Pulmonary leukostasis can be associated with acute lung injury. We studied lung peroxidase activity using myeloperoxidase (MPO) as a granulocyte marker to quantitate pulmonary leukostasis in rabbits. Lungs were homogenized in detergent, freeze-thawed, sonified, and centrifuged, and supernatants were assayed for MPO. Seven extractions were performed, and greater than 80% of cumulative MPO was found in the first three extractions. By use of a three-extraction procedure, the mean lung MPO (delta A X min-1 X g tissue-1) was determined in normal [20.9 +/- 5.2 (SE)], granulocyte-depleted (6.5 +/- 2.0), saline-injected (22.2 +/- 5.6), and pneumococcus (PNC)-challenged (69.7 +/- 10.6) animals. Lung MPO was significantly decreased in granulocyte-depleted compared with normal animals (P less than 0.005) and significantly increased in PNC-challenged compared with saline-injected animals (P less than 0.001). MPO extracted from granulocytes and lungs from normal as well as PNC-challenged animals were all biochemically identical. Lung extract did not inhibit MPO, and no MPO was detected in bronchoalveolar lavage fluid obtained from leukostatic lungs. Lung MPO significantly (P less than 0.01) correlated with intravascular intrapulmonary granulocytes. Determination of lung MPO is a relatively simple quantitative method that can be used to detect pulmonary leukostasis.
Trimetrexate and BW301U (piritrexim isethionate), lipid-soluble inhibitors of dihydrofolate reductase, are potent inhibitors of the growth of Pneumocystis carinii in culture with WI-38 cells. Inhibition was observed with 0.1 ,Ig of trimetrexate or BW301U per ml. Trimethoprim is ineffective at 100 ,ug/ml in this culture system. Both trimetrexate and BW301U were effective as prophylactic agents against P. carinii pneumonia in rats; trimetrexate at 7.5 mg/kg protected 9 of 10 rats, and BW301U at 5 mg/kg protected 4 of 10.Drugs approved for treatment or prophylaxis of pneumonia caused by Pneumocystis carinii are unsatisfactory for many patients. Trimethoprim in combination with sulfamethoxazole or other sulfonamides often causes adverse reactions in acquired immunodeficiency syndrome patients (10, 13). The other commonly prescribed drug, pentamidine, causes a variety of mild to severe side effects (16). Other drugs and drug combinations have been used for treatment and prophylaxis of P. carinii pneumonia, but the numbers of patients treated have been small and the response to drugs has been difficult to evaluate in many instances. Pyrimethamine with sulfadoxine (Fansidar) is being evaluated (19), and difluoromethylornithine has had limited success in a small number of patients (17). Dapsone plus trimethoprim caused clinical improvement in 15 patients but still caused significant adverse reactions (14).P. carinii does not grow in continuous culture but will undergo several replicative cycles when grown in contact with certain mammalian cell lines (4, 7). The organisms grow extracellularly, some remaining attached to the host cells and some being released into the medium. Growth is less predictable than for many other cultured organisms, but with appropriate controls the system is useful for studying drug effects on P. carinii. Previously we have been able to identify drugs with activity against P. carinii by incorporating drugs into medium on cultures of WI-38 cells infected with P. carinii from rats (2, 3). This culture system has now been used to identify two folate inhibitors that strongly inhibit proliferation of P. carinii: trimetrexate and BW301U (piritrexim isethionate). Both drugs were originally developed as anticancer agents (5,11,15,18) and are believed to inhibit dihydrofolate reductase in a manner similar to that of methotrexate but are proposed to enter cells more readily because these new agents are more lipid soluble than methotrexate. These and the other drugs reported in this study were selected for testing because drugs with similar mechanisms of action (trimethoprim and pyrimethamine) are among the most useful agents for treating infections caused by P. carinii. * Corresponding author. MATERIALS AND METHODSCultures of Pneumocystis carinii were prepared and evaluated as previously described (2-4). Briefly, human embryonic lung fibroblastic cells (WI-38) were cultured in 12-well tissue culture plates with minimum essential medium containing 10% fetal calf serum. Confluent monolayers were inoculated ...
Rats free of latent Pneumocystis carinii organisms were immunosuppressed with adrenal corticosteroids and transtracheally injected with P. carinii. These animals subsequently developed P. carinii pneumonia. Infection was accomplished by using organisms from infected rat lung or from culture. Diffuse infection was produced with no significant differences in the numbers of organisms found in various lobes of the lungs. Infections progressed over time so that by 6 weeks postinoculation all animals were heavily infected. Infection by transtracheal injection has three advantages over current models. First, transtracheal injection provides a reliable model which is not dependent on naturally occurring latent Pneumocystis infection. Second, transtracheal injection allows the perpetuation of specific Pneumocystis strains. Third, transtracheal injection is a more rapid and economical means of producing severe Pneumocystis pneumonia.
The combination of primaquine with clindamycin is effective in both in vitro and in vivo models of Pneumocystis infection. Primaquine alone at concentrations from 10 to 300 ,ug/ml reduced the numbers of organisms in cultures to less than 7% of control. Significant inhibition was observed down to 0.1 ,ug/ml. Clindamycin at 5 ,ug/ml was ineffective alone. Combinations of clindamycin and primaquine in culture at various concentrations were effective, but there was no evidence of true synergy. In rats with established Pneumocystis pneumonia, clindamycin alone at 5 or 225 mg/kg was ineffective. Primaquine alone at 0.5 or 2 mg/kg did not significantly affect the numbers of organisms remaining. The combination of 0.5 mg of primaquine per kg and 225 mg of clindamycin per kg was effective for therapy, lowering the numbers of organisms in the lungs by about 90%. The combination of 2 mg of primaquine per kg and 225 mg of clindamycin per kg was more effective, lowering the numbers of organisms by almost 98%. In the in vivo prophylaxis model, primaquine at 0.1 or 0.2 mg/kg did not prevent the development of Pneumocystis pneumonia in immune-suppressed rats. Clindamycin at 50 mg/kg had a modest effect alone, but at 5 mg/kg all animals became heavily infected. At 0.5 mg/kg, primaquine alone reduced the severity of infection, but seven of eight rats were still infected. In contrast, the combination of 5 mg of clindamycin per kg and 0.5 mg of primaquine per kg prevented infection in 8 of 10 rats; 2 rats had minimal infection. These studies suggest that the combination of clindamycin and primaquine should be tested in therapy or prophylaxis of Pneumocystis infections in humans.Treatment or prophylaxis of pneumonia caused by Pneumocystis carinii in patients with acquired immune deficiency syndrome (AIDS) has been hampered by the lack of effective, nontoxic agents. Pentamidine causes a variety of side effects which may limit therapy, and relapses are frequent (29,34). Trimethoprim with sulfamethoxazole causes a surprisingly high incidence of side effects in AIDS patients, including severe hypersensitivity reactions that limit use for treatment (29,33,34). Other experimental agents have been tested with variable results. Difluoromethylornithine is effective in some patients, but thrombocytopenia may be a limiting toxicity (9, 34). Pyrimethamine with sulfadoxine (Fansidar) or dapsone with trimethoprim may be effective in individual patients, but significant adverse reactions still occur with both combinations (10, 21, 34). Trimetrexate, a lipid-soluble analog of methotrexate, is being investigated (1).In the course of screening antimicrobial agents for potential effectiveness against P. carinii, we have evaluated combinations as well as individual agents. One of the combinations we chose to test was clindamycin with primaquine. This selection was based on the observation by Schmidt (25) that the curative action of primaquine in malaria was enhanced by mirincamycin. Mirincamycin, like clindamycin, is a lincosamide but has not been exte...
Tumor necrosis fattor a (TNF-a) mediates components of the acute-phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied whether human recombinant TNF-a (rTNF-a) could. increase pulmonary edema formation and pulmonary vascular permeability. Rabbits preinfused with '251-albumin were administered rTNF-a or saline. Animals were sacrificed, and lung wet/dry weight ratios as well as bronchoalveolar lavage fluid and plasma 125I activities were determined. rTNF-a increased lung wet/dry weight ratios by 151% (P < 0.02) and bronchoalveolar lavage fluid/plasma 1251 activity ratios by 376% (P < 0.01) compared with values for saline controls. Electron microscopy of lung sections demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural permeability tracer. To demonstrate that rTNF-a could directly increase pulmonary vascular endothelial permeability in vitro, we studied albumin transfer across cultured porcine pulmonary artery endothelial cell monolayers. rTNF-a induced time-dependent dose-response increments in transendothelial albumin flux in the absence of granulocyte effector cells. These observations suggest that rTNF-a can provoke acute pulmonary vascular endothelial injury in vivo as well as in vitro.
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