the diagnosis of tuberculous meningitis

Background: Diagnosis of tuberculous meningitis (TBM) is hampered by the lack of rapid and accurate diagnostic tools. We evaluated the immunological response to Mycobacterium tuberculosis anti-A60 antibodies in cerebrospinal fluid (CSF) in comparison to adenosine deaminase (ADA) determination, for the diagnosis of TBM. Methods: A total of 63 CSF samples were analyzed by indirect ELISA for the detection of anti- A60 IgG, IgM and IgA. These include samples from 17 patients with confirmed TBM and 46 control patients with other infections. Results: The mean individual anti-A60 IgM, IgG and IgA CSF antibody titers were significantly higher in TBM in comparison with control groups (p < 0.01). The best discriminatory CSF antibody for confirming TBM diagnosis was IgM, with an area under the receiver operating characteristic curve of 0.928 (95%CI 0.834-0.978), compared to 0.863 (95% CI: 0.752-0.936) for ADA testing (p = NS). The sensitivity of anti- A60 IgM CSF antibody titers (cutoff > 0.06 U/ml) was 94.1% compared to 88.2% for ADA (cutoff > 6.2 U/ml), p = NS. Both anti A60 IgM and ADA showed the same moderate specificity (80.4%). Two cases of TBM were correctly identified by anti-A60 IgM but missed by ADA. Conclusion: The ELISA test for anti-antigen A60 antibodies (IgM) is a rapid and sensitive tool for the rapid diagnosis of TBM that can be a complement to ALDA determination. The specificity of both tests is still a limitation in TBM diagnosis.

Antecedentes: El diagnóstico de meningitis tuberculosa (MTBC) se ve limitado por la ausencia de técnicas diagnósticas rápidas y precisas en líquido cefalorraquídeo (LCR). En este estudio evaluamos la respuesta inmunoló-gica de anticuerpos anti-antígeno A60 de Mycobacterium tuberculosis en LCR en comparación a la determinación de adenosina deaminasa (ADA). Métodos: Un total de 63 muestras de LCR fueron estudiadas mediante ELISA indirecto para detección de IgG, IgM e IgA anti-A60. Estas muestras incluyeron 17 casos de MTBC confirmada y 46 controles con otras infecciones. Resultados: Los títulos de IgG, IgM e IgA anti A-60 resultaron significativamente superiores en casos de MTBC versus controles (p > 0,01). El anticuerpo con mej or poder discriminatorio resultó IgM, con un área bajo la curva ROC de 0,928 (95%IC 0,8340,978), comparado a 0,863 (95% IC: 0,752-0,936) para ADA (p = NS). La sensibilidad de IgM anti-A60 (nivel de corte > 0,06 U/ml) fue de 94,1% versus 88,2% para ADA (nivel de corte > 6,2 U/ml), p = NS. Ambos IgM anti-A60 y ADA presentaron la misma especificidad baja-moderada (80,4%). Dos casos de MMTBC fueron correctamente identificados por IgM anti-A60 pero no por ALDA. Conclusión: La detección de anticuerpos anti-A60 (IgM) puede ser de ayuda en el diagnostico de MTBC en forma complementaria a la determinación de ALDA. La baja especificidad de ambos tests constituye su principal limitante.

Profile of pregnant women and children treated at a reference center for congenital toxoplasmosis in the northern state of Minas Gerais, Brazil / Perfil de mulheres grávidas e crianças atendidas em um centro de referência para toxoplasmose congênita no norte do estado de Minas Gerais, Brasil

INTRODUCTION: To describe the clinical and epidemiological profile of pregnant women and children treated at a reference outpatient clinic for congenital toxoplasmosis. METHODS: Pregnant women potentially exposed to Toxoplasma gondii were observed. Diagnoses were made using serologic tests compatible with acute toxoplasmosis. Children presenting with: Toxoplasma-specific antibodies (IgM or IgA or ascending IgG titers higher than maternal titers in the first 3 months of life) coupled with toxoplasmosis symptoms; intracranial calcifications (by transfontanelar ultrasound or cephalic segment tomography); or retinochoroiditis (by fundoscopy examination) in the first 8 months of life were also included in the study. RESULTS: Fifty-eight mother-child pairs were observed (mean age of the mothers was 22.1 years). Most patients lived in urban areas (86.2 percent) and had attended less than 8 years of school (51.7 percent). Diagnosis was made after birth in 19 (32.8 percent) children. Thirty-four (58.6 percent) women received some type of treatment during pregnancy. Most (72.4 percent) of the children did not present with clinical alterations at birth. The main findings were ophthalmological: 20 (34.5 percent) children with retinochoroiditis, 17 (29.3 percent) with strabismus, and 7 (12.1 percent) with nystagmus. Of the children with retinochoroiditis, 9 presented with subnormal vision. Ten (32.3 percent) out of 31 children presented with intracranial calcifications by cephalic segment congenital toxoplasmosis, and 9 (42.9 percent) children presented with delayed psychomotor development. CONCLUSIONS: Our results highlight a critical situation. Protocols for follow-up of pregnant women and their children must be created to improve medical care and minimize sequelae.

INTRODUÇÃO: Descrever o perfil clínico e epidemiológico de gestantes e crianças atendidas em um centro de referência para toxoplasmose congênita. MÉTODOS: Gestantes potencialmente expostas ao Toxoplasma gondii foram estudadas. Os diagnósticos foram feitos através de testes sorológicos compatíveis com toxoplasmose aguda. Crianças que apresentaram anticorpos específicos (IgM e IgA ou títulos ascendentes de IgG, superiores aos títulos maternos nos primeiros três meses de vida), juntamente com os sintomas da toxoplasmose, calcificações intracranianas (ultrassom transfontanela ou tomografia do segmento cefálico), ou retinocoroidite (fundoscopia) nos primeiros oito meses de vida, também foram estudadas. RESULTADOS: Foram estudados 58 pares mãe-filho (a média de idade das mães foi de 22,1 anos). A maioria (86,2 por cento) das mães residia em áreas urbanas e tinham até 8 anos de escolaridade (51,7 por cento). O diagnóstico foi feito após o nascimento para 19 (32,8 por cento) crianças; 34 (58,6 por cento) mulheres receberam algum tipo de tratamento durante a gravidez. A maioria (72,4 por cento) das crianças não apresentou alterações clínicas ao nascimento. Os principais achados foram oftalmológicos: 20 (34,5 por cento) crianças com retinocoroidite, 17 (29,3 por cento) com estrabismo e 7 (12,1 por cento) com nistagmo. Das crianças com retinocoroidite, 9 apresentaram visão subnormal. Calcificações intracranianas estiveram presentes em 10 (32,3 por cento) das 31 crianças que realizaram toxoplasmose congênita de segmento cefálico e 9 (42,9 por cento) crianças apresentaram atraso do desenvolvimento psicomotor. CONCLUSÕES: Os resultados mostram uma situação crítica. Protocolos para acompanhamento de gestantes e seus filhos devem ser criados para melhorar o atendimento médico e minimizar sequelas.

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.

Serological monitoring of a Toxoplasma infection after hematopoietic stem cell transplantation / Monitoramento sorológico de uma infecção toxoplásmica após transplante de células progenitoras hematopoiéticas

We report a primary response to Toxoplasma gondii following a hematopoietic stem cell transplantation in a patient with multiple myeloma. The primary response to T. gondii was supported by IgM, IgG and IgA seroconversion. The patient was promptly treated and there were no complications related to toxoplasmosis in the subsequent months.

Esse relato de caso descreve uma resposta primária ao Toxoplasma gondii após transplante de células progenitoras hematopoiéticas em paciente com mieloma múltiplo. A resposta primária para o T. gondii foi evidenciada pela soroconversão observada na resposta de anticorpos IgM, IgG e IgA. O paciente foi prontamente tratado e complicações relacionadas à toxoplasmose não foram observadas nos meses subseqüentes.

Laboratorial atopy markers in children with human immunodeficiency virus

Changes in immune system functions are one of the most important consequences of human immunodeficiency virus (HIV) infection. Studies have reported a higher prevalence of disease mediated by immunological hypersensitivity mechanisms in HIV-positive patients. This study aims to observe how immunological changes in HIV-infected children interfere in atopy determinants. Fifty-seven HIV-positive children were studied between June 2004-August 2005 to evaluate the possible modifications in atopy diagnosis from prick test environmental allergen reactivity. Patients were subjected to two evaluations: on both occasions, atopic and non-atopic groups were correlated with immunological (CD4+ and CD8+ lymphocyte concentrations and serum levels of IgA, IgM, IgG and IgE) and viral parameters (HIV viral load). The percent atopy was 20.05 in the first and 29.82 in the second evaluation and atopy was diagnosed in patients without immunosuppression or with moderate immunosuppression. Six patients changed from a negative to a positive atopy profile. One patient with a decreased CD4+ T lymphocyte concentration failed to demonstrate prick test positivity between evaluations. Multivariate analysis showed that the variables associated with atopy diagnosis included a personal history of allergic diseases as well as elevated IgE for age and elevated IgE levels. Atopy development in HIV-infected children seems to be modulated by genetic and environmental factors as well as immunological condition.

Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic). The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs) were absorbed by nitrocellulose membrane and blocked with a 5 percent skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

Anticuerpos antinucleares, imágenes y características obtenidas por inmunofluorescencia: Importancia de los isotipos IgA, IgM e IgG / Antinuclear antibodies, patterns and characteristics obtained by immunofluorescence: The importance of the IgA, IgM and IgG isotypes

La técnica de elección para el screening de anticuerpos antinucleares (ANA) es la inmunofluorescencia indirecta que utiliza como sustrato una línea de células epiteliales de carcinoma de laringe humano (IFI-HEp2), y como antisuero, anti-IgG o anti-Ig totales. Los ANA-IgG son los más importantes para el diagnóstico y monitoreo de las enfermedades del tejido conectivo (ETC), mientras los ANA-IgM son de menor relevancia clínica en estos pacientes. Sin embargo, poco se sabe de los ANA-IgA ya que estos Ac han sido menos investigados. El objetivo de este trabajo fue estudiar la prevalencia de los diferentes isotipos de inmunoglobulinas de anticuerpos antinucleares en los pacientes con ETC y evaluar la conveniencia de utilizar conjugados monovalentes o polivalentes. Se procesaron 100 sueros de pacientes con diversas ETC empleando IFI-HEp2, en los cuales se detectó 38% de ANA-IgA (títulos ≥ 1:80) y 12% de ANA-IgM (títulos ≤ 1:160). En 29 casos se detectó IgA en ausencia de IgM, en 3 casos IgM en ausencia de IgA. En todos los casos los ANA-IgG estuvieron presentes. En 6 sueros se observó un cambio de imagen con conjugado anti-IgA y en 3 con conjugado anti-IgM. Debido a la alta prevalencia de ANA-IgA detectada por IFI-HEp2, se destaca la conveniencia de utilizar conjugado anti-Ig totales en lugar de anti-IgG, mientras se desconozca la relevancia de los ANA-IgA en el diagnóstico, pronóstico y seguimiento de las enfermedades reumáticas sistémicas.

The indirect immunofluorescence with epitelial cell line from human laryngeal carcinoma as substrate (IIF-HEp2) and anti-IgG or anti-total Ig as antisera, is the technique currently used for the detection of antinuclear antibodies. The most important antibodies for the diagnosis and follow-up of connective tissue diseases (CTD) are the IgG-ANA, while the IgM-ANA have no clinical relevance. However the IgA-ANA have not been thoroughly investigated so far. The aim of this work was to study the prevalence of different ANA isotypes of Ig antibodies in CTD patients and to evaluate the convenience of the use of monovalent or polyvalent conjugate. We examined the sera of 100 patients with different CTD by IIF-HEp2 and detected a prevalence of 38% IgA-ANA (titles ≥ 1:80) and 12% IgM-ANA (titles ≤ 1:160). In twenty nine cases we detected IgA-ANA in absence of IgM-ANA, and in 3 cases IgM-ANA in absence of IgA-ANA. In all the cases IgG-ANA were present. In 6 sera a change in the immunofluorescence pattern was observed while using anti-IgA conjugate, whereas in 3 the change was observed with the use of anti-IgM conjugate. Because of the high prevalence of ANA-IgA detected by IIF-HEp2, we emphasize the convenience of employing anti-total Ig in spite of anti-IgG conjugated until the role of ANA-IgA is dilucidated in CTD patients, in order to establish its relevance in the diagnosis, prognosis and follow-up of systemic rheumatic diseases.

Immunoactivation and immunopathogeny during active visceral leishmaniasis / Imunoativação e imunopatogenia durante leishmaniose visceral ativa

Visceral leishmaniasis is caused by protozoan parasites of the Leishmania donovani complex. During active disease in humans, high levels of IFN-γ and TNF-α detected in blood serum, and high expression of IFN-γ mRNA in samples of the lymphoid organs suggest that the immune system is highly activated. However, studies using peripheral blood mononuclear cells have found immunosuppression specific to Leishmania antigens; this poor immune response probably results from Leishmania antigen-engaged lymphocytes being trapped in the lymphoid organs. To allow the parasites to multiply, deactivating cytokines IL-10 and TGF-β may be acting on macrophages as well as anti-Leishmania antibodies that opsonize amastigotes and induce IL-10 production in macrophages. These high activation and deactivation processes are likely to occur mainly in the spleen and liver and can be confirmed through the examination of organ samples. However, an analysis of sequential data from studies of visceral leishmaniasis in hamsters suggests that factors outside of the immune system are responsible for the early inactivation of inducible nitric oxide synthase, which occurs before the expression of deactivating cytokines. In active visceral leishmaniasis, the immune system actively participates in non-lymphoid organ lesioning. While current views only consider immunocomplex deposition, macrophages, T cells, cytokines, and immunoglobulins by diverse mechanism also play important roles in the pathogenesis.

A leishmaniose visceral é causada por protozoários do gênero do complexo Leishmania donovani. Durante a doença ativa no homem são detectados altos níveis de IFN-γ e de TNF-α no soro, e elevada expressão de mRNA de IFN-γ em amostras de órgãos linfóides sugerindo um estado intensamente ativado do sistema imunológico. A visão atual, no entanto, refere-se à imunossupressão específica aos antígenos de Leishmania com base em estudos utilizando células mononucleares do sangue periférico; a explicação para sua resposta deficiente seria provavelmente porque os linfócitos compometidos com antígeno de Leishmania são sequestrados nos órgãos linfóides. Para permitir a proliferação do parasito, citocinas desativadoras IL-10 e TGF-β atuariam nos macrófagos, bem como os anticorpos anti-Leishmania opsonizando amastigotas e induzindo a produção IL-10 pelos macrófagos. Estes processos de intensa ativação e desativação provavelmente ocorreriam no baço e fígado, principalmente, e confirmados com amostras de órgãos. No entanto, analisando dados seqüenciais obtidos na leishmaniose visceral no hamster, sugere-se provável presença de fatores fora do sistema imunológico como responsável pela inativação inicial de sintase induzível do óxido nítrico que ocorre antes da expressão de citocinas desativadoras. Na leishmaniose visceral ativa o sistema imunológico participa ativamente na lesão de órgãos não linfóides. Contrária à visão existente que considera somente mecanismos de deposição de imunocomplexos, observa-se na patogenia a participação de macrófagos, células T, citocinas e imunoglobulinas por mecanismo alternativo.

Experimental lagochilascariosis in X-chromosome-linked immunodeficient mice / Lagochilascariose experimental em camundongos com imunodeficiência ligada ao cromossomo X

Lagochilascaris minor is the etiological agent of lagochilascariosis, a disease that affects the neck region and causes exudative abscesses, with eggs, adult parasites and L3/L4 larvae in the purulent exudates. Mice are now considered to be intermediate hosts for the parasite. To determine the pattern of infection in B1 cell-deficient mice, experimental lagochilascariosis was studied in BALB/c and X-chromosome-linked immunodeficient (xid) mice. BALB.xid-infected mice showed lower numbers of larvae. Third-stage larvae, fourth-stage larvae and adult parasites were found in both strains. BALB/c mice produced IgM, IgG, IgA and IgE against the crude extract and secreted/excreted antigens of the parasite. On the other hand, BALB.xid mice did not produce IgM and produced lower levels of IgG and IgA, and similar quantities of IgE.

Lagochilascaris minor é o agente etiológico da lagochilascariose, uma doença que afeta a região de pescoço provocando abscessos exudativos contendo ovos, parasitas adultos e larvas L3/L4 nos exudates purulentos. Atualmente, camundongos são considerados hospedeiros intermediários do parasita. Para determinar o padrão de infecção em camundongos deficientes de células B1, a lagochilascariose experimental foi estudada em camundongos BALB/c e em camundongos com imunodeficiência ligada ao cromossomo X (xid). Camundongos BALB.xid infectados mostraram menor número de larvas. Larvas L3, L4 e parasitas adultos foram encontrados em ambas as linhagens. Camundongos BALB/c produziram IgM, IgG, IgA e IgE contra o extrato bruto e antígenos secretados/excretados do parasita; por outro lado, camundongos BALB.xid não produziram IgM, produziram baixos níveis de IgG e IgA, e quantidades semelhantes de IgE.

sICAM-1 intrathecal synthesis and release during the acute phase in children suffering from Coxsackie A9 and S. pneumoniae meningoencephalitis / Sintesis intratecal de sICAM-1 y liberación durante la fase aguda en niños con meningoencefalitis por Coxsackie A9 y S pneumoniae

The intercellular adhesion molecule is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Serum and cerebrospinal fluid (CSF) soluble intercellular adhesion molecule 1 (sICAM-1) from normal control children as well as from children with Guillain-Barré syndrome (GBS), with Coxsackie A9 virus meningoencephalitis and with Streptococcus pneumoniae meningoencephalitis were studied. sICAM-1 was quantified using an immunoenzimatic assay and albumin using the immunodiffusion technique in both biological fluids. Increased sICAM-1 values in CSF in patients with GBS correspond to an increase of the albumin CSF/serum quotient. In contrast, in inflammatory diseases like S. pneumoniae and Coxsackie A9 virus meningoencephalitis an increased brain-derived fraction was observed. In particular cases these values are 60-65 percent and 70-75 percent respectively. The results indicate an additional synthesis of sICAM-1 in subarachnoidal space during central nervous system (CNS) inflammatory process. An important role of sICAM-1 in the transmigration of different cell types into CSF during CNS inflammation in children with S. pneumoniae and Coxsackie A9 meningoencephalitis may be suggested.

La molécula de adhesión intercelular es una glicoproteína que pertenece a la superfamilia de las inmunoglobulinas. Se estudiaron los niveles de molécula de adhesión intercelular tipo 1 soluble (sICAM-1) en suero y líquido cefalorraquídeo (LCR) de niños con meningoencefalitis por Streptococcus pneumoniae y por Coxsackie A9 al igual que en niños con sindrome de Guillain-Barré (SGB). sICAM-1 fue cuantificado por ensayo inmunoenzimático y la albúmina por inmunodifusión en ambos líquidos biológicos. Los valores incrementados de sICAM-1 en LCR en los pacientes con GBS corresponden a valores aumentados de razón LCR/suero de albúmina. En contraste, en las enfermedades inflamatorias como las meningoencefalitis por S. pneumoniae y por Coxsackie A9 se observa un incremento en la fracción derivada del cerebro. En casos particulares los valores se incrementan hasta un 60-65 por ciento y 70-75 por ciento respectivamente. Los resultados indican una síntesis adicional de sICAM-1 en el espacio subaracnoideo durante el proceso inflamatorio del sistema nervioso central (SNC). Esto puede sugerir un importante papel del sICAM-1 en la transmigración de diferentes tipos celulares en el LCR durante la inflamación del SNC en niños con meningoencefalitis por S pneumoniae y coxsackie A9.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate / Reatividade de anticorpos de soros humanos a antígenos de Paracoccidioides brasiliensis tratados com metaperiodato sódico

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

Neste trabalho, foi avaliado o perfil de isotipos de imunoglobulinas anti-Paracoccidioides brasiliensis em soros de pacientes com formas crônica e aguda de paracoccidiodomicoses usando antígeno total e tratado com meta-periodato. Todos os tipos de imunoglobulinas presentes nos soros de pacientes com formas aguda e crônica apresentaram alta reatividade ao antígeno total quando comparado ao tratado com meta-periodato (P < 0,05). Houve maior reatividade de IgG e IgM anti-antígeno tratado com meta-periodato em soros de pacientes com forma aguda da doença (P < 0,05), enquanto IgA foi mais reativa em soros da forma crônica (P < 0,05). Houve maior reatividade de IgG1 e IgG2 com antígeno total e tratado com meta-periodato em soros de pacientes comparados aos com outras parasitoses (P < 0,05). Além disso, IgG1 de pacientes com a forma aguda reconhecem antígenos de 19kDa, 27kDa e 31kDa por western blot. Assim, os resultados sugerem que alterações nos epitopos de antígenos de Paracoccidioides brasiliensis podem auxiliar no aprimoramento do imunodiagnóstico da paracoccidioidomicose.

Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and ß2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0 percent) hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95 percentCI: 21.5-25.4 MPL), only three carriers (<3 percent) had IgG anticardiolipin antibodies (median, 23 GPL; 95 percentCI: 20.5-25.5 GPL). Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004). IgA anti-ß2-glycoprotein-I antibodies were detected in 29 of 109 (27.0 percent) hepatitis C carriers (median, 41 SAU; 95 percentCI: 52.7-103.9 SAU). Twenty patients (18.0 percent) had IgM anti-ß2-glycoprotein I antibodies (median, 27.6 SMU; 95 percentCI: 23.3-70.3 SMU), while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU). Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-ß2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.