Prevalencia del antígeno D débil y su clasificación fenotípica en donantes voluntarios de sangre / Prevalence of "weak" D antigen and its phenotypic classification in voluntary blood donours / Prevalência do antígeno D "fraco" e sua classificação fenotípica em doadores voluntários de sangue

En el campo de la medicina transfusional la correcta identificación de los fenotipos del sistema Rh y en especial del antígeno D debe ser de manera inequívoca por su relevancia clínica. El antígeno D tiene variantes denominadas D parcial, D débil y DEL, las que se producen por mutaciones de los alelos RHD/RHCE o por una supresión en la expresión fenotípica. Se trató de un estudio descriptivo, retrospectivo de corte transversal en el que se realizó una revisión de registros primarios durante el período 2011-2014 validados de acuerdo con el protocolo de Hernández-Sampieri R. Se utilizó estadística descriptiva mediante la aplicación del software informático SPSS versión 22.0 y se estableció la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado. Se determinó una prevalencia de donantes RhD negativos de 1,8 a 2,5% y RhD débil de 1,79 a 2,28%. La fenotipificación serológica permitió identificar que los tipos 2 y 5 eran los más frecuentes. También se estableció la existencia de aloinmunización por anti-D, anti-C y anti-E. Se estableció de esta manera la existencia de D débil y una importante aloinmunización en la población de donantes de sangre tipificados como D negativo y D débil, por lo que se recomienda implementar un algoritmo de identificación del antígeno D en servicios de medicina transfusional.

In the field of transfusion medicine, the correct identification of the phenotypes of the Rh system and especially of the D antigen must be unequivocal for clinical relevance. The D antigen has variants called partial D, weak D and DEL. These are produced by mutations of the RHD/RHCE alleles or a suppression in phenotypic expression. The objective of this study was to establish the frequency of weak D antigen in the population of blood donours from 17 Ecuadorian states and their phenotypic combinations. It was a descriptive, retrospective cross-sectional study performed during the 2011-2014 period and validated with primary records in accordance with the Hernández-Sampieri R protocol. A descriptive statistics through the application of SPSS computer software version 22.0 was used and the relationship between independent variables through the Chi-square statistic method was established. A prevalence of RhD negative donours from 1.8 to 2.5% and weak D 1.79 to 2.28% was observed The serological phenotyping made it possible to identify that type 2 and 5 were the most frequent. The presence of alloimmunization by anti-D, anti-C and anti-E was also established. Besides, the presence of weak D types and significant alloimmunization in the donour population of blood typed as D negative and weak was established, so it is recommended to implement an algorithm for the identification of D antigen in transfusional medicine services.

No campo da medicina transfusional, a correta identificação dos fenótipos do sistema Rh e especialmente do antígeno D deve ser inequívoca devido a sua relevância clínica. O antígeno D tem variantes chamadas de D parcial, D fraca e DEL, as quais são produzidos por mutações dos alelos RHD/RHCE ou por uma supressão na expressão fenotípica. O objetivo deste estudo foi estabelecer a frequência do antígeno D fraco em uma população de doadores de sangue de 17 províncias equatorianas e suas combinações fenotípicas. Foi uma estudo descritivo, retrospectivo de corte transversal em que se realizou uma revisão dos registros primários validados de acordo com o Protocolo Hernández-Sampieri R durante o período 2011-2014. Utilizou-se estatísticas descritivas através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes através da análise estatística de qui-quadrado. Foi determinada uma prevalência de doadores RhD negativos de 1,8 a 2,5% e RhD fraco de 1,79 a 2,28%. A genotipagem serológica permitiu identificar que os tipos 2 e 5 são os mais frequente. A existência de alo imunização por anti-D, anti-C e anti-E também foi estabelecida. A existência de D fraco e uma alo imunização significativa na população de doadores de sangue tipificados como D negativo e fraco, por isso é recomendado implementar um algoritmo de identificação do antígeno D em serviços de medicina transfusional.

Inmunogenicidad de los concentrados de Factor VIII doblemente inactivados producidos en UNC-Hemoderivados / Immunogenecity of double inactivation FVIII concentrates manufactures by UNC-Hemoderivados

El tratamiento de elección en la hemofilia A es la administración de concentrados de factor VIII y hasta un 33 % de estos pacientes pueden desarrollar inhibidores dirigidos contra el factor infundido. Varias causas pueden estar involucradas en este proceso inmune siendo la intensidad de la terapia y tipo de concentrados empleados uno de los más estudiados. Por lo tanto, el análisis cuali / cuantitativo de diferentes proteínas que hacen a un concentrado más inmunogénico que otros cobra vital importancia. En este trabajo se evaluaron los niveles de factor VIII antígeno (FVIII:Ag), factor von Willebrand (FvW) funcional e inmunológico, factor de crecimiento transformador ß1 (TGF-ß1), fibrinógeno e inmunoglobulinas G, A y M en diferentes lotes de concentrados de factor VIII manufacturados en UNC-Hemoderivados y fueron comparados con otros productos comerciales de similares características. Se estudiaron 6 lotes de 250 UI y 2 de 500 UI producidos en UNC-Hemoderivados, dos lotes de 250 UI producidos por las firmas A y B y un lote de 500 UI comercializado por la firma C. Para las determinaciones de factor VIII/factor de von Willebrand, funcional y antigénico, se emplearon métodos estándares y para el TGF-ß1 se utilizó un ELlSA comercial. Los resultados obtenidos mostraron que las relaciones entre FVIII:C/FVIII:Ag y FVIII:C/FvW funcional fueron en promedio 0.60 y 0.52 para el producto de UNC-Hemoderivados y 0.50 y 0.38 para los productos de origen comercial. Los valores TGF­ß1 y la actividad específica arrojaron mejores resultados en el producto de UNC-Hemoderivados que los productos A, B y C y en las otras proteínas analizadas no hubo diferencias. Por lo tanto podemos concluir que los concentrados de FVIII producidos en UNC-­Hemoderivados presentan propiedades que potencialmente indicarían una menor respuesta inmune contra el FVIII infundido.

Replacement therapy using plasma factor VIII (FVIII) concentrates is currently the major mean of preventing and controlling bleeding in hemophilia A patients. However. approximately a 33% of these patients may develop inhibitors to the substituted FVIII. Several causes may be involved in the immune process being the intensity of therapy and type of concentrate used one of the most studied. Therefore. the study quali/quantitative analysis of different proteins which make a concentrated more immunogenic than other takes a vital importance. In this study we evaluated FVIII antigen (FVIII: Ag), von Willebrand factor (vWF) functional and immunological, TGF-Beta1, fibrinogen and immunoglobulins G, A and M levels in FVIII concentrates manufactured in UNC-Hemoderivados and compared with other commercial products of similar characteristics. We studied 6 lots of 250 IU and 2 lots of 500 IU manufactured in UNC-Hemoderivados, 2 lots of 250 IU produced by two pharmaceutical companies named A and B and 1 batch of 500 IU marketed by C. For the determinations of F VIII/FvW functional and antigenic we used standards methods and the TGF-ß1 was assayed by ELlSA test. The results show that the relationship between FVIII/FVIII:Ag and FVIII/vWF functional were in average 0.6 and 0.52 for the product of UNC-Hemoderivados and 0.5 and 0.38 for products from commercial sources. TGF-ß1 levels and the specific activity showed upper values in UNC-Hemoderivados compared to commercial products and none difference was observed in the other proteins assayed. Therefore we can conclude that FVIII concentrates produced at UNC-Hemoderivados have properties that indicate a potentially lower immune response against the infused FVIII.

Evaluación de neoantígenos en concentrados de factor VIII analizados por enzimoinmunoensayo / Neoantigens evaluation on F VIII concentrates analyzed by enzymeimmunoassay (EIA)

El propósito de este estudio fue evaluar la inmunogenicidad de concentrados de factor VIII producidos en UNC-Hemoderivados antes y después del tratamiento térmico empleando un enzimoinmunoensayo (EIE) desarrollado en nuestro laboratorio. Materiales y método: Para ese propósito se obtuvieron anticuerpos contra factor VIII calentado y no-calentado por inmunización de conejos y la inmunoglobulina G específica fue aislada por afinidad a Proteína A-Sepharosa. El EIE fue realizado empleando como antígeno de captura el factor VIII con o sin tratamiento térmico (0.5 ug/pocillo), los sitios inespecíficos bloqueados con albúmina al 2 por ciento y el anticuerpo revelado con un anti-IgG de conejo conjugada a peroxidasa. La presencia de neoantígenos fue estudiada por incubar durante 2 hs a 37°C y luego a 4°C toda la noche, con cantidades crecientes de factor VIII antes y después del tratamiento térmico (0.03 a 600 ug de proteínas), con cantidades adecuadas de IgG anti-factor VIII calentado y no-calentado. Luego de centrifugar la muestra para la separación de los inmunocomplejos, se valoró la presencia de anticuerpos en el sobrenadante por EIE tanto para factor VIII calentado y no calentado. Resultados: Los resultados obtenidos permitieron observar que para ambos anticuerpos (calentado y no calentado) se neutralizaba la misma cantidad de factor VIII calentado y no calentado (0.30 ug) y además, se pudo comprobar que esta conducta se repetía cuando se empleaban en el EIE como antígeno de captura, factor VIII con y sin tratamiento térmico. Conclusiones: Los datos obtenidos de este estudio nos permiten concluir que el tratamiento térmico aplicado a los concentrados de factor VIII elaborados en la UNC-Hemoderivados no producen ninguna formación de neoantígenos. A juzgar por el sensible y específico EIE desarrollado en nuestro laboratorio.

Introduction: with the purpose of improving viral security, plasma-derived products are generally subjects of solvent/detergent and heating (100°C for 30 minutes) treatment which are introduced during the manufacturing process. The formation of neoantigens in factor VIII concentrates produced after the heat treatment could trigger an immune response against the modified protein and the native protein. Objetives: the purpose of this study was to evaluate the immunogenicity of factor VIII concentrates produced in UNC-Hemoderivados before and after heat treatment, using an EIA developed in our laboratory. Materials and methods: antibodies against factor VIII with and without heating treatment were obtained by rabbit immunization. The specific IgG was isolated by protein-A-Sepharose affinity chromatograpy. Two EIA plates were coated (0.5 ug/well) one with factor VIII heated (H) and the other one with factor VIII no-heat (no-H) and the antibodies detection were performed using rabbit anti-lgG peroxidase conjugated. The neoantigens were studied by incubation of increasing concentrations of factor VIII (0.03-600 ug of proteins) before and after heating treatment during 2 hours at 37 °C and overnight at 4 °C with adequate concentrations of anti-factor VIII IgG (with and without treatment). The immunocomplexes were removed by centrifugation and the free antibodies in the supernatant were measured by EIA in plates H and no-H. Results: the EIA showed a linear behavior between antibodies dilution ranged from 1/8000 to 1/512000. With these results we can conclude that the same concentration of factor VIII heated and unheated (0.30 ug) were neutralized with either antibodies (heated and unheated). These results were similar in both EIA plates coated with factor VIII H and no-H...

O desfecho perinatal da aloimunização eritrocitária não-relacionada ao antígeno Rhd / The outcome of perinatsl erythrocyte alloimmunization unrelated to the RhD antigen

Analisar o desfecho perinatal da aloimunização eritrocitária não-relacionada ao antígeno RhD. De forma complementar, confrontar os resultados perinatais com os da aloimunização RhD e com aqueles de gestantes Rh-não-sensibilizadas...

Effect of Schistosoma mansoni infection on physiological gastrointestinal transit and contractility

Schistosoma mansoni [S. mansoni] is a major health problem; a large proportion of infected individuals suffer from motility-related gastrointestinal disturbances. However, the exact relationship between the gastrointestinal motility changes, the enteric nervous system neuronal excitability, and the complexity of the resulting symptoms, in both the acute and chronic phases of inflammation are still not clear. The work was designed to investigate the effect of two different intensities of S. mansoni infection on gastrointestinal transit and contractility of the colonic muscles, in both acute and chronic phases of inflammation of experimentally infected mice; and to asses the relation between gastrointestinal transit, contractility, and serum antigen and antibody levels. The study was conducted on 60 mice divided into 2 groups a control group consisting of 12 mice, and S. mansoni infected group consisting of 48 mice. The infected group was further subdivided into two subgroups, each infected by a different intensity of cercaria [50 and 200 cercaria/mouse]. Gatrointesintal transit and contractility were recorded from the colon of each group and were analyzed at the acute [8[th] week] and chronic phase [12[th] week] of inflammation. In addition, the immunological changes of the host were assessed in both infected subgroups of mice, at the same studied durations. At 8weeks postinfection, in both infected subgroups, gastrointestinal transit was significantly decreased, in concurrent with significant increase in the colonic muscle contractility, compared to the control group. At that time, the serum antigen was absent, while the serum antibody was detectable at low titre. However, at twelve weeks postinfection, there was a further statistically significant decrease in gastrointestinal transit, and increase in the colonic muscle contractility. These alterations were coinciding with absence of serum antigen and increase in the antibody titre. All changes were more pronounced in the second infected subgroup [200 cercaria/mouse] than the first infected one [50 cercaria/mouse], Indicating that the increased host immunity at the 12[th] week postinfection did not improve the motility disorder of the colon. We conclude that intestinal schistosomiasis is associated with disturbances in gastrointestinal transit and contractility, which are related to the time course and the intensity of the infection, and may be involved in the pathogenesis of the motility disorder associated with the disease. The hypercontractility in the acute phase can be explained by functional changes in the excitability of enteric nervous system neurons due to the release of mediators, and in the chronic phase due to granuloma formation leading to structural changes in the enteric nervous system. Elucidation of the mechanism whereby inflammation alters enteric nervous control of gastrointestinal function may lead to novel treatments of motility disorders of the disease

Hereditary angioedema: a family study.

Hereditary angioedema (HAE) is a rare, life-threatening, autosomal dominant disease characterized by recurrent episodes of angioedema, and caused by a deficiency of the plasma protein C1-esterase inhibitor (C1-INH). Clinical manifestation of HAE may first develop during childhood but typically presents around puberty with nonpruritic and non-pitting edema of the subcutaneous and mucosal tissues. Up to now, there has been no published report of HAE case in Taiwan. We reported a 33 year-old female patient who had recurrent painful swelling of face and hands since 27 years of age. She first suffered from sudden onset of painful swelling of the eyelids and lips in August 1998 when she was pregnant for the first time. Subsequently, similar episodes recurred for a few times. Her blood test disclosed that her C3 and C4 were 125 mg/dl and 6 mg/dl, respectively. Her uncle died of laryngeal edema at the age of 30 years. Her father and elder brother also had the similar history of recurrent facial and hand swelling. The C4 levels of her elder brother were 6 mg/dl and 13.3 mg/dl on two separate occasions. The C1-INH antigen serum level and functional assay of the index patient and ten other family members were studied. A total of seven members of the family were confirmed to have type 1 HAE as evidenced by the low C4 and low C1-INH antigenic level and functional activity. Two of the seven cases were asymptomatic up to the date of our report.

Von Willebrand factor (vWF) antigen levels and function in healthy Thais.

Type I von Willebrand disease (vWD) is very common in caucasians. Its genetic basis is possibly heterogenous, lying both within and out of the vWF gene locus. We sought to investigate vWF levels in the Thai population, to compare with those of western countries. The vWF antigen and activity were measured using ELISA and Collagen Binding Assay (CBA), respectively, in 311 healthy Thai volunteers. The mean age was 32.3, ranging from 18 to 75 years. Fifty-four percent were female. Low vWF antigen and activity (below 50 U/dl) were found in 3.5% and 10.2%, respectively. Around 75% and 20% of these cases had O and A blood groups, respectively. Three (0.96%) had definitely low levels of vWF (vWF antigen level below 35 U/dl), suggesting the diagnosis of vWD. Similar to previous studies, vWF levels were lowest in subjects with group O blood. We found that subjects with blood group A had higher vWF levels than group O subjects, but significantly lower vWF levels than those with group B. The average ratio between the vWF activity and antigen was 0.96, ranging from 0.66 to 1.66. These ratios were inversely correlated with age (p=0.047), suggesting a decline in vWF activity per vWF protein with advancing age. Low levels of vWF are common in healthy Thais. Clinicians should be aware of vWD in bleeding patients and beware low levels of vWF in therapeutic plasma products, especially from blood groups O and A.

Utilidad de la detección de células citomegálicas circulantes en pacientes inmunosuprimidos VIH negativos en el control de la infección por citomegalovirus / Presence of circulating cytomegalic cells in HIV negative immunosuppressed individuals following cytomegalovirus infection

Objetivo. Buscar la correlación entre la presencia de células citomegálicas circulantes con la antigenemia pp65 para citomegalovirus y el desarrollo de complicaciones clínicas inherentes a infección y enfermedad por citomegalovirus. Materiales y métodos. Entre diciembre de 2002 y julio de 2003 se procesaron 110 muestras de sangre periférica, obtenidas de 46 pacientes inmunosuprimidos. La antigenemia pp65 y la presencia de células citomegálicas circulantes se determinaron mediante inmuno-fluorescencia indirecta utilizando un estuche comercial para la detección del antígeno pp65 de citomegalovirus. En leucocitos de sangre periférica, la antigenemia fue positiva cuando se encontró una o más células con tinción fluorescente, multilobulada y homogénea en el núcleo celular. Se determinó la presencia de células citomegálicas circulantes cuando se observaron células de gran tamaño (35 a 50 mm) con un patrón de tinción fluorescente extendido en todo el citoplasma celular en células mononucleares de sangre periférica. Resultados. Se encontraron ocho pruebas con antigenemia positiva, procedentes de siete pacientes (15 por ciento). De éstos, cuatro (57 por ciento) también presentaron células citomegálicas circulantes, tres habían recibido un trasplante renal y uno un trasplante hepático. El número de células positivas en la antigenemia fue mayor en los pacientes con trasplante renal que en el resto de pacientes inmunosuprimidos (457 vs.1,96; p<0,005). No se encontró asociación entre la presencia de células citomegálicas circulantes, la morbilidad, la mortalidad y el desarrollo de enfermedad injerto contra huésped (GVHD) (p<0,001). Discusión. No encontramos correlación entre la detección de células citomegálicas circulantes, la antigenemia y la mortalidad en estos pacientes. Sin embargo, es necesario realizar estudios prospectivos con un mayor número de individuos para determinar mejor dicha correlación y definir su utilidad clínica en pacientes inmunosuprimidos

Comparison of prevalence of anti-CMV antibodies [IgM and IgG] and CMV Ag in renal transplant recipients

Human cytomegalovirus [HCMV] is a DNA virus, approximately 200nm in diameter, belonging to the herpes virus family. CMV infection in immunocompromised patients including organ transplantation recipients, patients with AIDS patients under immunosuppressive therapy, and in developing fetus may result in either localized or disseminated diseases. Patients are at both primary CMV infection and reactivation of latent infection CMV can be transmitted through blood transfusion and organ transplantation. In this descriptive study, 62 recipients of kidney transplant [26 females, 41.9% and 36 male, 58.1%] ranging from 2-58 years of age [mean 34 +/- 15] were analysed to detect CMV antibodies by ELISA technique; CMV antigen was also evaluated by Indirect Immunofluorescence Technique. All patients were CMV IgG positive, 10 [16.1%] were CMV IgM positive and 7 [11.4%] were at borderline. 23 [37.1%] of recipients were CMV Ag positive. Statistical analysis showed no relation between CMV Ab and CMV Ag. In spite of the presence of anti-CMV IgG and IgM antigenemia appears in several patients. There is not a strong correlation between antibodies against cytomegalovirus and the detection of CMV antigen in patients with acute infections

Evaluation of capillaria philippinensis coproantigen in the diagnosis of infection

In order to overcome the false negative diagnosis of infection with C. philippinensis in the absence of eggs in stool, coproantigen prepared from the stools of infected patients was evaluated serologically. This antigen was able to detect anti-Capillaria antibodies in the sera of infected cases at the same OD level produced with Capillaria crude worm antigen using indirect ELISA technique. C. Philippinensis coproantigen did not cross-react with the sera from patients with Schistosomiasis mansoni, Fascioliasis or Strongyloidiasis at 1:00 serum dilution. Laboratory-prepared hyperimmune sera versus crude worm antigen of C. philippinensis succeeded in capturing Capillaria antigen prepared from the stools of infected patients and did not cross-react with the coproantigens prepared from the stool samples of cases infected with S. Mansoni or Fasciola using sandwich ELISA technique

Identification of immunoreactive proteins of Trichinella spiralis aduit and adut excretory/secretory [E/S] antigens in sera of human and animals

In this study, blood samples were collected from 16 symptomatic patients at hospital and 12 asymptomatic individuals working at the swine slaughter house. Blood samples were also obtained from 75 pigs at two abattoirs and experimentally infected rats [day 25 PI]. There was no great difference in the seroprevalence of trichinellosis between symptomatic [56%] and asymptomatic [50%] individuals. ELISA results recorded that 13% of swine was seropositive, while 44% were suspected to be infected with T. spiralis. Immunoblotting profiles of T. spiralis adult antigen against human, swine and rat sera showed common reactive bands at 95.00 and 64.466 kDa [human and swine] and 35.554 kDa [human and rat]; while, the blotting patterns of adult E/S products against human, swine and rat antibodies recognized two trichinellosis-specific determinants between human and swine [87.619 and 74.136 kDa] as well as between human and rat [98.00 and 16.535 kDa]

von Willebrand factor antigen levels in plasma of patients with malignant breast disease

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 + or - 78 U/dl in patients with cancer, 148.4 + or - 59 U/dl in patients with benign disease and 130.6 + or - 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 + or - 113 U/dl, stage IIIB = 194.0 + or - 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 + or - 65 U/dl, stage IIA = 146.9 + or - 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression

Nuevas estrategias para el análisis genético y molecular del sistema Rh / New strategies for molecular and genetic analysis of Rh system

El locus RH está compuesto, en cada cromosoma de individuos RhD positivo, por dos genes estructurales, adyacentes y homólogos, denominados RHCE y RHD, que codifican las proteínas RhCcEe y RhD respectivamente, mientras que en individuos RhD negativo se encuentra únicamente el gen RHCE. La determinación de las bases moleculares asociadas con los antígenos y fenotipos de este sistema permite investigar el gran polimorfismo del locus RH. El objetivo de este trabajo es estudiar nuevas estrategias para el análisis genético y molecular del sistema Rh. Investigamos la organización genética general del locus RH en individuos pertenecientes a la población de Rosario con distintos fenotipos Rh. En muestras de ADN genómico de dadores voluntarios analizamos dos regiones diferentes del gen RHD mediante el diseño de una estrategia de PCR multiplex basado en el polimorfismo de longitud del intrón 4 y en la presencia de una secuencia específica en la región 3' no codificante del gen RHD. Los resultados obtenidos con esta estrategia molecular presentaron una estricta correlación con los hallados por métodos serológicos. Los estudios realizados en la población analizada en este trabajo indicaron que todos los individuos RhD positivo poseen los dos genes RH, mientras que las personas RhD negativo tendrían solamente el gen RHCE. Se estudiaron 15 muestras RhD positivo débil, observándose siempre el patrón de bandas característico de las muestras RhD positivo. Estos resultados permitieron descartar fenotipos RhD negativo en las muestras con aglutinación muy débil y la presencia, en estos individuos, de genes híbridos responsables del fenotipo D.

Padronizaçäo da técnica antigenemia para a detecçäo do citomegalovirus / Padronization of antigenemia metodology for detection of citomegalovirus

Antigenemia para citomegalovirus (CMV) é um importante marcador de evoluçäo de doença e eficácia de tratamento em pacientes imunocomprometidos. O objetivo desse estudo foi comparar diferentes técnicas de processamentos e de imunomarcaçäo para a detecçäo da proteína da matrix do CMV pp65 em leucócitos do sangue periférico. Amostras de sangue coletadas de pacientes submetidos ao transplante de medula óssea (TMO) foram processadas e imunomarcadas por diferentes metodologias. Separou-se leucócitos de sangue periférico, utilizando-se duas técnicas, sedimentaçäo espontânea a 37§C (Processamento 1) e a sedimentaçäo com Dextran (Processamento 2), após a lise eritrocitária procedeu-se a contagem dos leucócitos, ajuste da densidade celular e o preparo das lâminas que continham (2x10 a quinta potência) células, por citocentrifugaçäo. As lâminas, obtidas através das diferentes técnicas de processamentos, foram coradas, utilizando-se a metodologia de Imunoperoxidase (IP) e os resultados obtidos foram analisados de acordo com parâmetros qualitativos e quantitativos. Também avaliou-se duas diferentes técnicas de imunomarcaçäo: IFI Imunofluorescência Indireta) e IP onde comparou-se o número de células positivas. Obteve-se lâminas de melhor qualidade pelo processamento 1 e um maior número de célu;as positivas com técnica de IP

Contribution in the serodiagnosis of visceral leishmaniasis in Syria by using the recombinant antigen[rK39]

The diagnosis of visceral leishmaniasis [VL] remains problematic for physicians in Syria, because the appearance of this disease is new and its clinical criteria are similar to Malaria, Tuberculosis, and Toxoplasmosis symptoms. For this reason, we used the specific antigen [rK39] of Leishmania donovani complex, in order to certify the appearance of visceral leishmaniasis in some patients. Then we followed the infected cases with VL during treatment [15 days] and after treatment [10 days] with Glucantime. We noted an improvement in clinical criteria and a significant decrease in the parasite numbers as well as in the specific L. donovani antibodies. These data demonstrate the utility of this specific antigen [rK39] in the serodiagnosis of VL. For this reason, we suggest the application of this antigen for sensitive and specific serodiagnosis in the endemic areas in Syria, in order to treat the infected patients early and rapidly

Padronização do teste ELISA baseado em antígeno capsular purificado dos sorotipos 3, 5 e 7 de Actinobacillus pleuropneumoniae / Standarization of ELISA test based on purified capsular antigen from serotypes 3, 5 and 7 of Actinobacillus pleuropneumoniae

Foram padronizados testes de ELISA (Enzymelinked immunosorbent assay) baseados em antígeno capsular purificado de Actinobacillus pleuropneumoniae sorotipos 3, 5 e 7, prevalentes no Brasil. Para a padronizaçäo foram utilizadas amostras de soro provenientes de leitöes inoculados com os três sorotipos do agente em estudo, dos quais se colheram amostras de sangue semanais, durante 15 semanas para estudo da dinâmica da síntese de anticorpos. O controle negativo dos testes constituiu-se de uma mistura de 130 soros de animais livres de Actinobacillus pleuropneumoniae (App). Os antígenos também foram testados com amostras de soro de animais infectados com outros agentes causadores de doenças respiratórias e vacinados contra rinite atrófica. Os antígenos produzidos foram eficientes na detecçäo de animais infectados com App, permitindo determinar densidades óticas superiores à média dos soros controles negativos acrescida de quatro desvios-padröes. Os testes de ELISA para os sorotipos 3, 5 e 7 apresentaram especificidade de 100 por cento e sensibilidade de 92, 88 e 90 por cento respectivamente. Näo ocorreram reaçöes cruzadas com outros sorotipos, assim como com soros de animais inoculados com ooutros agentes causadores de problemas respiratórios. Os resultados foram analisados através da análise discriminante de ANDERSON (1958), utilizando-se o programa Statistical Analysis System. Conclui-se que os antígenos testados säo adequados para sorotipar animais que tenham sido submetidos ao screening através de um teste de ELISA polivalente baseado em LPS-LC.

Diagnostico serologico de la enfermedad celiaca: anticuerpos anti-pepticos de sintesis de gliadina y anti-transglutaminasa de tejido / Serological diagnosis of disease: anti-gliadin peptide antitibodies and tissue anti-transglutaminase

Los marcadores serológicos comúnmente utilizados en el diagnóstico de la enfermedad celíaca son los anticuerpos antigliadina (AG) y antiendomisio (AE). Recientemente (1997) se identificó a la transglutaminasa de tejido (tTG) como el principal autoantígeno de los anticuerpos AE. El objetivo de este trabajo fue determinar la sensibilidad y especificidad de testes de ELISA desarrollados en base a la utilización de estructuras moleculares definidas como antígenos de captura para los anticuerpos AG y AE. Como antígenos inmovilizados para los anticuerpos AG se ensayaron tres péptidos de sínteses correspondientes a la región amino terminal de la alfa gliadina y para los AE, la transglutaminasa de hígado de cobayo. Se examinaron un total de 80 sueros correspondientes a: pacientes celíacos, no tratados y tratados, controles enfermos no celíacos y controles sanos. Rango de edad: 7 meses a 14 años. Se obtuvo una sensibilidad del 97 por ciento y una especificidad 86 por ciento para la IgG determinada utilizando como antígeno uno de los tres péptidos de síntesis (correspondiente a los residuos 31-55 de la alfa gliadina). Este péptido aparece como un antígeno altamente sensible y más específico que la gliadina. El mejor resultado, con un 100 por ciento de especificidad y sensibilidad, se obtuvo en la determinación de la IgA anti-tTG, lo que destaca la relevancia de estos anticuerpos como marcadores serológicos de la enfermedad celíaca.