Rat Pneumocystis carinii grown on lung-derived cell lines in tissue culture flasks and multiwell plates was tested for susceptibility to four antimicrobial agents currently being used in the treatment of human pneumocystosis. Standard criteria for organism quantitation, replication, viability, and inoculum size were established. Trimethoprim-sulfamethoxazole inhibited P. carinii growth at a concentration ratio of 1:19 ,ug/ml, and pentamidine isethionate was active at 0.1 ,ug/ml. a-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, inhibited P. carinii at a concentration of 1 mM once erythrocytes (which are high in polyamine content) were removed from the inoculum; this effect could be overcome by the polyamine putrescine. Dapsone suppressed P. carinii replication at a dose of 0.1 ,ug/ml, but this effect was lost after 72 h in culture. Overall, the reduction in P. carinii numbers with these drugs was relatively modest (45 to 84%), which is consistent with their lack of lethal effects on the organism in vivo. Thus, the system presented here should be helpful in developing new anti-P. carinii agents and in elucidating their mechanism of action.Over the past two decades, Pneumocystis carinii has been recognized as a leading cause of pneumonia in immunosuppressed patients. With the emergence of the acquired immune deficiency syndrome (AIDS), the number of cases of pneumocystosis has greatly increased (5). Not only is AIDS the major predisposing host factor in the development of pneumocystosis, but P. carinii is also the most frequent opportunistic infection and a leading cause of death in AIDS. Trimethoprim-sulfamethoxazole (TMP-SMX) and pentamidine isethionate have been the principal drugs used in the therapy of pneumocystosis (17,34,39). The high recurrence rate of pneumocystosis and high frequency of adverse reactions to TMP-SMX in patients with AIDS emphasize the need to develop new forms of treatment (12, 14, 21, 30).The search for new drugs active against P. carinii in humans has rested primarily on naturally infected rodents. Rats administered corticosteroids for 8 weeks spontaneously develop pneumocystosis with histopathologic features identical to those in humans (9,35). Although the system is time consuming and has permitted the testing of relatively few antimicrobial agents, there has been a high correlation between drug efficacies in rats and humans (9,18,20). The most recent application of this form of drug development is dapsone (19), which is currently undergoing clinical trials. An exception seems to be a-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis; this agent has been ineffective in the rat model (19) but has shown promising activity in small numbers of P. carinii patients (11, 31). Thus, there is a need for alternative methods of drug evaluation.One approach to this problem has involved in vitro systems. Several different techniques of cell culture have been used for the cultivation of P. carinii, but these studies have been limited by a lack of reproducibili...

Pneumocystis carinii obtained from infected rats and patients was cultured in the A549 cell line, a presumptive alveolar type 2 cell line derived from a human lung carcinoma. Standard criteria were established for organism sampling, quantitation, and growth. The trophozoite form of P. carinii was a more sensitive indicator of growth than was the cyst. Rat P. carinii increased 10-fold in primary culture and could be serially passed three additional times to new cultures; success in growing human P. carinii was limited and appeared to be related to the quality of the specimen received for culture. Growth pattern experiments suggested that close interaction of P. carinii with the cell monolayer is an important step in the life cycle of the organism. Thus, the A549 culture system should be useful for in vitro studies of the immunobiology of P. carinil.

Pneumocystis carinii pneumonia was induced in rats by the administration of corticosteroids, and histologic and quantitative techniques were compared in the evgluation of the severity of the disease and response to therapy. A highly significant correlation was found between the histologic score of the extent of alveolar involvement (the standard method of assessment) and the number of P. carinii cysts and nuclei in lung homogenates, lung weight, and lung weight/body weight ratio. Clear differences were noted between rats which responded well and rats which responded poorly to therapy by all techniques. Quantitation of P. carinii cysts and nuclei revealed a 104-fold reduction in organism burden with successful treatment. Thus, these techniques should be helpful in the development of testing of new antimicrobial agents in the rat model of pneumocystosis.Pneumocystis carinii, an organism of low virulence, is an important cause of pneumonia in the immunocompromised host, particularly malnourished infants, children with immunodeficiency disorders, and patients of all ages receiving immunosuppressive therapy (20). With the emergence of the acquired immunodeficiency syndrome, the number of cases of pneumocystosis has risen dramatically (1). The problems in the treatment of P. carinii pneumonia in patients with acquired immunodeficiency syndrome (i.e., slow response, frequent relapse, high rate of adverse drug reactions) (8, 16) have stimulated efforts to develop new forms of therapy (3,7,13).Drug development for pneumocystosis has mainly been conducted in animal models in which rats administered corticosteroids for about 8 weeks spontaneously develop the disease (5, 10-13, 15, 22). Assessment of drug efficacy has been based on histopathologic examination of the extent of P. carinii pneumonia in the lungs. While this process has been useful, it is largely subjective and has not permitted the development of uniform or quantitative standards. Thus, it has been difficult to compare the results of one investigator with those of another even if the same treatment regimen was used.We have previously found a high degree of correlation between a semiquantitative histologic scoring system of the intensity of P. carinii pneumonia and the number of P. carinii cysts in lung homogenates of infected animals (21). More recently, we have developed the ability to quantitate P. carinii nuclei in lung homogenates and in tissue culture (2-4); this technique has allowed evaluation of cysts, trophozoites, and intermediate stages in the life cycle of P. carinii and has been a sensitive indicator of organism replication in vitro.In the present study, we applied these methods to the evaluation of drug treatment of experimental pneumocystosis by comparing the histologic score with P. carinii cyst and nuclei counts, lung weight, and lung weight/body weight ratio. * Corresponding author. MATERIALS AND METHODSExperimental protocol. The animals evaluated here were part of a larger study designed to compare the efficacy of different inhibitors of fol...

The major ester‐linked fatly acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid‐trealed rats were 16:0, 18:0, 18:1,18:2 and 20:4. Others detected included 14:0,16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0,22:0,24:0 and 24:1; others included 14:0,18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Recent analyses of rRNA have suggested a taxonomic relationship of Pneumocystis carinii with fungi. We investigated various liquid and solid media without feeder cells for the ability to support growth of P. carinii. Organisms were obtained from the lungs of immunosuppressed rats and separated from host cells by treatment with hypotonic solutions and a series of microfiltrations. The growth of P. caring was quantitated by staining nuclei with Diff-Quik. Optimal replication was observed in media with neopeptone and N-acetylglucosamine at low pH. Increases of at least 8to 10-fold occurred during the first 24 to 72 h. P. carinii replication was influenced by pH, temperature, inoculum size, and anti-P. carinii drugs. The viability of organisms was confirmed by incorporation of radiolabeled uracil, methionine, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitable compounds showed that [35S]methionine was incorporated into specific P. carinùi proteins. Thus, P. carinii can metabolize and replicate in the absence of mammalian cells. This system should have applications for in vitro drug screening and other molecular and metabolic analyses. * Corresponding author. National Cancer Institute were immunosuppressed with weekly injections of 4 mg of methylprednisolone acetate (Depo-Medrol; The Upjohn Co., Kalamazoo, Mich.) for 6 to 8 weeks (9). P. carinii was obtained by homogenization of minced lungs with a Stomacher Lab Blender (Tekmar, Cincinnati, Ohio) as previously described (10-12). Hypotonic lysis and exhaustive filtrations through 10-,um-poresize Mitex filters (Millipore Corp., Bedford, Mass.) were used to provide a preparation free of intact host cells. Inocula were plated on Mueller-Hinton and Sabouraud dextrose agars for the detection of microbial contaminants. In addition, ail inocula were evaluated for the presence of host and microbial contaminants by the following criteria: (i) examination of 10 oil immersion fields (magnification, x 1,000) of each of three 10-,ul drops (for a total of 30 oil immersion fields) stained with Diff-Quik; (ii) examination of an unstained 1-,ul drop by phase-contrast microscopy; and (iii) examination of 10 oil immersion fields of a 10-pul drop stained with Gram stain. Fungal and bacterial surveillance cultures of all inocula used for culture studies were negative. Organism numbers were assessed by enumeration of P. carinii nuclei per milliliter (9-12; see below). The viability of preparations was always >95% by erythrosin B dye exclusion (12). Culture media and conditions. The following media and additives were evaluated for support of P. carinii growth: Dulbecco minimal essential medium (DMEM), yeast extractpeptone-dextrose (YEPD) broth and agar, yeast extract-malt extract (YM) broth, brain heart infusion broth and agar, wort broth and agar, Sabouraud dextrose broth and agar, modified Sabouraud dextrose broth and agar, and Vogel and Johnson agar. Solid agars were tested at pH 4.0 and 7.0. Additives were purchased from...