RESUMO
Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidiodomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated form patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.
Assuntos
Pneumopatias Fúngicas/microbiologia , Fungos Mitospóricos/patogenicidade , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inoculações Seriadas , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis.
Assuntos
Citotoxicidade Imunológica , Ativação de Macrófagos , Macrófagos/parasitologia , Fungos Mitospóricos/crescimento & desenvolvimento , Paracoccidioides/crescimento & desenvolvimento , Fagocitose , Animais , Fracionamento Celular , Sistema Livre de Células , Células Cultivadas , Meios de Cultura , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/imunologia , Cavidade Peritoneal , Alvéolos PulmonaresRESUMO
The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.
Assuntos
Blastomyces , Interferon gama/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Fungos Mitospóricos , Paracoccidioides , Alvéolos Pulmonares/citologia , Animais , Antioxidantes/farmacologia , Concanavalina A/farmacologia , Radicais Livres , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Fungicidal activity of murine peritoneal macrophages for the yeast form of the dimorphic fungal pathogen P. brasiliensis was studied. Killing was assessed by reduction of colony forming units (CFU) using a new medium which has a good plating efficiency. Resident peritoneal macrophages phagocytosed but did not kill P. brasiliensis. Macrophages treated overnight with recombinant gamma-interferon (IFN), lymph node cells plus concanavalin A (Con A) or Con A-stimulated spleen cell culture supernatants (Con A Sup) reproducibly killed three different isolates of P. brasiliensis (35 - 55%, P less than 0.05 - P less than 0.001). This is the first demonstration of killing of this organism by macrophages. Activated macrophages did not show enhanced phagocytosis of P. brasiliensis. Activation of macrophages for killing by IFN was dose-dependent and, varying with the isolate, 100 - 10,000 U/ml was required for inducing significant fungicidal effects against P. brasiliensis. Activation of macrophages by IFN or Con A Sup was abrogated by anti-IFN antibody. These results suggest that immune modulation may be an approach to therapy of paracoccidioidomycosis. Killing was not significantly inhibited in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethylsulfoxide (300 mM) or azide (1 mM). This indicated that killing mechanism(s) did not depend upon products of the oxidative burst. These results show that P. brasiliensis can be significantly killed by activated macrophages without products of the oxidative burst.