Pneumolysin-induced complement depletion during experimental pneumococcal bacteremia.

To quantify complement depletion by pneumolysin during Streptococcus pneumoniae bacteremia, cirrhotic and control rats were infected intravenously with one of three isogenic mutant strains of S. pneumoniae expressing different forms of pneumolysin. Outcome measures included clearance of the organisms from the bloodstream, alterations in 50% serum hemolytic complement (CH(50)) activity and complement C3 levels during infection, and serum opsonic capacity at 18 h postinfection. Cirrhotic rats had significantly lower CH(50) and C3 levels than control rats, both before and after infection. However, initial complement levels did not predict bacterial load after 18 h of infection. Changes in CH(50) and C3 levels over the 18-h period correlated with numbers of H+C+ but not H+C- or PLY- organisms in the bloodstream at 18 h postinfection. The sera of cirrhotic rats infected with the H+C+ strain had significantly decreased levels of C3 and showed significantly lower opsonizing activity for S. pneumoniae than sera from H+C+-infected control rats. These studies suggest that under limiting concentrations of complement, the expression of pneumolysin by pneumococci has a significant, negative effect on serum complement levels and reduces serum opsonic activity.

Use of rat models to mimic alterations in iron homeostasis during human alcohol abuse and cirrhosis.

With alcoholism, there are marked disturbances in iron homeostasis that are linked to alterations in serum transferrin and ferritin concentrations. This study identifies rat models of alcohol abuse that closely mimic these disturbances. Male rats were placed in one of the following three protocols: (1) pair-feeding of liquid diets for 1-8 weeks; (2) agar-block feeding for 8 weeks; or (3) generation of cirrhosis with CCl(4). Serum samples were analyzed for ferritin, transferrin, and iron levels, and the transferrin iron saturation and ferritin/transferrin ratios were calculated. Liver iron concentrations were also determined. Serum transferrin levels were elevated in animals fed alcohol for 8 weeks in pair-feeding and agar-block feeding protocols, but reduced in rats with cirrhosis. Serum ferritin concentration was reduced in rats fed ethanol in the liquid diet, but increased in rats consuming ethanol in agar blocks, in rats pair-fed the liquid control diet, and in rats with cirrhosis. This finding was mirrored by liver nonheme iron concentrations in all experimental groups, but not in the corresponding control groups. Serum iron levels were significantly elevated only in rats fed the liquid control diet. There was a progressive decrease in transferrin iron saturation and ferritin/transferrin ratios for animals fed ethanol in the liquid diet, but not when ethanol was ingested from agar blocks. The development of cirrhosis resulted in elevated liver iron concentrations and doubled ferritin/transferrin ratios. It is concluded that these models may be used to study disturbances in iron homeostasis that occur during alcohol abuse and the (subsequent) development of liver disease.

Pneumococcal vaccine response in cirrhosis and liver transplantation.

Cirrhosis is a major risk factor for severe pneumococcal infection, and patients evaluated for liver transplantation routinely receive pneumococcal vaccine. This study followed serologic antibody levels of 45 adults evaluated for transplantation and 13 age-matched control subjects. All received 23-valent pneumococcal polysaccharide vaccine (PPS). Serum anti-PPS levels and antibodies specific for capsular types 3 and 23 were measured by ELISA before and 1 and 6 months after vaccination. Antibody levels for the 25 patients who received transplants also were measured immediately before and 3 months after transplantation. Control subjects had higher IgG responses to the whole vaccine, whereas patients appeared to produce more IgM and IgA. IgA, and possibly IgM levels, also declined faster in patients than in control subjects. All anti-PPS levels were at or below prevaccination baselines by 3 months after transplantation. These data suggest that vaccination with PPS may not be effective for patients during and after liver transplantation.

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.

Role of Pneumolysin's complement-activating activity during pneumococcal bacteremia in cirrhotic rats.

We investigated the role of pneumolysin's complement-activating activity during Streptococcus pneumoniae bacteremia in a hypocomplementemic, cirrhotic host. Isogenic mutant pneumococcal strains, in which pneumolysin was expressed from a plasmid, were used. These strains included H+C+, expressing wild-type pneumolysin with both cytolytic and complement-activating activity; PLY-, carrying the plasmid without the pneumolysin gene; and, H+C-, expressing pneumolysin with cytolytic activity only. In control rats, intravenous infection with 2.0 x 10(7) CFU of H+C+ per ml of blood resulted in a decrease in bacteremia of 3.5 log units by 18 h postinfection and 55% mortality. By contrast, cirrhotic rats infected similarly with the H+C+ strain demonstrated a 0.2-log-unit increase in bacteremia by 18 h postinfection and 100% mortality. Both control and cirrhotic rats cleared the PLY- strain more effectively from their bloodstreams by 18 h postinfection (6.2 and 5. 6 log unit decreases, respectively). Infection with the PLY- strain also resulted in low mortality (0 and 14%, respectively) for control and cirrhotic rats. When infected with the H+C- strain (without complement-activating activity), both groups cleared the organism from their bloodstreams nearly as well as they did the PLY- strain. Furthermore, the mortality rate for control and cirrhotic rats was identical after infection with the H+C- strain. These studies suggest that pneumolysin production contributes to decreased pneumococcal clearance from the bloodstream and higher mortality in both control and cirrhotic rats. However, pneumolysin's complement-activating activity may uniquely enhance pneumococcal virulence in the hypocomplementemic, cirrhotic host.

Fellowship training in infectious diseases: a report from the regional and national meetings of infectious diseases division chiefs and program directors.

During the 2-year period April 1995 to April 1997, six regional meetings and one national meeting of division chiefs and program directors of adult infectious diseases programs in the United States were held to review fellowship training. Herein, we report data on job availability and job selection for recently graduated fellows. We summarize discussions on decreasing the number of fellows in training, and we outline suggested components of a core clinical curriculum and of three training tracks--clinician track, clinical investigator track, and basic investigator track.

Career opportunities for infectious diseases subspecialists.

Few data are available regarding trends in career opportunities for subspecialty physicians. A review of advertisements for infectious diseases physicians in the 1990, 1993, and 1995 issues of the New England Journal of Medicine revealed a significant decline in the number of openings between 1990 and 1995. In each year the number of private practice advertisements exceeded those in all other categories combined, and this ratio increased over time. Both private practice and academic advertisements commonly listed opportunities or requirements for teaching or research. Expertise in internal medicine, rarely mentioned in advertisements for academic infectious diseases physicians, became the most frequently cited private practice subclassification in 1995. Both private practice and academic settings offered numerous positions related to the care of patients with human immunodeficiency virus infection. The demand for expertise in epidemiology, intravenous therapy, travel medicine, transplantation, or sexually transmitted diseases remained low. Most positions were in heavily populated states or geographic areas.

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.

Vaccination with protein-conjugated and native type 3 capsular polysaccharide in an ethanol-fed rat model of pneumococcal pneumonia.

A chronic ethanol-fed rat model was used to determine the effect of alcohol ingestion on production of antibody to type 3 pneumococcal capsular polysaccharide. Sprague-Dawley rats were fed a liquid diet containing 36% of calories as ethanol (ethanol-fed), an isocaloric diet containing dextrin-maltose (pair-fed) or standard rat chow (chow-fed). After 7 days of feeding, the rats were vaccinated subcutaneously with placebo or with either 25 microg of type 3 pneumococcal capsular polysaccharide (SpnCP) or 5 microg of SpnCP linked to the protein carrier CRM197 (SpnCP/CRM197). Rats given the conjugated vaccine received a booster injection 14 days later. Maximum antibody titers were observed six days postvaccination for rats given SpnCP alone and 21 days postvaccination for rats given SpnCP/CRM197. All rats were infected transtracheally with 2-3 times the expected lethal dose50 for each feeding group of type 3 Streptococcus pneumoniae on the day of peak antibody titers. Mortality was recorded for a 10-day period. Vaccination with SpnCP increased survival of ethanol- and chow-fed, but not pair-fed rats. This protection was only statistically significant in the chow-fed group (p < 0.01). Vaccination with SpnCP/CRM197 moderately increased survival of rats in all three feeding groups, but this was not statistically significant in any of them.

Ethanol ingestion impairs neutrophil bactericidal mechanisms against Streptococcus pneumoniae.

Ethanol ingestion impairs both killing of selected pneumococcal strains by rat polymorphonuclear leukocytes (PMNL) in vitro and clearance of these same strains from experimentally infected rat lungs. To determine the mechanism(s) of this impairment, we isolated neutrophils (PMNL) by a magnetic cell sorting technique from the peripheral blood of chow-fed rats (C-PMNL) or rats pair fed for 7 days with a liquid diet providing 36% of its calories as either ethanol (E-PMNL) or dextrin-maltose (P-PMNL). Phagocytosis of fluorochrome-labeled bacteria and oxygen radical production, as determined by oxidation of dihydrorhodamine 123, were measured by flow cytometry. Degranulation, as determined by lysozyme release, was measured as lysis of a suspension of Micrococcus lysodeikticus. E-PMNL, P-PMNL, and C-PMNL were equivalent in their ability to phagocytose pneumococci and to produce an oxidative burst in response to stimulation by opsonized zymosan. However, E-PMNL produced fewer oxygen radicals and released less lysozyme than either P-PMNL or C-PMNL when stimulated by exposure to S. pneumoniae. There was no difference in oxygen radical production by E-PMNL and P-PMNL when stimulated with phorbol myristate acetate, but both cell types mounted a significantly reduced response in comparison to C-PMNL. These data suggest that ingestion of ethanol for 7 days significantly reduces both the oxidative burst and degranulation of rat PMNL in response to S. pneumoniae, thereby compromising anti-pneumococcal activity.

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.

The effect of ethanol ingestion on killing of Streptococcus pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis by rat neutrophils.

The precise effects of chronic ethanol ingestion on the role of neutrophil function in pneumococcal pneumonia have not been fully delineated. In this study, the bactericidal capacity of polymorphonuclear leucocytes (PMNL) from rats pair-fed an ethanol-containing liquid diet or a liquid control diet was compared both in vitro and in vivo. The PMNL were allowed to phagocytose several bacterial species in vitro, and intracellular killing after 1h was determined by plate counts. There was no significant difference in killing of Streptococcus pneumoniae types 6B or 37, Staphylococcus aureus or Staphylococcus epidermidis by PMNL from ethanol-fed and pair-fed rats (E-PMNL and P-PMNL, respectively). However, E-PMNL killed significantly less of Streptococcus pneumoniae types 10A, 14 and 19F. To corroborate these results in vivo, rats were infected transtracheally, and quantitative lung counts were performed 1 week post infection. Streptococcus pneumoniae types 6B and 37, as well as Staphylococcus aureus, were effectively cleared from the lungs of both groups of rats. Streptococcus pneumoniae types 10A, 14 and 19F, however, were cleared well from the lungs of pair-fed but not ethanol-fed animals. These data suggest that chronic ethanol ingestion induces a strain-specific deficit in neutrophil bactericidal activity against certain S. pneumoniae which does not extend to commonly encountered staphylococci.

Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir.

Characterization of a ganciclovir-resistant cytomegalovirus strain from a patient with AIDS showed a histidine-to-glutamine change at residue 520 of UL97 (Q520 mutation). In anabolism studies, Q520 was associated with impaired phosphorylation of ganciclovir. Transfer of Q520 to a recombinant virus resulted in a ganciclovir-resistant phenotype.

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.

Granulocyte colony-stimulating factor protects control rats but not ethanol-fed rats from fatal pneumococcal pneumonia.

A model of chronic ethanol ingestion was used to study the effects of granulocyte colony-stimulating factor (G-CSF) on the pathogenesis of pneumococcal pneumonia in intoxicated rats. G-CSF or 5% dextrose in water (D5W) was administered subcutaneously to ethanol-fed and pair-fed control rats on days 6 and 7 of pair feeding. Rats were infected transtracheally with type 3 Streptococcus pneumoniae on day 8. In pair-fed control rats, G-CSF significantly increased the total number and percentage of polymorphonuclear leukocytes (PMNL) in the peripheral blood (P < .001), augmented PMNL recruitment to infected lungs (P < .01), and significantly increased survival from pneumococcal pneumonia (P = .01). In contrast, treatment of ethanol-fed rats with G-CSF did not enhance pulmonary PMNL delivery and did not increase survival following experimental pneumococcal pneumonia, despite a significant increase in the total number and percentage of circulating PMNL (P < .001). These data suggest that despite increasing the numbers of circulating PMNL, G-CSF is unable to provide protection against fatal pneumococcal pneumonia in ethanol-fed rats.

Ethanol impairs neutrophil chemotaxis in vitro but not adherence or recruitment to lungs of rats with experimental pneumococcal pneumonia.

The effect of 7 days of ethanol ingestion on circulating neutrophil (PMNL) counts and PMNL adherence, chemotaxis, and recruitment was investigated. Pair-feeding of rats resulted in a significant decrease in PMNL counts in both ethanol-fed and control rats. The mean number of PMNL exhibiting chemotaxis in a modified Boyden chamber in response to lipopolysaccharide-activated normal rat serum was significantly decreased in ethanol-fed rats compared with controls. The percentage of adherence to nylon wool columns, however, was similar in both groups. To measure pulmonary PMNL recruitment, rats were infected transtracheally with 10(5) Streptococcus pneumoniae and sacrificed. Bronchoalveolar lavage fluid from both groups contained similar numbers of PMNL 8 h after infection. By 24 h, PMNL numbers in lavage fluid from ethanol-fed rats exceeded those in controls. PMNL recruitment continued in the ethanol-fed rats at 48 and 72 h, whereas values in controls had returned to baseline. Thus, the impaired pulmonary defense against S. pneumoniae in ethanol-fed rats is not due to defective PMNL recruitment.

Treatment of Strongyloides stercoralis hyperinfection syndrome with thiabendazole administered per rectum.

There is a rising interest in Strongyloides stercoralis infection due to the expanding population of immunosuppressed patients. Currently the drug of choice for both enteric and tissue forms of infection with this organism is oral thiabendazole. We report a patient with a small bowel obstruction due to S. stercoralis hyperinfection who was unable to take thiabendazole orally. Thiabendazole was administered rectally, and the hyperinfection syndrome resolved. Peak serum concentrations of thiabendazole were achieved 4 hours after rectal administration, and drug levels were sustained longer than previously reported with oral dosing. In addition, elevated levels of thiabendazole metabolites in the patient's urine further confirmed significant absorption. Rectal administration of thiabendazole should be considered for patients unable to take the medication orally.