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.

[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.

Macrophage maturity and modulation of response to Junín virus in infected rats.

Replication of Junín virus in peritoneal macrophages from newborn rats was greater for prototype strain XJ than for strain XJC13, whereas in cells from adult animals viral multiplication proved minimal. Transfer of peritoneal adherent cells from normal adult to strain XJ-infected newborn rats lowered mortality significantly. Silica blockade of macrophages protected two-day-old strain XJ-infected animals and depressed brain titers of virus significantly, whereas treatment had no effect on strain XJC13-infected rats. Macrophages from infected two-day-old rats caused illness and death in recipient animals. Silica blockade rendered macrophage-mature 11-day-old rats partially susceptible to infection with Junín virus. Thus pathogenicity of strain XJ in the two-day-old rat by the intraperitoneal route depends on the ability to replicate in peritoneal macrophages, whereas strain XJC13 is nonlethal because it fails to multiply efficiently in these cells. Macrophage maturity seems essential to inhibit viral replication and thus confer protection on the adult host.

[Role of macrophages in the dissemination of the Junin virus in the rat]. / Papel del macrófago en la diseminación del virus Junín en la rata.

The 2-day-old rat is known to be susceptible to infection by ip route with the XJ strain of Junin virus but resists inoculation with XJC13 strain (85% vs 15% mortality). In order to determine whether peritoneal macrophages play a role in modulating the course of infection, viral replication in adherent peritoneal cells infected with either strain was studied. XJ was found to replicate 30-fold as regards XJC13 at day 3 pi. Besides, silica blockage of peritoneal macrophages was also evaluated. Following silica treatment, 60% survival was recorded for XJ-infected animals vs 15% for untreated infected controls. No significant differences were recorded for silica-treated XJC13-infected rats vs untreated infected controls. These preliminary findings indicate that, upon replication within the host's macrophages at the peritoneal inoculation site, virus spreads readily to reach the target organs. Effective silica blockage lends support to this pathway, as shown by significantly greater survival in XJ-infected rats.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the former's greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.

Papel del macrófago en la diseminación del virus Junín en la rata / [Role of macrophages in the dissemination of the Junin virus in the rat]

The 2-day-old rat is known to be susceptible to infection by ip route with the XJ strain of Junin virus but resists inoculation with XJC13 strain (85

mortality). In order to determine whether peritoneal macrophages play a role in modulating the course of infection, viral replication in adherent peritoneal cells infected with either strain was studied. XJ was found to replicate 30-fold as regards XJC13 at day 3 pi. Besides, silica blockage of peritoneal macrophages was also evaluated. Following silica treatment, 60

survival was recorded for XJ-infected animals vs 15

for untreated infected controls. No significant differences were recorded for silica-treated XJC13-infected rats vs untreated infected controls. These preliminary findings indicate that, upon replication within the host’s macrophages at the peritoneal inoculation site, virus spreads readily to reach the target organs. Effective silica blockage lends support to this pathway, as shown by significantly greater survival in XJ-infected rats.

Attenuation parameters for Junín virus in the newborn rat

Para caracterizar una cepa viral como atenuada es necesario contar con criterios biológicos y bioquímicos. En el caso del virus Junín la rata de 2 días de vida resultó ser un marcador biológico de atenuación con respecto a la moratalidad. En el presente trabajo se investigaron otros aspectos que amplían lo ya conocido sobre el comporatamiento de la cepa prototipo XJ y la atenuada XJC13 en este huésped experimental. En el binomio rata-virus Junín se determinó la acción de las cepas mencionadas a distintos tiempos pi sobre parámetros: a) hematológicos: recuento de leucocitos, hematíes, plaquetas y fórmula leucocitaria. b) esplénicos: Nro. de células e índice de bazo y c) respuesta inmune humoral hacia glóbulos rojos de carnero (GRC). Para la determinación de las células formadoras de anticuerpos (CFA) los animales recibieron 10**9 GRC a los 16 días de vida, realizando el ensayo a los 4 y 6 días post-inmunización, dado que en las ratas controles al día + 4 se obtiene la máxima respuesta, declinando al día + 6. La cepa XJ cualquiera sea la vía de infección produce una significativa leucocitosis con respecto a los animales normales, sin modificación en los otros parámetros hemáticos; la cepa XJC13 no provocó alteraciones (Cuadro 1). Además la cepa XJ produjo una significativa esplenomegalia con incremento del índice esplénico, no así la XJC13 (Cuadros 2 y 3). Con respecto a la respuesta humoral la cepa XJ disminuyó el número de CFA hacia GRC no observándose esta acción con XJC13 (Cuadro 4). Estas diferencias encontradas entre la cepa patógena y atenuada podrían deberse a la mayor replicación y diseminación que presenta la cepa XJ con respecto a la XJC13. La disminución en CFA con la cepa XJ podría deberse a una disfunción general del bazo o bien a una acción del virus sobre una determinada población celular que interviene en la respuesta inmune hacia GRC

Attenuation parameters for Junín virus in the newborn rat

Para caracterizar una cepa viral como atenuada es necesario contar con criterios biológicos y bioquímicos. En el caso del virus Junín la rata de 2 días de vida resultó ser un marcador biológico de atenuación con respecto a la moratalidad. En el presente trabajo se investigaron otros aspectos que amplían lo ya conocido sobre el comporatamiento de la cepa prototipo XJ y la atenuada XJC13 en este huésped experimental. En el binomio rata-virus Junín se determinó la acción de las cepas mencionadas a distintos tiempos pi sobre parámetros: a) hematológicos: recuento de leucocitos, hematíes, plaquetas y fórmula leucocitaria. b) esplénicos: Nro. de células e índice de bazo y c) respuesta inmune humoral hacia glóbulos rojos de carnero (GRC). Para la determinación de las células formadoras de anticuerpos (CFA) los animales recibieron 10**9 GRC a los 16 días de vida, realizando el ensayo a los 4 y 6 días post-inmunización, dado que en las ratas controles al día + 4 se obtiene la máxima respuesta, declinando al día + 6. La cepa XJ cualquiera sea la vía de infección produce una significativa leucocitosis con respecto a los animales normales, sin modificación en los otros parámetros hemáticos; la cepa XJC13 no provocó alteraciones (Cuadro 1). Además la cepa XJ produjo una significativa esplenomegalia con incremento del índice esplénico, no así la XJC13 (Cuadros 2 y 3). Con respecto a la respuesta humoral la cepa XJ disminuyó el número de CFA hacia GRC no observándose esta acción con XJC13 (Cuadro 4). Estas diferencias encontradas entre la cepa patógena y atenuada podrían deberse a la mayor replicación y diseminación que presenta la cepa XJ con respecto a la XJC13. La disminución en CFA con la cepa XJ podría deberse a una disfunción general del bazo o bien a una acción del virus sobre una determinada población celular que interviene en la respuesta inmune hacia GRC (AU)

Papel del macrófago en la diseminación del virus Junín en la rata. / [Role of macrophages in the dissemination of the Junin virus in the rat]

The 2-day-old rat is known to be susceptible to infection by ip route with the XJ strain of Junin virus but resists inoculation with XJC13 strain (85

vs 15

mortality). In order to determine whether peritoneal macrophages play a role in modulating the course of infection, viral replication in adherent peritoneal cells infected with either strain was studied. XJ was found to replicate 30-fold as regards XJC13 at day 3 pi. Besides, silica blockage of peritoneal macrophages was also evaluated. Following silica treatment, 60

survival was recorded for XJ-infected animals vs 15

for untreated infected controls. No significant differences were recorded for silica-treated XJC13-infected rats vs untreated infected controls. These preliminary findings indicate that, upon replication within the hosts macrophages at the peritoneal inoculation site, virus spreads readily to reach the target organs. Effective silica blockage lends support to this pathway, as shown by significantly greater survival in XJ-infected rats.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the formers greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.