Ethanol feeding does not affect the efficacy or pharmacokinetics of azithromycin, trovafloxacin, or ceftriaxone in a rat model of pneumococcal pneumonia.

A rat model of ethanol feeding was used to study the effects of ethanol on antibiotic therapy of pneumococcal pneumonia. Male Sprague-Dawley rats (150 g) received a liquid diet containing 36% of total calories as ethanol. Controls were pair-fed a liquid diet without ethanol or received rat chow. Diets began 7 days pre- and continued postinfection. Rats were infected transtracheally with type 3 Streptococcus pneumoniae and then treated with azithromycin (50 mg/kg), trovafloxacin (50 mg/kg), or ceftriaxone (100 mg/kg) injected subcutaneously twice daily for 5 days. Antibiotic levels in serum, lung cells, and lavage fluid were measured by HPLC. Ethanol- and pair-fed rats had depressed baseline peripheral neutrophil counts but were able to generate adequate numbers of peripheral and pulmonary polymorphonuclear leukocytes early in the course of their infection. Ethanol feeding did not alter the pharmacokinetics of azithromycin, trovafloxacin, or ceftriaxone. All three antibiotics were equally effective in curing experimental pneumococcal pneumonia, and survival rates were similar in treated ethanol-fed and control rats.

Use of magnetic cell sorting to isolate blood neutrophils from rats.

The difficulty in obtaining sufficient numbers of neutrophils from rat blood limits the usefulness of this species in studies involving neutrophil function. To increase the neutrophil yield from rats drinking alcohol on a long-term basis, which further decreases neutrophil yield, we developed a magnetic cell-sorting technique. The rats were exsanguinated and neutrophils were isolated, using either traditional density gradient centrifugation or magnetic cell sorting. In the latter method, the leukocytes were labeled with biotinylated anti-rat granulocyte antibodies, followed by addition of streptavidin-conjugated superparamagnetic microbeads. The labeled cell suspension was applied to a steel wool column suspended within a magnetic field. Unlabeled cells were washed through the column. Retained, labeled neutrophils were eluted after the column was removed from the magnetic field. Compared with density gradient centrifugation, magnetic cell sorting yielded two- to fivefold higher neutrophil numbers per rat with increased purity. Viability was comparable for neutrophils isolated by the two techniques. Magnetic cell sorting is a rapid, gentle method for isolation of rat blood neutrophils and enhances the potential usefulness of rats in neutrophil-related research.

Effects of granulocyte colony-stimulating factor in cirrhotic rats with pneumococcal pneumonia.

A rat model was used to study the effects of granulocyte colony-stimulating factor (G-CSF) on the pathogenesis of pneumococcal pneumonia in cirrhosis. G-CSF or 5% dextrose in water was administered subcutaneously to cirrhotic and control rats before or after transtracheal infection with type 3 Streptococcus pneumoniae. In both groups, G-CSF significantly increased the total number and percentage of polymorphonuclear leukocytes (PMNL) in peripheral blood (P < .002) and bronchoalveolar lavage fluid (P < .01). An in vivo phagocytosis assay revealed no increase in uptake of pneumococci by PMNL within the lungs of cirrhotic or control rats receiving G-CSF. G-CSF administered before infection did not protect cirrhotic or control rats, but G-CSF treatment after infection significantly reduced mortality in control (P = .04) but not cirrhotic rats. These data suggest that despite increasing numbers of circulating and pulmonary PMNL, G-CSF does not protect against fatal pneumococcal pneumonia in cirrhotic rats.

Phagocytosis of Streptococcus pneumoniae measured in vitro and in vivo in a rat model of carbon tetrachloride-induced liver cirrhosis.

Both humans and rats with liver cirrhosis have increased morbidity and mortality from pneumococcal pneumonia. By use of a rat model of carbon tetrachloride-induced liver cirrhosis, uptake of fluorochrome-labeled Streptococcus pneumoniae by polymorphonuclear leukocytes (PMNL) and alveolar macrophages (AM) was examined by flow cytometry. Peripheral blood PMNL from cirrhotic rats showed no defect in phagocytic or bactericidal capacity for type 10A S. pneumoniae in vitro. However, in vivo, fewer type 3 S. pneumoniae were engulfed by PMNL in the lungs of cirrhotic rats with a concomitant increase in the number of organisms taken up by their AM in comparison with controls. These studies indicate the importance of using more relevant in vivo methodologies for assessing bacterial phagocytosis. In addition, the reduction in uptake of type 3 pneumococci by PMNL within the microenvironment of the cirrhotic rat lung could help to explain the increased susceptibility of cirrhotic rats to pneumococcal pneumonia.

Effect of cirrhosis on the production and efficacy of pneumococcal capsular antibody in a rat model.

We sought to study the immunogenicity of Type 3 pneumococcal capsular polysaccharide (PCP) antigen and the protective efficacy of Type 3 PCP antibodies in a rat model of cirrhosis. Cirrhosis with ascites was induced in male Sprague-Dawley rats by weekly gavage with CCl4. Cirrhotic and age-matched control rats were vaccinated with 25 micrograms of Type 3 PCP. Serum antibodies against Type 3 PCP were determined before vaccination and on postvaccination Days 5, 7, 10, 14, 21, 28, and 42 by radioimmunoassay. Maximum concentrations occurred at 7 days in cirrhotic rats and 10 to 14 days in control rats. Geometric mean Type 3 PCP antibody levels (ng AbN/ml) were higher in cirrhotic versus control rats before vaccination (75.9 versus 33.8; p = 0.011) and on post-vaccination Day 5 (626 versus 158; p = 0.008) and Day 7 (1,755 versus 493; p = 0.002). Postvaccination antibody from immunized control and cirrhotic animals provided passive immunity to Type 3 Streptococcus pneumoniae infection in mouse protection studies. Sham-immunized and PCP-immunized control and cirrhotic rats were challenged with 10(7) cfu Type 3 S. pneumoniae. Immunization was associated with a greater reduction in postchallenge mortality in control rats (91% reduced to 36%; p = 0.02) compared with cirrhotic rats (100% reduced to 83%; p = 1.0). Thus, the increased serum concentrations of functional, type-specific anticapsular antibody in vaccinated cirrhotic rats does not reverse their impaired resistance to Type 3 pneumococcal pneumonia.

Pulmonary recruitment, adherence, and chemotaxis of neutrophils in a rat model of cirrhosis and pneumococcal pneumonia.

Cirrhotic rats have decreased pulmonary bactericidal activity and increased bacteremia after experimental pneumococcal pneumonia. To determine if this finding is due to impaired pulmonary recruitment of polymorphonuclear leukocytes (PMNL), bronchoalveolar lavage (BAL) was done on cirrhotic and normal rats after transtracheal challenge with pneumococcal types 3 and 1. Mean absolute numbers of recruited PMNL in BAL fluid (BALF) at 2, 4, 6, 8, and 24 h after 10(7) cfu of type 3 challenge were similar in cirrhotic and normal rats. In both groups, lower numbers of PMNL were recruited after challenge with 10(5) cfu of type 3. Type 1 pneumococci stimulated recruitment of similar mean absolute numbers of PMNL (x10(7] in BALF (cirrhotics vs. normals) at 24 h after challenges with 10(5) cfu (0.3 +/- 0.1 vs. 0.3 +/- 0.1) and 10(7) cfu (2.9 +/- 1.3 vs. 2.8 +/- 0.7). Peripheral blood PMNL from cirrhotic and normal rats did not differ in adherence to nylon wool columns or in chemotaxis toward lipopolysaccharide-activated normal rat serum. Thus the impaired pulmonary defense against pneumococcal pneumonia in cirrhosis is not due to deficient pulmonary PMNL recruitment.