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 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...

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The three nuclei of the cat lateral lemniscus (dorsal, intermediate, and ventral) were distinguished by their immunoreactivities for the putative inhibitory transmitters, gamma-aminobutyric acid (GABA) and glycine. Each nucleus had a distinct pattern of somatic and perisomatic labeling. The dorsal nucleus contained mostly GABA-immunoreactive neurons (85%), with moderate numbers of GABA- and glycine-immunoreactive puncta along their somata. The remaining neurons were nonimmunoreactive (15%). The intermediate nucleus contained mostly nonimmunoreactive neurons (82%), and these had numerous glycine-immunoreactive and few GABA-immunoreactive perisomatic puncta. The remaining neurons were immunoreactive for GABA only (10%), glycine only (2%), or both (6%). The ventral nucleus contained mostly glycine-immunoreactive neurons (81%), and about half of these were also GABA-immunoreactive. The remaining neurons were either nonimmunoreactive (8%) or GABA-immunoreactive only (11%). Neurons in the ventral nucleus had fewer immunoreactive perisomatic puncta than neurons in either the dorsal or the intermediate nuclei. These differences in neuronal immunoreactivity and in the relative abundance of GABA-and glycine-immunoreactive perisomatic puncta among the three nuclei of the lateral lemniscus support connectional and electrophysiological evidence that each nucleus has a different functional role in auditory processing. In particular, this study demonstrates that the intermediate nucleus of the cat is cytochemically distinct from the dorsal and ventral nuclei in terms of the somatic and perisomatic immunoreactivity of its neurons for these two important inhibitory transmitters and may provide novel inputs to the inferior colliculus.

One of the most prominent features of S-antigen induced uveitis is the massive infiltration of polymorphonuclear leukocytes (PMNs) and mononuclear cells in the ocular tissues and fluids. These inflammatory cells generate reactive oxygen metabolites as microbicidal agents and release these oxidants into the surrounding tissues. Using the cerium perhydroxide method, we have localized subcellular hydrogen peroxide in various inflamed ocular tissues. Most notably, the positive electron-dense granules were seen in the plasma membranes of PMNs that were infiltrating in the retina and uvea. These deposits were noted also in PMNs located within the extravascular spaces. For the intravascular PMNs, the positive reaction products were seen in much lower concentrations. A direct demonstration of substantial concentrations of hydrogen peroxide in experimental autoimmune uveitis, therefore, suggests the possibility that this reactive metabolite is an inflammatory mediator in this condition.