Glibenclamide-pregnenolone derivative has greater hypoglycemic effects and biodistribution than glibenclamide-OH in alloxan-rats.

AIM: The present study was designed to investigate the activity of two glibenclamide derivatives on glucose concentration. An additional aim was to identify the biodistribution of glibenclamide derivatives in different organs in a diabetic animal model. METHODS: The effects of two glibenclamide derivatives on glucose concentration were evaluated in a diabetic animal model. In addition, glibenclamide derivatives were bound to Tc-99m using radioimmunoassay methods. To evaluate the pharmacokinetics of the glibenclamide derivatives over time (15, 30, 45 and 60 min) the Tc-99m-glibenclamide conjugates were used. RESULTS: The results showed that glibenclamide-pregnenolone had greater hypoglycemic activity than glibenclamide or glibenclamide-OH. The data also showed that the biodistribution of Tc-99m-glibenclamide-OH in all organs was less than that of the Tc-99m-glibenclamide-pregnenolone derivative. CONCLUSIONS: The glibenclamide-pregnenolone derivative had greater hypoglycemic effects and its biodistribution was wider than glibenclamide-OH. The data suggest that the steroid nucleus may be important to the hypoglycemic activity of the glibenclamide-pregnenolone derivative and this could be related to the degree of lipophilicity induced by the steroid nucleus in the chemical structure of glibenclamide-pregnenolone.

An inexpensive antigen for serodiagnosis of Chagas' disease.

UNLABELLED: In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas' disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas' patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a "gold standard" test for Chagas, we tested 566 sera from an endemic area. RESULTS: sensitivity and specificity were 100%; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa = 0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas' disease. Further validations of this antigenic formulation in other countries are necessary.

An inexpensive antigen for serodiagnosis of Chagas’ Disease

In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas’ disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas’ patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a “gold standard” test for Chagas, we tested 566 sera from an endemic area. Results: sensitivity and specificity were 100 percent; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa=0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas’ disease. Further validations of this antigenic formulation in other countries are necessary.

Este estudio fue realizado para evaluar las características de sensibilidad y especificidad de una formulación antigénica (AgA), producida a bajo costo, para detectar anticuerpos dirigidos a Trypanosoma cruzi, en muestras de suero de pacientes con enfermedad de Chagas. El AgA fue producido por el efecto combinado de estrés nutricional y autoclave de los parásitos. La especificidad y sensibilidad fueron evaluadas en dos estudios separados, con 152 sueros de individuos sanos y 175 de pacientes Chagásicos. La reactividad cruzada con 289 sueros de pacientes con diagnóstico parasitológico de enfermedades con anticuerpos que reaccionan con antígenos de T. cruzi. Estos sueros fueron evaluados con AgA-ELISA y con tres estuches comerciales. 566 muestras de suero provenientes de un área endémica, fueron empleadas para estudiar la concordancia entre nuestro diagnostico y una prueba designada por nosotros como patrón oro estándar. Resultados: la sensibilidad y especificidad fue de 100 por ciento. El AgA presento el más bajo porcentaje de reactividad cruzada, respecto a los estuches comerciales evaluados. La concordancia con la prueba patrón oro, en Venezuela, fue excelente (kappa=0,92). En conclusión, Aga-ELISA, presentó niveles de sensibilidad, especificidad y de reactividad cruzada, comparables o superiores a los obtenidos por los tres estuches comerciales mas empleados en el país, pero con costos de producción al menos 10 veces menor. Este balance conveniente, favorece su potencial uso para el despiste en los bancos de sangre de los países pobres y endémicos para la enfermedad de Chagas. Futuras validaciones de esta formulación en otros países es necesaria.

A simple method for the production of anti-C3d monoclonal antibody.

Production of monoclonal antibodies to C3d usually involves the purification of protein. Our method does not require C3 purification; it relies on attachment of C3b to mouse erythrocytes by activation of alternative pathways and further conversion in C3d. We prepared human complement-coated mouse red cells and sensitized mice of the same strain with our own schedule of immunization and applied the classical methods to obtain a mouse monoclonal antibody. We obtained a clone called BMS-11 which produces a monoclonal antibody of IgM class, to C3d with a title of 1:500000. The monoclonal antibody obtained has shown that it is suitable for use as an antiglobulin reagent.

[Production of monoclonal antibody with anti-B specificity, to be used as reagent for blood classification]. / Producción de anticuerpo monoclonal (AcMo) con especificidad anti B, para ser utilizado como reactivo de hemoclasificación.

The purpose of this research was to produce murine monoclonal antibodies with anti B specificity to be used as reagents in red cells typing. Balb/c mice were immunized with substance B from saliva of a donor secreting B antigen, in order to obtain spleen cells or splenocytes producing antibodies with anti B specificity. These cells were fused with mouse mycloma cells P3-X63.Ag8.653 by using the PEG technique. After seeded, the hybridomas secreting the putative antibodies were cloned through the limiting dilution technique to obtain monoclonality. Afterwards, the antibodies were evaluated using immunological and immunochemical methods. BMS-4 clone secreting antibodies IgM-kappa with anti B specificity were obtained, which displayed high potency, excellent avidity, great sensibility and long time stability. We think that this clone can be used to produce a red cell typing reagent.

Producción de anticuerpo monoclonal (AcMo) con especificidad anti B para ser utilizado como reactivo de hemoclasificación / Production of monoclonal antibodies with anti B specificity used as a blood typing reagent

El objetivo de este trabajo fue producir un AcMo murino con especificidad anti B, el cual pudiera ser utilizado como reactivo de hemoclasificación. Ratones bald/c fueron inmunizados con sustancia antigénica de saliva de donante secretor, con el fin de obtener linfocitos esplénicos o esplenocitos productores de anticuerpos con especificidad anti B. Estos fueron fusionados con células de mieloma de ratón P3-X63.Ag8.653 utilizando PEG. Después de sembradas, las células híbridas o hibridomas resultantes que tuvieron la capacidad de secretar el anticuerpo requerido, fueron sometidas a clonación por dilución límite para asegurar su monoclonalidad. Posteriormente los anticuerpos fueron sometidos a evaluaciones inmunohematológicas e inmunoquímicas. Se obtuvo la clona BMS-4 secretora de anticuerpos IgM Kappa, con especificidad anti B, los cuales demostraron tener una alta potencia, excelente avidez, gran sensibilidad y estabilidad en el tiempo. Dado el comportamiento demostrado por los anticuerpos en las evaluaciones realizadas, pensamos que esta clona puede ser utilizada para producir reactivo para la hemoclasificación

Molecular and serological evaluation of surface antigen negative hepatitis B virus infection in blood donors from Venezuela.

Surface antigen negative hepatitis B virus (HBV) infection was evaluated in Venezuela, by molecular characterization of blood samples positive for antibodies to core antigen (anti-HBc) and negative for surface antigen (HBsAg) in blood donors (residual infections). HBV DNA was found in 11/258 samples (4.3%), and was significantly associated with high levels of anti-HBc antibodies (>25 UI/ml, P < 0.05), while no correlation was found between the presence of HBV DNA and the levels of anti-HBs. Synonymous and non-synonymous mutations were found in the HBV surface region (but not vaccine escape mutants) and in the precore/core region (precore mutants in 2/7 samples and 33-45 bp deletions near the N-terminal core region in 4/19 samples). While HBV genotype F prevails among HBsAg positive samples from blood donors in Venezuela, residual infection isolates were mainly genotypes A and D. Phylogenetic analysis of viral surface and core region revealed discrepancies in genotype designation in 6/9 samples, suggesting the presence of mixed infection or recombination. In conclusion, HBV residual infection in Venezuela does not seem to be frequently observed in HBV genotype F. This type of infection is frequently associated with variants exhibiting mutations in the surface gene that might be affecting the correct recognition by commercial tests, with precore mutants and with core internal deletions. These variants do not seem to cause severe liver disease, and on the contrary, were found circulating at low viremia.

Clade analysis and surface antigen polymorphism of hepatitis B virus American genotypes.

Eight genotypes (A-H) of hepatitis B virus (HBV) have been described, HBV genotypes F and H being autochthonous to America. HBV genotype F has been classified in four clusters. The objective of this study was to gain insight into the molecular epidemiology of HBV American genotypes, as well as to analyze the genotype-related polymorphism in some functional domains of the surface proteins. The sequences of the S region of 106 isolates genotype F and H were analyzed, out of which 47 isolates genotype F circulated in different Venezuelan populations. Most of the Venezuelan isolates genotype F were grouped in cluster III (n = 39) and 7 in cluster II. One isolate obtained from a blood donor could not be classified in any clade and harbored amino acid substitutions characteristic of a vaccine escape mutant (G145R) and a stop codon in the surface antigen. Amino acid analysis of the PreS and S gene products showed unique genetic characteristics in genotype F and H sequences in some important domains involved in the early steps of infection. Out of 30 available sequences, two complete genome sequences of HBV genotype F from Venezuela were obtained. Phylogenetic analysis of these complete genomes confirmed the presence of four clusters inside genotype F, differing in more than 4% nucleotide divergence. Our extended analysis showed that genotype F clades Ia, III, and IV exhibit a restricted geographic distribution (Central America, the North and the South of South America, respectively) while clades Ib and II are found in all the Americas except in the Northern South America and North America respectively.

[Seropositivity for human T-lymphotropic virus types I and II among donors at the Municipal Blood Bank of Caracas and associated risk factors]. / Seropositividad al virus linfotrópico de células T humanas tipos I y II en donantes del Banco Municipal de Sangre de Caracas y factores de riesgo asociados,

OBJECTIVE: To conduct research at the Municipal Blood Bank of Caracas (MBBC) and find out the proportion of blood units discarded for being seropositive for human T-lymphotropic virus (HTLV) types I and II, the prevalence of that infection among their donors, and the probable risk factors for that infection among those HTLV-positive donors. METHODS: ELISA serological testing was done with 23 413 donors seen at the MBBC between July 2000 and April 2001. Samples that were repeat reactive (RR) with the ELISA underwent supplementary Western blot (WB) testing. Donors who had a positive or indeterminate WB result were scheduled for counseling in order to carry out confirmatory testing using nucleic acid amplification (NAA), to collect data on their risk background, and to advise them concerning their HTLV status. RESULTS: Of the 23 413 MBBC donors, 48 of them (0.2%) had a donation that was RR. Of those 48, 25 of them (52.1%) were positive on the WB (23 for HTLV-I and 2 for HTLV-II), 2 of them (4.1%) were indeterminate on the WB, 14 of them (29.2%) were negative, and 7 (14.6%) could not be evaluated. Of the 27 donors scheduled for counseling, 16 of them actually attended (14 WB-positive for HTLV-I, 1 WB-positive for HTLV-II, and 1 indeterminate). All 16 of them were positive with the confirmatory NAA testing. When these 16 seropositive donors were compared with a control group of seronegative donors, no significant differences were found with regard to age, sex, type of donation, number of previous donations, history of transfusions, and sexual behavior. However, significant differences were found in two areas: the seropositive donors were more likely to have used non-intravenous drugs (P < 0.05), and the seropositive donors were much more likely to have had an extended breast-feeding period (more than 2 years) as a child (P < 0.001). To assess the probability of mother-to-child transmission, six of the mothers of seropositive donors who had had an extended breast-feeding period were tested, and all six of those mothers were also found to be seropositive. With the 16 seropositive donors who were counseled, the spouse or partner of 13 of them was also tested; only 1 of those 13 was positive, but the oldest son of that couple was also HTLV-positive. CONCLUSIONS: Of the donated blood, 0.2% of the units were discarded for being positive for HTLV-I or HTLV-II, and the prevalence found among the donors was 0.11%. Sexual transmission between an HTLV-positive donor and a partner or spouse was less frequent than was mother-to-child transmission. At present in Venezuela, blood banks are not required to screen donations for HTLV. Given our results at the MBBC, we believe serious consideration should be given to implementing serological screening for HTLV I/II among blood donors throughout Venezuela.

Seropositividad al virus linfotrópico de células T humanas tipos I y II en donantes del Banco Municipal de Sangre de Caracas y factores de riesgo asociados / Seropositivity for human T-lymphotropic virus types I and II among donors at the Municipal Blood Bank of Caracas and associated risk factors

OBJETIVOS: Conocer la proporción de sangre descartada por seropositividad al virus linfotrópico de células T humanas (HTLV) tipos I y II, la prevalencia de dicha infección y los probables factores de riesgo en donantes del Banco Municipal de Sangre de Caracas (BMSC). MÉTODOS: Se evaluaron serológicamente mediante ensayos de inmunoadsorción enzimática (ELISA) 23 413 donantes atendidos entre julio del año 2000 y abril de 2001 en el BMSC. Las muestras repetidamente reactivas (RR) se estudiaron por inmunoblot de Western (WB), como prueba suplementaria. Los donantes positivos o indeterminados por WB fueron citados a la consejería para realizar la confirmación mediante la amplificación de ácidos nucleicos por reacción en cadena de la polimerasa (PCR), recoger datos sobre sus antecedentes de riesgo y asesorarlos acerca de su estado. RESULTADOS. El 0,2 por ciento de las donaciones resultaron RR; de ellas 52,1 por ciento resultaron positivas en el WB (23 a HTLV I y 2 a HTLV II); 4,1 por ciento indeterminadas por WB; 29,2 por ciento negativas; y el 14,6 por ciento no pudo ser evaluado. Asistieron a la consejería 16 donantes (14 WB positivos a HTLV I, 1 a HTLV II y 1 indeterminado). Todos resultaron positivos en la RCP. No se encontraron diferencias significativas con el grupo control en cuanto a edad, sexo, tipo de donación, número de donaciones previas, antecedentes de transfusiones y comportamiento sexual. Se observaron diferencias significativas según los antecedentes de consumo de drogas no intravenosas (P < 0,05), y altamente significativas (P < 0,001) según los antecedentes de lactancia materna larga. Las madres estudiadas de seis de los donantes positivos que manifestaron haber tenido una larga lactancia materna resultaron positivas, al igual que el hijo mayor de la única pareja positiva de las 13 evaluadas. CONCLUSIONES. Se descartó el 0,2 por ciento de la sangre por resultar positiva al HTLV I/II. La prevalencia entre los donantes fue de 0,11 por ciento. En el 37,5 por ciento de los casos se pudo determinar la probabilidad de transmisión de madre a hijo. La transmisión sexual resultó menos frecuente. Se debe considerar seriamente la implementación del tamizaje serológico del HTLV I/II en los donantes de sangre de Venezuela

High prevalence of GB virus C/hepatitis G virus genotype 3 among autochthonous Venezuelan populations.

GB virus C or hepatitis G virus (GBV-C/HGV) is highly prevalent among population groups at risk of parenterally transmitted viral agents, but it has also a worldwide distribution in other non-risk population groups. GBV-C/HGV RNA and antibodies against its envelope protein (anti-E2 Abs) were found in 3/86 (3%) and 7/89 (8%) of biomedical science personnel (BSP), in 31/453 (7%) and 37/200 (19%) of blood donors (BD), and in 6/64 (9%) and 26/59 (44%) of hemodialysis patients (HD) from Caracas, Venezuela. A significant gradient of GBV-C/HGV exposure (anti-E2 Abs and/or GBV-C/HGV RNA) was found between BSP (lowest prevalence), BD, and HD (P < 0.001). GBV-C/HGV RNA and anti-E2 Abs were also found in 2/69 (2.9%) and 2/44 (4.5%) of individuals from a rural community, in 9/162 (5.5%) and 2/40 (5%) of West Amerindians, and in 14/56 (25%) and 4/53 (7.5%) of South Amerindians. Socioeconomic and cultural factors may have contributed to the relatively high risk of exposure to GBV-C/HGV in BD and Amerindians. Whereas GBV-C/HGV genotypes 1 (n = 1), 2 (n = 6), and 3 (n = 22) were present in Venezuela, only the Asiatic genotype 3 was found infecting Amerindians and rural populations (n = 16). Genotype assignment based on the 5' noncoding region of the GBV- C/HGV genome was corroborated in some isolates by genetic analysis of the E2 region. This report confirms the circulation of the Asiatic genotype of GBV-C/HGV among Amerindians, suggesting an old origin of GBV-C/HGV. This might be associated with the apparently low pathogenesis of this virus.

Cómo reducir la prevalencia de donantes de sangre VIH positivos / How to decrease the prevalence of HIV positive blood donors

A pesar de haber abolido la entrega de los resultados serológicos a los donantes de sangre, como recurso para promover la donación de sangre y de haber mejorado el interrogatorio pre-donación, nuestra prevalencia de VIH en donantes se mantiene elevada. En el presente trabajo, hemos tratado de analizar las causas por las cuales fueron aceptados un grupo de donantes VIH positivos que acudieron a nuestra consejería desde enero de 1995 a diciembre de 1996. Para la evaluación se utilizó una encuesta en la que se recogieron datos de identificación, tipo y frecuencia de la donación, antecedentes de riesgo, conocimiento previo de su estado inmunoserológico, motivación hacia la donación, autoconsideración como persona de riesgo u conocimiento de las implicaciones de transfundir sangre contaminada. Para el tamizaje serológico se utilizaron los reactivos Abbott HIV/HIV2-3ra generación plus EIA, Abbott Diagnostic y HIV-1 Western Blot Cambridge Biotech, Worcester, MA. Durante los dos años de evaluación, se atendieron 53.338 donantes de los cuales 130 (0,24 por ciento) fueron confirmados positivos. Sólo 18/130 (13,84 por ciento) acudieron a la consejería. La edad promedio fue de 33.27 ñ 5,35 años, todos del sexo masculino, 15 solteros y 3 con pareja fija. 6/18 (33,33 por ciento) pudieron haber sido descartados por su apariencia y por su ocupación. La donación "voluntaria" fue mayor que en el grupo control (p=0,0001). La homo/bisexualidad y la promiscuidad (p=0,0003) fueron los antecedentes que predominaron, presentándose en el 55,55 por ciento de dichos donantes más de un antecedente de riesgo juntos. La donación con marcadores de VHB y de sífilis fue significativamente mayor (p=0,0001 y p=0,0005). 5/18 (27,77 por ciento) refirieron conocer previamente su problema, 3/18 (16,66 por ciento) lo sospechaban y 10/18 (55,55 por ciento) lo ignoraban, aunque los antecedentes de riesgo se distribuyeron en forma similar en ambos grupos. Los motivos para la donación en los que contestaron afirmativo/sospechas: Para realizar nuevamente la prueba: 6; Falla en el interrogatorio: 1; Presión familiar: 1, De los que ignoraban estar positivos: No se consideraron de riesgo: 6; Poca confianza en el interrogador: 3; Falla en el interrogatorio: 1. El 61,11 por ciento sabía que la prueba se práctica a todas las donaciones