A slight modification of the chemically defined medium of McVeigh and Morton resulted in an excellent substratum for the cultivation of Paracoccidioides brasiliensis yeast phase.

Recently, data have been reported suggesting natural killer (NK) cells may function in natural resistance against a fungus, Cryptococcus neoformans. The primary objective of this study was to examine the reactivity of murine splenic cells against another fungus, Paracoccidioides brasiliensis. Levels of NK activity in effector cell pools were varied by: (i) removing nylon wool-adherent cells, (ii) fractionating splenic cells on Percoll discontinuous gradients, (iii) using old and young effector cell donor mice, (iv) using donors from different strains, and (v) pretreating donors with NK-augmenting and-depressing agents. The various effector cell pools were simultaneously used in the 4-h 5tCr release assay with YAC-1 targets to determine the NK reactivity and in the in vitro growth inhibition assay against P. brasiliensis yeast phase targets. In each case, the level of NK reactivity correlated with the ability of the effector cells to inhibit the in vitro growth of P. brasiliensis. NK activity and P. brasiliensis growth-inhibiting ability could be augmented by fractionation of splenic cells through nylon wool or Percoll gradients. The effector cells responsible for the NK activity and P. brasiliensis growth inhibition were characterized as being nylon wool nonadherent, being found in the low-density fractions from Percoll discontinuous gradients, and having no detectable Thy-i antigen or immunoglobulin but having asialo GM1 on their surface. These data support the contention that NK or NK-like cells are responsible for limiting the in vitro growth of P. brasiliensis.

The ability of P. brasiliensis yeast cells to withstand microaerophilic-conditions was investigated in a liquid medium distributed in tall columns in screw-capped tubes. Young cells of three isolates were inoculated on top of the medium, and the tubes were incubated aerobically and anaerobically at 36 °C for 28 days. The viability of cells that had sedimented to the bottoms of the tubes was studied by fluorescent micro.~6opy and by their capacity to resume growth when transferred to fresh medium undercontinuous agitation. The proportion of viable cells in the sediments diminished with time of incubation. However, after 28 days, 27 % of the cells were still viable and fully capable of active growth when placed under adequate aeration. On the other hand, drastic reduction of oxygen access elicited an accelerated death rate, with no survival after 7 days of incubation.The yeast and the mycelial phase of Paracoccidioides brasiliensis both require a generous supply of oxygen for growth. This is particularly true when cultures are attempted in liquid media. Irrespective of the quality of the medium, no growth is obtained in stationary liquid cultures, while continuous agitation results in adequate development of the fungus I-4, 8, 9].This stringent oxygen requirement is difficult to reconcile with the long periods of latency known to occur in clinical paracoccidioidomycosis, as illustrated by cases diagnosed outside of the endemic areas, the majority of which have involved patients who developed paracoccidioidomycosis 15 or more years after their return from endemic countries to non-endemic zones [5]. This extreme latency of infection indicates that the fungus can persist dormant within closed lesions, in a manner similar to Mycobacterium tuberculosis [13,14]. Furthermore, the finding of residual nodular lesions containing the characteristic P. brasiliensis cells in the lung and the adrenals of apparently normal individuals [1], as well as the description of a healed primary pulmonary lymph node complex 1-12], point to the reticuloendothelial system as the most probable site of residence of the fungus during, the latency period.We investigated the survival of the yeast (tissue) phase of P. brasiliensis under conditions of oxygen deprivation in order to assess the possibility of accommodation to the environment operating in walled-off lesions. MATERIALS AND METHODSThree isolates of the fungus, recovered from patients with chronic progressive paracoccidioidomycosis were employed: C81, isolated in 1962, LA in 1970 and CG in 1980. They have been maintained in the mycelial phase by bi-monthly transfers on Sabouraud dextrose agar (BBL, Cockeysville, Maryland, U.S.A.) at 24-26 °C.