Defective priming of the phagocyte oxidative burst in a child with recurrent intracellular infections.

Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms. Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation. This process is known as priming. We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms (Mycobacterium bovis, BCG, and Salmonella typhimurium). No T- or B-cell quantitative or qualitative defects were found. Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease. Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H(2)O(2) production after pretreatment with LPS (5 microg/mL for 30 min), TNFalpha (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10(-6) M for 5 min). In these conditions, H(2)O(2) production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H(2)O(2). Moreover, monocytes from the patient showed an impaired capacity to kill S. typhimurium in vitro. Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst. This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens.

Drug evaluation of concurrent Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium complex infections in a rat model.

We present a new experimental model for the simultaneous evaluation of the activities of drugs against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium complex infections. Rats latently infected with P. carinii were challenged with the MO-1 strain of M. avium and then immunosuppressed with corticosteroids for 7 weeks. At week 5 the RH strain of T. gondii was intraperitoneally injected. Organs were examined for the three pathogens after death or killing of the animals at week 7. Without treatment, rats challenged with T. gondii died with pulmonary P. carinii infection and disseminated T. gondii and M. avium infections. In order to assess the value of the model for evaluation of the activities of drugs, we administered by oral gavage for 7 weeks drugs or combinations of drugs selected for their individual efficacies against at least one pathogen. We found that clarithromycin with sulfamethoxazole, clarithromycin with atovaquone, roxithromycin with sulfamethoxazole or dapsone, and rifabutin with atovaquone were effective against the three infections, whereas PS-15 with dapsone and trimethoprim with sulfamethoxazole were active against Toxoplasma and Pneumocystis infections only. This triple-infection rat model offers a new tool for the simultaneous evaluation of the activities of drugs against three of the major opportunistic infections occurring in immunosuppressed individuals.

Influence of Pneumocystis carinii on nitrite production by rat alveolar macrophages.

Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by NG-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by NG-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. carinii-naive rats do not generate nitrite whereas those of latently infected rats do.

Differential nitric oxide (NO) production by macrophages from mice and guinea pigs infected with virulent and avirulent Legionella pneumophila serogroup 1.

L-arginine-dependent reactive nitrogen intermediates have been identified as macrophage cytotoxic effector molecules against intracellular pathogens. To determine its role, ex vivo production of NO by peritoneal macrophages of C3H/HeN mice and Dunkin-Hartley guinea pigs infected intraperitoneally with a virulent and isogenic avirulent Legionella pneumophila serogroup 1 strain was compared with bacterial clearance from the lungs. While the virulent strain was cleared from mice lungs, the guinea pigs died within 96 h. In vivo infection with both strains resulted in the production of NO by mouse peritoneal macrophages ex vivo. In contrast, guinea pig macrophages did not produce detectable NO. In addition, infection by the avirulent strain led to the production of significantly more NO by mouse macrophages than the virulent parent strain, irrespective of stimulation with lipopolysaccharide (LP) and/or interferon-gamma (ifn-gamma). These results suggest that resistance to Leg. pneumophila infection may depend on the production of NO by host macrophages.

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila serogroup 1.

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.

Comparative postantibacterial activities of pefloxacin, ciprofloxacin, and ofloxacin against intracellular multiplication of Legionella pneumophila serogroup 1.

The inhibitory and postantibacterial activities of pefloxacin, ciprofloxacin, and ofloxacin against virulent Legionella pneumophila serogroup 1 were evaluated in cell-free and cellular models. In the absence of macrophages (with the tissue culture medium alone), bacterial numbers remained unchanged at 24 h in the presence of 0.1 microgram of pefloxacin, ciprofloxacin, or ofloxacin per ml and 1.0 microgram of pefloxacin per ml, whereas they were reduced in the presence of 1.0 microgram of ciprofloxacin or ofloxacin per ml. Experiments to evaluate the postantibacterial effects of these drugs were therefore performed with concentrations of 0.1 microgram/ml. In the cell-free model, brief exposure (1 h) of bacteria to each antimicrobial agent resulted in a transient decrease in numbers followed by logarithmic growth. In the cellular model, all three drugs (at 0.1 and 1.0 microgram/ml) inhibited the intracellular multiplication of L. pneumophila. The intracellular postantibacterial effects of 0.1 microgram of pefloxacin, ciprofloxacin, and ofloxacin per ml, which were left in contact with L. pneumophila-infected human macrophages for 24 h, were evaluated at various times after removal of the drugs. Pefloxacin was found to exhibit a significant inhibitory effect at 72 h, whereas following the removal of ciprofloxacin and ofloxacin, rapid bacterial multiplication occurred, leading to the destruction of the macrophage monolayer within 48 h. Thus, while pefloxacin, ciprofloxacin, and ofloxacin all inhibited the multiplication of L. pneumophila in human monocyte-derived macrophages, only pefloxacin exhibited a prolonged postantibacterial effect.