Increased Fas-mediated apoptosis in polymorphonuclear cells from HIV-infected patients.

Neutrophils represent an important line of innate host defence against invading microorganisms and their functional detriment during HIV infection, including accelerated spontaneous cell death, has been shown to contribute to AIDS development. Neutrophils are susceptible to apoptosis via Fas and an interaction between Fas and FasL was suggested originally as a mechanism to explain constitutive neutrophil apoptosis. We have explored some intracellular pathways leading to PMN apoptosis from 28 HIV-infected patients and 24 healthy volunteers. As previously reported, accelerated spontaneous apoptosis was observed in HIV+ patients, but this did not correlate with viral load. Furthermore, an increase in the level of spontaneous apoptosis was detected in neutrophils from HIV-infected patients following inhibition of ERK, suggesting an impairment of this kinase pathway during the early stages of infection which may contribute to PMN dysfunction. An elevated susceptibility to undergo apoptosis was observed following cross-linking of Fas, which correlated both with viral load and co-expression of Fas/FasL surface molecules. Different mechanisms for spontaneous and Fas-induced apoptosis are proposed which together contribute to the neutropenia and secondary infections observed during the progression to AIDS.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope.

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 microm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 æm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique

[Immunodiagnosis of neurocysticercosis: comparative study of antigenic extracts from Cysticercus cellulosae and Taenia crassiceps]. / Inmunodiagnóstico de la neurocisticercosis: estudio comparativo de extractos antigénicos de Cysticercus cellulosae y Taenia crassiceps.

Different antigenic extracts of Taenia solium and Taenia crassiceps were evaluated in connection with the detection of antibodies in patients with neurocysticercosis aimed at selecting immunorelevant antigens for the diagnosis of neurocysticercosis by means of the immunoenzymatic assay and immunoblotting. The vesicular fluid of T. crassiceps proved to be more sensitive (100%) and specific (86%). On using the immunoblotting technique it was also observed that this extract was the most sensitive and specific. Within the protein profile of the antigen the band of 18 kDa was mostly recognized by the serum and cerebrospinal fluid of patients with neurocysticercosis. The vesicular fluid of T. crassiceps represents an alternative in the optimization of the diagnosis of neurocysticercosis in the serum and cerebrospinal fluid and in the substitution of T. solium antigens due to its high sensitivity and specificity and to its easy obtention under controlled laboratory conditions.

Nuevas fracciones antigénicas para el inmunodiagnóstico de la neurocisticercosis / New antigenic fractions for neurocysticercosis diagnosis

La neurocisticercosis (NCC) es la enfermedad parasitaria más frecuente del sistema nervioso central causada por la forma larvaria enquistada de la Taenia solium, su diagnóstico se basa en la integración de datos clínicos imagenológicos y serológicos. La detección de anticuerpos específicos usando el ensayo inmunoenzimático (ELISA) es una útil estrategia para confirmar el diagnóstico de la NCC. Estudios previos demuestran que la Taenia crassiceps y la Taenia solium tienen similitud estructural y antigénica; en este trabajo evaluamos antígenos de ambos parásitos frente a 68 líquidos cefalorraquídeos y 40 sueros pertenecientes a casos confirmados de NCC y a controles. Para cumplir tal objetivo enfrentamos en ELISA las muestras de los pacientes contra cada uno de tres extractos antigénicos de cada tenideo: el fluido vesicular, la membrana externa y el extracto total. El análisis estadístico demostró una alta sensibilidad y especificidad de los antígenos de la T.crassiceps, especialmente el fluido vesicular; el cual dio una sensibilidad de 90,3 por ciento, una especificidad de 94,5 por ciento y valores predictivos positivos y negativos de 93,3 por ciento y 92,1 por ciento frente a los LCR. Estos atributos hacen de esta fracción parasitaria una herramienta de gran utilidad en el diagnóstico de la NCC, pudiendo sustituir los antígenos procedentes de carcasas de cerdos infectados naturalmente con T.solinum

Prevalencia de enteroparásitos en una comunidad rural venezolana / Prevalence of enteroparasites in a venezuelan rural community

Se realizó un estudio epidemiológico en una región rural de los Andes venezolanos, (El Dividive, Edo. Trujillo), con la finalidad de dterminar el perfil de enteroparásitos. Se seleccionaron aleatoriamente 332 viviendas y se procedió a encuestar y realizar exámenes parasitológicos de las muestras fecales de 1.124 personas. La mayoría de las viviendas contaba con servicio de acueducto intradomiciliario muy irregular (69,9 por ciento), el 30 por ciento no poseía instalaciones sanitarias ni cloacas, el 40,7 por ciento no tenía sistema de recolección de basura intradomiciliaria. El promedio de edad fue de 23 años; solo el 26,8 por ciento de los habitantes había concluido la educación primaria, el 13,7 por ciento tenía empleo fijo, el resto se dedicaba a oficios domésticos, actividades agrícolas y al comercio informal. En el 72,3 por ciento de las muestras se pudo detectar la presencia de al menos un tipo de parásito; el poliparasitismo se evidenció en el 66 por ciento de los sujetos y sólo el 27,8 por ciento fue negativo al examen de heces. Se observó predominio de los protozoarios (83,7 por ciento) sobre los helmintos (16,3 por ciento). Los parásitos más comunes fueron: Blastocystis hominis 38 por ciento, Endolimax nana 28,6 por ciento, Entamoeba hystolitica 24,2 por ciento, Entamoeba coli 14,3 por ciento, Entamoeba hartmanni 11,3 por ciento, Giardia lamblia 9,3 por ciento, Ascaris lumbricoides 10,3 por ciento, Trichuris trichuria 6,0 por ciento y otros 8,0 por ciento. Se evidenció en esta población de precarias condiciones socioeconómicas: elevada presencia de parasitosis (72,3 por ciento) y alto grado de poliparasitismo. Se concluye que, en esta región, el escaso saneamiento ambiental está estrechamente asociado al nivel de parsitosis

Relationship between red cell folate and cervical intraepithelial neoplasia and cervical human papillomavirus infection.

OBJECTIVE: The aim of this study was to determine the relationship between red cell folate levels and the presence of cervical intraepithelial neoplasia (CIN) or cervical human papillomavirus infection (HPV) (or both). For that purpose, we designed a case-control study. MATERIALS AND METHODS: One hundred three asymptomatic women who were between the ages of 20 and 76 and attending the gynecological clinic of the Military Hospital in Bogota, Colombia, were selected. Their mean age was 37.9 years. Inclusion criteria combined a colposcopic examination and a cervicovaginal Papanicolaou smear; accordingly, patients were divided into women without CIN or HPV (55 women, the control group) and women with CIN or HPV (or both) (48 women, the study group). Red cell folate levels were determined by radioimmunoassay. RESULTS: Statistically significant differences in folate levels were found between cases [2.55 ng/ml; standard deviation, 0.75; 95% confidence interval, 2.34-2.73] and controls (2.96 ng/ml; standard deviation, 0.77; 95% confidence interval, 2.75-3.14; p < .008). The odds ratio between the folate levels and CIN or HPV (or both) was 0.49 (p = .01). Red cell folate levels of the individuals participating in this study were not found to be associated with parity, the use of oral contraceptives, cigarette smoking, or age. CONCLUSIONS: High red cell folate levels appear to provide a protective effect against the development of CIN or HPV (or both).

Evaluación inmunoquímica de cepas de entamoeba hystolitica venezolanas / Immunochemical evaluation of venezuelan strains of entamoeba hystolitica

La identificación de la patogenicidad de la Entamoeba hystolítica ha sido útil como punto de partida en la búsqueda de antígenos relevantes para el inmunodiagnóstico y la inmunoprofilaxia. Recientemente se describió el aislamiento y axenización de las dos primeras cepas de E.hystolítica venezolanas: IULA:1092:1 (MMM) e IULA:0593:2 (NER). En el presente estudio evaluamos los siguientes parámetros: perfiles electroforéticos, reactividad frente a anticuerpos monoclonales y policlonales y la virulencia cuando son inoculadas en animales de laboratorio y los comparamos con la cepa patógena de referencia (HM-1). Los extractos amibianos fueron cromatografiados en geles de poliacrilamida SDSPAGE, se electrotransfirieron a membranas de nitrocelulosa y se enfrentaron a sueros humanos y de animales positivos negativos. La capacidad de las cepas para producir abscesos, se evaluó, inoculándolas en el hígado de hamsters. El análisis de los resultados nos permitieron concluir que las dos cepas en estudio presentan un perfil electroforético similar a la cep patógena de referencia internacional (HMI); que son de alta virulencia, demostrada por la producción de abscesos hepáticos amibianos en todos los animales inoculados y que presentan un complejo patrón de reactividad frente a los sueros positivos y que en ellas está presente un antígeno marcador de patogenicidad reconocido por el monoclonal HU-511. Con base en estos criterios y los anteriormente evaluados podemos concluir que las dos cepas son alto potencial patógeno, pudiendo ser usadas como antígeno en los ensayos de inmunodiagnóstico e inmunoprofilaxis

Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody.

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immunoenzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Isolation and axenization of two entamoeba histolytica strains.

We describe the isolation and axenization of two E. histolytica strains, obtained from the stools of two patients with the clinical diagnosis of dysentery. We used Pavlova's medium for initial polixenic culture, and TYI-S-33 (Diamond's) medium for monoxenic and axenic cultures. In order to eliminate the microorganism contaminating the stools the following antibiotics were used: penicillin, streptomycin, ampicillin, chloramphenicol, kanamycin, nistatin, ceftriaxone and amphoterycin B. Both strains grew in similar culture conditions with a yield of 2 x 10(6) microorganism per tube of 15 ml. Both strains belong to pathogenic zymodemes, and virulence was determined by the capacity for producing hepatic abscesses in 100% of the hamsters inoculated intrahepatically.

Entamoeba histolytica: detection of coproantigens by purified antibody in the capture sandwich ELISA.

A sensitive and specific Capture Sandwich ELISA (CSE) was developed using polyclonal purified rabbit antibodies against three different axenic strains of Entamoeba histolytica: CSP from Brazil and HM1 - IMSS from Mexico, for the detection of coproantigens in fecal samples. Immunoglobulin G (IgG) against E. histolytica was isolated from rabbits immunized with throphozoites whole extract in two stages: affinity chromatography in a column containing E. histolytica antigens bound to Sepharose 4B was followed by another chromatography in Sepharose antibodies 4B-Protein A. A Capture Sandwich ELISA using purified antibodies was able to detect 70ng of amebae protein, showing a sensitivity of 93% and specificity of 94%. The combination of microscopic examination and CSE gave a concordance and discordance of 93.25% and 6.75%, respectively. It was concluded that CSE is highly specific for the detection of coproantigens of E. histolytica in feces of infected patients, is quicker to perform, easier and more sensitive than microscopic examination.

Entamoeba histolytica: antigenic characterization of axenic strains from Brazil

Trofozoitos obtidos de cultura de Entamoeba histolytica isoladas e axenizadas no Brasil (ICB-CSP, ICB-462 e ICB-32) foram utilizados para a producao de soros imunes em coelhos e para a caracterizacao de antigenos atraves dos seus perfis eletroforetico e glicoproteico, em paralelo com uma cepa padrao isolada e axenizada nos Estados Unidos (HK-9). Obtiveram-se soros hiperimunes, reativos frente aos antigenos homologos e heterologos. As quatro cepas em estudo apresentaram perfis eletroforetico e glicoproteico complexos e semelhantes, compostos por polipeptideos com pesos moleculares variando de 200 kDa a menos de 29 kDa. Nao se detectaram diferencas significativas entre as cepas patogenicas e nao patogenicas.

Entamoeba histolytica: antigenic characterization of axenic strains from Brazil.

Trophozoites from cultures of Entamoeba histolytica strains isolated and grown axenically in Brazil (ICB-CSP, ICB-462 and ICB-32) were used for immune sera production and for characterization of their antigens by using electrophoretic and glycoproteic profiles, in parallel with a standard strain isolated and kept under axenic conditions in USA (HK-9). Hyperimmune sera, presenting high antibody titers with homologous and heterologous antigens, were obtained. The four strains in study revealed similar and complex electrophoretic and glycoproteic profiles showing polypeptides with molecular weights ranging from 200 to less than 29 kDa. No significant differences were detected between the pathogenic and non-pathogenic strains.