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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.

Summary To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10–15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

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.

A new attenuation marker to distinguish a virulent strain (XJJV) from an attenuated strain (XJC13JV or XJOJV) of Junin virus by means of the humoral and cellular responses to unrelated antigens was studied in guinea pigs. Strain XJJV suppressed the humoral immune response, as shown by the lower titers of precipitating antibody to ovalbumin. The concomitant decrease in serum complement level contributed to a milder Arthus cutaneous reactivity. In contrast, the attenuated strains did not decrease the humoral response. The pathogenic strain suppressed cell-mediated immunity, as demonstrated by decreased contact sensitivity to 2,4-dinitro-1-fluorobenzene and by depression of delayed skin reactions to tuberculin purified protein derivative. When attenuated strains were used, such suppressive effects were not observed. For virulent strain XJJV, virus replication in lymphoid organs and immunosuppressive effects were correlated. These findings provide a further means to differentiate between virulent and attenuated strains of Junin virus for the purpose of vaccine control of Argentine hemorrhagic fever.

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.

Coccidioidomycosis 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 559r 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.

Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non-suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups.

This study was carried out to determine the pathways along which two strains of Junin virus (JV), the pathogenic XJV and the attenuated XJC13V, reach the CNS following IP inoculation of 2-day-old rats. A sequential study of infectivity and antigen distribution in peritoneal macrophages, spleen, and brain was performed. Mortality was 85% with the former strain, but only 15% with the latter. At 4-7 days PI, XJV-infected animals had viral antigen in 10% of peritoneal macrophages. Viremia and spleen virus lasted for 10-15 days. Low brain titers were detected at day 7, with a peak at day 15. Brain antigen correlated with virus titers. In contrast, XJC13V-infected rats, macrophage antigen appeared later and to a lesser degree (1% of cells). Viremia and spleen virus were transient, while both the titer of brain virus and the viral antigen proved lower. Antibody titers were over twofold higher for XJ-infected animals. It is suggested that the different replication rate at the inoculation site could account for the greater ability of the XJV strain to reach the CNS. A greater antigen mass and/or more numerous antigenic determinants presented by the macrophage could explain the higher antibody titers found in XJ-injected rats, which were unable, however, to prevent viral spread.

2-day-old Wistar rats intracerebrally infected with the XJC13 strain of virus exhibited a 5% survival rate which rose to 71% after immune serum treatment. Brain viral titers were relatively unaffected by this treatment. Histologic studies showed necrosis in the cerebellum and brain cortex with mononuclear cell infiltration in both treated and nontreated groups. Beginning on day 16 postinfection, however, intracerebral perivascular gliosis foci and mild meningeal congestion were minimal in the treated animals. These findings imply that passively transferred humoral immunity leads to prompt recovery in this experimental model.

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.