[Effect of cyclophosphamide in experimental histoplasmosis in the rat]. / Acción de la ciclofosfamida en la histoplasmosis experimental de la rata.

Histoplasmosis is a fungal disease caused by the dimorphous fungus Histoplasma capsulatum (Hc). Cyclophosphamide (Cy) was used as an immunomodulator capable of modifying the course of the disease, as well as of regulating the mechanisms involved in T-lymphocyte mediated immune response. Rats were subjected to intracardiac inoculation of Hc followed by a fractionated treatment with a 100 mg/kg body weight dose of Cy on days +4, +5, +6, +7 and +11 pi. Until day 26 pi, treatment with Cy caused 85% mortality whereas no mortality was observed among animals only inoculated with Hc. On day 14 pi, the group of Hc animals showed a delayed hypersensitivity test (DH) of 26.60 + 13.96 as determined by the swelling of the leg. Conversely, DH was significantly depressed in rats inoculated with Hc and treated with Cy: 3.88 +/- 1.00 (p < 0.01). Colony forming units count in this group was 2020 CFU/g of spleen, and 24 CFU/g of spleen (p < 0.01) in controls. A macroscopic study of the organs revealed that the animals in the Hc+Cy group had spleenomegaly and lungs with granuloma and hemorrhagic spots. The controls only presented small lung abscesses. These findings lead to the conclusion that Cy causes a deterioration of cell mediated immune response which results in the manifestation of an acute, fatal experimental mycosis.

Obtención de un exoantígeno de Paracoccidioides brasiliensis a partir de cultivo en medio sólido / [Isolation of an Paracoccidioides brasiliensis exoantigen from solid culture media]

The goal of this work was to develop in solid medium a fast method to obtain Paracoccidioides brasiliensis (Pb) with a high yield. Four culture media were assayed: Sabouraud honey-agar, Sabouraud dextrose-agar, tomato -agar-medium (TOM) and a medium based on grape pulp. The most exhuberant growth was observed in medium based on grape pulp. Antigen was prepared in microscale at 6, 10 and 15 days incubation of solid cultures and the crude product concentrated by means of Centriplus tubes (Helena, France). Isolated antigens were subjected to polyacrylamide gel electrophoresis, followed by immunolabelling and detection of the characteristic gp45 antigen employing human and Pb-infected rat sera. Best results were observed after 10 days culture in grape medium. None of the other three media afforded comparable results.

[Isolation of an Paracoccidioides brasiliensis exoantigen from solid culture media]. / Obtención de un exoantígeno de Paracoccidioides brasiliensis a partir de cultivo en medio sólido.

The goal of this work was to develop in solid medium a fast method to obtain Paracoccidioides brasiliensis (Pb) with a high yield. Four culture media were assayed: Sabouraud honey-agar, Sabouraud dextrose-agar, tomato -agar-medium (TOM) and a medium based on grape pulp. The most exhuberant growth was observed in medium based on grape pulp. Antigen was prepared in microscale at 6, 10 and 15 days incubation of solid cultures and the crude product concentrated by means of Centriplus tubes (Helena, France). Isolated antigens were subjected to polyacrylamide gel electrophoresis, followed by immunolabelling and detection of the characteristic gp45 antigen employing human and Pb-infected rat sera. Best results were observed after 10 days culture in grape medium. None of the other three media afforded comparable results.

Cyclophosphamide effect on Paracoccidioidomycosis in the rat

La paracoccidioidomicosis es una enfermedad endemica fungica ampliamente distribuida en Latino-America. La ciclofosfamida ha sido usada como potente imunosupresor para modular la respuesta inmune, en um modelo experimental infectado con Paracocidioides brasiliensis. Ratas macho buffalo/sim endocriadas de 250-300 gr. de peso, fueron inoculadas por via intracardiaca con 5.10 elevado a sexta potencia celalas de P. brasiliensis en fase levaduriforme...

Cyclophosphamide effect on paracoccidioidomycosis in the rat.

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.

Obtención de un exoantígeno de Paracoccidioides brasiliensis a partir de cultivo en medio sólido. / [Isolation of an Paracoccidioides brasiliensis exoantigen from solid culture media]

The goal of this work was to develop in solid medium a fast method to obtain Paracoccidioides brasiliensis (Pb) with a high yield. Four culture media were assayed: Sabouraud honey-agar, Sabouraud dextrose-agar, tomato -agar-medium (TOM) and a medium based on grape pulp. The most exhuberant growth was observed in medium based on grape pulp. Antigen was prepared in microscale at 6, 10 and 15 days incubation of solid cultures and the crude product concentrated by means of Centriplus tubes (Helena, France). Isolated antigens were subjected to polyacrylamide gel electrophoresis, followed by immunolabelling and detection of the characteristic gp45 antigen employing human and Pb-infected rat sera. Best results were observed after 10 days culture in grape medium. None of the other three media afforded comparable results.

[Evaluation of alternative hemoculture tests for the diagnosis of early neonatal sepsis]. / Evaluación de pruebas alternativas al hemocultivo en el diagnóstico de la sepsis neonatal precoz.

In order to evaluate the diagnostic usefulness of coadjuvant tests such as external auditory canal swab culture and cultures from nasopharyngeal and gastric aspirates, and to determine the incidence and etiology of early neonatal sepsis (ENS) at our Unit, 90 newborn cases whose mothers experienced premature rupture of the membranes (PRM) were studied prospectively. Although a firm diagnosis requires positive blood cultures, the difficulty in recovering microorganisms and the trauma induced by sample collection in the baby justify the search for alternative diagnostic tests. Out of 2293 childbirths during 1991, 90 mothers (4%) had PRM more than 24 hours pre-partum, while 6.9/1000 (16/2293) developed ENS. In newborns from PMR mothers, ENS percentage was 3.3%, but increased to 5.5% in association with chorionamnionitis and reached 8.8% in premature cases. Among etiological ENS agents, Gram-positive microorganisms predominated (Table 1), particularly Staphylococcus aureus. Despite the finding that none of the coadjuvant assays (Table 2) had sufficient sensitivity or positive predictive value to identify all septic cases, they may prove useful to pinpoint newborns at high risk due to amniotic fluid exposure to infection or to chorioamnionitis.

Evaluación de pruebas alternativas al hemocultivo en el diagnóstico de la sepsis neonatal precoz. / [Evaluation of alternative hemoculture tests for the diagnosis of early neonatal sepsis]

In order to evaluate the diagnostic usefulness of coadjuvant tests such as external auditory canal swab culture and cultures from nasopharyngeal and gastric aspirates, and to determine the incidence and etiology of early neonatal sepsis (ENS) at our Unit, 90 newborn cases whose mothers experienced premature rupture of the membranes (PRM) were studied prospectively. Although a firm diagnosis requires positive blood cultures, the difficulty in recovering microorganisms and the trauma induced by sample collection in the baby justify the search for alternative diagnostic tests. Out of 2293 childbirths during 1991, 90 mothers (4

) had PRM more than 24 hours pre-partum, while 6.9/1000 (16/2293) developed ENS. In newborns from PMR mothers, ENS percentage was 3.3

, but increased to 5.5

in association with chorionamnionitis and reached 8.8

in premature cases. Among etiological ENS agents, Gram-positive microorganisms predominated (Table 1), particularly Staphylococcus aureus. Despite the finding that none of the coadjuvant assays (Table 2) had sufficient sensitivity or positive predictive value to identify all septic cases, they may prove useful to pinpoint newborns at high risk due to amniotic fluid exposure to infection or to chorioamnionitis.

Experimental coccidioidomycosis in the immunosuppressed rat.

C. immitis inoculated rats are known to develop infection restricted to lung whereas cyclophosphamide (CY) treatment leads to widespread dissemination with considerable mortality. In this study, an attempt was made to elucidate the mechanisms involved in such behaviour. With this aim, spleen cells were transferred from infected CY-treated to infected untreated rats, achieving significant specific inhibition in footpad swelling to coccidioidin in recipients, attributable to a suppressor T cell subpopulation induced by greater fungal antigen concentration arising from widespread C. immitis dissemination in immunosuppressed animals. NK activity proved similar regardless of CY treatment. Lastly, chronically infected rats presented increased colony forming units count after several weekly doses of CY, as happens in immunosuppressed patients harbouring a previous infection.

Virus sincicial respiratorio en menores de 5 años hospitalizados con infección respiratoria aguda baja / Respiratory syncitial virus in children under 5 years of age with acute lower respiratory infections

Entre julio 1988 y septiembre 1989, durante el período invernal, se investigó la presencia de Virus Sincicial Respiratorio (VSR) en 150niños menores de 5 años con infección respiratoria aguda (IRA) del tracto inferior (bronquiolitis: 56 por ciento; neumonías: 23 por ciento; bronquitis: 21 por ciento) por medio de un método rápido (inmunofluorescencia indirecta) sobre células del aspirado nasofaríngeo obtenido por succión con bomba de vacío. Se detectó antígeno de VSR en el 21,33 por ciento de los casos estudiados. El 25 por ciento de las bronquiolitis, el 17,6 por ciento de las neumonías y el 15,6 por ciento de las bronquitis resultaron positivas para VSR. La mayor frecuencia de positividad en bronquiolitis fue estadísticamente significativa (p<0,01). En neumonías se observó una disminución de la positividad a medida que aumentaba la edad. La distribución por grupo etáreo de los resultados positivos, independientemente del cuadro clínico, mostró una mayor frecuencia de VSR en menores de 23 meses. No se detectó diferencia significativa con respecto al sexo, estado nutricional y vacunación en pacientes con o sin VSR. En el grupo de 0-5 meses, el 42 por ciento de los casos positivos correspondió a menores de 30 días de vida. En 4/5 neonatos con VSR y en 3/12 menores de 12 meses con VSR se detectó la adquisición intrafamiliar de esta infección ya que se registró internación previa de hermanos con VSR en nuestro servicio. Se sugiere la necesidad de contar con métodos rápidos de estudio de la etiología viral en las IRAs a nivel hospitalario, lo que permitirá implementar estrategias más sólidas de control y tratamiento

Virus sincicial respiratorio en menores de 5 años hospitalizados con infección respiratoria aguda baja / Respiratory syncitial virus in children under 5 years of age with acute lower respiratory infections

Entre julio 1988 y septiembre 1989, durante el período invernal, se investigó la presencia de Virus Sincicial Respiratorio (VSR) en 150niños menores de 5 años con infección respiratoria aguda (IRA) del tracto inferior (bronquiolitis: 56 por ciento; neumonías: 23 por ciento; bronquitis: 21 por ciento) por medio de un método rápido (inmunofluorescencia indirecta) sobre células del aspirado nasofaríngeo obtenido por succión con bomba de vacío. Se detectó antígeno de VSR en el 21,33 por ciento de los casos estudiados. El 25 por ciento de las bronquiolitis, el 17,6 por ciento de las neumonías y el 15,6 por ciento de las bronquitis resultaron positivas para VSR. La mayor frecuencia de positividad en bronquiolitis fue estadísticamente significativa (p<0,01). En neumonías se observó una disminución de la positividad a medida que aumentaba la edad. La distribución por grupo etáreo de los resultados positivos, independientemente del cuadro clínico, mostró una mayor frecuencia de VSR en menores de 23 meses. No se detectó diferencia significativa con respecto al sexo, estado nutricional y vacunación en pacientes con o sin VSR. En el grupo de 0-5 meses, el 42 por ciento de los casos positivos correspondió a menores de 30 días de vida. En 4/5 neonatos con VSR y en 3/12 menores de 12 meses con VSR se detectó la adquisición intrafamiliar de esta infección ya que se registró internación previa de hermanos con VSR en nuestro servicio. Se sugiere la necesidad de contar con métodos rápidos de estudio de la etiología viral en las IRAs a nivel hospitalario, lo que permitirá implementar estrategias más sólidas de control y tratamiento (AU)

[Immunopathology induced in the rat by Junin virus]. / Inmunopatología inducida en la rata por virus Junín.

Intra-cerebral infection of the 10-day-old rat with the XJ prototype strain of Junin virus induces an immunopathological encephalitis with 100% mortality. In contrast with previous observations, our present work with antithymocyte serum (ATS) demonstrates a pathological role for the cellular immune response in this experimental model. As regards ATS treatment, 3 schedules were employed, the most efficient being daily 0.01 ml/g weight doses from day -1 to day +9, then +12, +14 and +16, taking day 0 as the time of virus infection. Survival reached 54% and the average day of death was delayed 12 days (Table 1). No differences were recorded in brain viral titres in treated vs untreated infected controls (Table 2). Lastly, splenocyte transfer from infected 10-day-old rats, to infected 2-day-old animals, which are known to develop persistence without death, led to 40% mortality in recipients vs 0% in 2-day-old non-transferred infected controls. Therefore, it may be concluded that: a) encephalitis in the 10-day-old rat is immunological in nature and b) transfer of lymphocytes to infected 2-day-old rats, induces disease and death.

Inmunopatología inducida en la rata por virus Junín. / [Immunopathology induced in the rat by Junin virus]

Intra-cerebral infection of the 10-day-old rat with the XJ prototype strain of Junin virus induces an immunopathological encephalitis with 100

mortality. In contrast with previous observations, our present work with antithymocyte serum (ATS) demonstrates a pathological role for the cellular immune response in this experimental model. As regards ATS treatment, 3 schedules were employed, the most efficient being daily 0.01 ml/g weight doses from day -1 to day +9, then +12, +14 and +16, taking day 0 as the time of virus infection. Survival reached 54

and the average day of death was delayed 12 days (Table 1). No differences were recorded in brain viral titres in treated vs untreated infected controls (Table 2). Lastly, splenocyte transfer from infected 10-day-old rats, to infected 2-day-old animals, which are known to develop persistence without death, led to 40

mortality in recipients vs 0

in 2-day-old non-transferred infected controls. Therefore, it may be concluded that: a) encephalitis in the 10-day-old rat is immunological in nature and b) transfer of lymphocytes to infected 2-day-old rats, induces disease and death.

Cyclophosphamide effect on coccidioidomycosis in the rat.

Cocidioidomycosis is a systemic mycosis, endemic in arid areas of the American continent. The rat was employed as an experimental host, since it had been shown to reproduce human lesions and present a chronic course of disease with granulomas mainly restricted to lungs. Given the influence of immunosuppressive therapy on the clinical course of human coccidioidomycosis, we studied the effect of cyclophosphamide (CY) in the experimental rat model. Accordingly, animals were inoculated with 400 Coccidioides immitis arthroconidia of the Acosta strain, by intracardiacal route. As single CY doses failed to alter the course of disease, three schedules were used: A) 4 daily doses of 20 mg/kg each, prior to C. immitis inoculation; B) 4 similar daily doses after infection; and C); 6 doses of 20 mg/kg each, given from day +1 to +4 then on days +8 and +9, post infection (pi), taking day 0 as the time of fungal inoculation. The first two schedules inhibited antibody formation up to day 28 pi, without modifying cellular response to coccidioidin as measured by foodpad swelling. Initially, there was greater fungal spread than in controls receiving C. immitis alone, which proved self-limiting in the latter. In contrast, schedule C led to 55% mortality with both humoral and cellular response abrogation, accompanied by extensive C. immitis dissemination. Histology disclosed significant alterations, such as the persistence of primary infection sporangia, corresponding to the acute stage of coccidioidomycosis in the absence of granuloma development. Therefore, the observed depression in cellular immunity seems responsible for the lack of inflammatory reaction capable of restricting sporangia proliferation in tissues which, in turn, enhances pathogen spread and mortality rate.

[Protection against encephalitis in rats caused by a pathogenic strain of the Junin virus, using peripheral inoculation of an attenuated strain]. / Protección contra la encefalitis producida en ratas por una cepa patógena de virus Junín por inoculación periférica con una cepa atenuada.

Argentine Hemorrhagic Fever manifests itself in man either subclinically or in hemorrhagic or neurological forms, mortality reaching 20%. Although Candid 1 strain is undergoing pilot trials, current therapy still resorts to convalescent serum administration. A neurological model was used to evaluate protection conferred by the attenuated XJC13 Junin virus strain. Newborn rats inoculated intraperitoneally (ip) prove resistant, whereas 8-12 day-old animals infected by intracerebral route with the XJ prototype strain suffer 100% mortality with neurological signs. The aim of this study was to achieve protection in this model and attempt to elucidate the mechanisms involved in resistance. It was observed that the longer the inoculation challenge interval, the greater was the survival percentage. In protected animals, brain viral titres were 3 log lower than in challenged controls, while XJC13 infected unchallenged controls presented low CNS values throughout. Neutralizing antibody levels were not significantly different in experimental versus challenged control groups, ruling out any secondary booster effect on protected rats. Neither the transfer of immunoserum nor of endogenous or exogenous interferon altered mortality. However, when splenocytes from rats infected 10 days previously were transferred prior to XJ challenge, survival was increased to 50%, but there was no gain in protection when cells were treated with antithymocyte serum plus complement. Consequently, protection in this neurological model can be attributed to a cellular immune response.

Protección contra la encefalitis producida en ratas por una cepa patógena de virus Junín por inoculación periférica con una cepa atenuada. / [Protection against encephalitis in rats caused by a pathogenic strain of the Junin virus, using peripheral inoculation of an attenuated strain]

Argentine Hemorrhagic Fever manifests itself in man either subclinically or in hemorrhagic or neurological forms, mortality reaching 20

. Although Candid 1 strain is undergoing pilot trials, current therapy still resorts to convalescent serum administration. A neurological model was used to evaluate protection conferred by the attenuated XJC13 Junin virus strain. Newborn rats inoculated intraperitoneally (ip) prove resistant, whereas 8-12 day-old animals infected by intracerebral route with the XJ prototype strain suffer 100

mortality with neurological signs. The aim of this study was to achieve protection in this model and attempt to elucidate the mechanisms involved in resistance. It was observed that the longer the inoculation challenge interval, the greater was the survival percentage. In protected animals, brain viral titres were 3 log lower than in challenged controls, while XJC13 infected unchallenged controls presented low CNS values throughout. Neutralizing antibody levels were not significantly different in experimental versus challenged control groups, ruling out any secondary booster effect on protected rats. Neither the transfer of immunoserum nor of endogenous or exogenous interferon altered mortality. However, when splenocytes from rats infected 10 days previously were transferred prior to XJ challenge, survival was increased to 50

, but there was no gain in protection when cells were treated with antithymocyte serum plus complement. Consequently, protection in this neurological model can be attributed to a cellular immune response.

Ribavirin effect on experimental Junin virus-induced encephalitis.

Junin virus, the etiological agent of Argentine hemorrhagic fever, produces in man a disease mainly characterized by hemorrhagic alterations, commonly accompanied by neurological symptoms, and leading to 10% mortality. Intracerebral inoculation in 10-day-old rats or intraperitoneal inoculation in 2-day-old rats leads to high mortality due to severe encephalitis. Here, the effect of Ribavirin on these experimental models was tested in order to evaluate the degree of protection achieved against neuropathological manifestations. In intracerebrally infected 10-day-old rats the drug was administered 2 hr before virus inoculation. Doses ranged from 30 to 90 mg/kg body weight. Protection reached 40% for the 60 and 90 mg doses. Intraperitoneally infected 2-day-old rats received the drug in five 30-mg daily doses, starting the same day as virus inoculation. Survival was 73%. Viral replication within peritoneal macrophages dropped markedly, leading to much lower CNS viral titres. Together with results reported in primates, our findings support further studies on Ribavirin, with a view to eventual trials in humans.

Macrophages are involved in age-dependent resistance of rats to Junin virus infection.

We attempted to correlate rat age with resistance to intraperitoneal infection with the XJ strain of Junin virus. Accordingly, mortality, viral replication in macrophages and brain, as well as neutralizing antibody (NA) levels were recorded in animals inoculated at 2, 5, 10 and 26 days of life. Two-day-old animals demonstrated both the greatest mortality (86%) and viral replication in macrophages, allowing virus to reach the brain where high titers were detected. This age group also had the highest NA titers. Mortality, viral multiplication and NA titers diminished with increasing age of the animals. The ability of peritoneal macrophages to support viral replication, therefore, seems to determine rat susceptibility to intraperitoneal infection with Junin virus.