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.

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.

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.

Comparison of serum responses in swine after vaccination and challenge exposure with Actinobacillus pleuropneumoniae serotype 1.

Clinical trials have shown that currently available commercial vaccines against porcine pleuropneumonia provide inconsistent, serotype-specific protection from the disease. Recovery from naturally acquired infection, however, provides solid, serotype cross-protective immunity. We examined various serum responses of pigs receiving 1 of 4 commercial vaccines or a cell extract, and compared the serologic responses of these pigs after challenge exposure with virulent Actinobacillus pleuropneumoniae serotype 1. Evaluation of serum included complement-mediated killing, opsonizing capacity, IgG titers to whole organisms, and cytotoxin neutralization titers. Pigs that received the cell extract had fewer clinical signs of pleuropneumonia than pigs in other vaccinated groups, and also were significantly (P < 0.05) better protected from development of lung lesions and death. Such vaccinates were the only pigs that developed significant (P < 0.05) serum antibody titers (ie, protective immune response) to whole-cell antigens and to cytotoxin.

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.

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.

Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants.

The leukotoxin of Pasteurella haemolytica is a major virulence factor of the organism. It is an unstable protein which has proven very difficult to purify using traditional techniques. Hybridomas secreting monoclonal antibodies (mAbs) to P. haemolytica leukotoxin were derived from spleen cells of a mouse immunized with crude culture supernatant. Five hybridomas secreting mAbs specific for the leukotoxin were stabilized. Each of the mAbs reacted with a protein of approximately 100 kDa in toxic culture supernatants, and two of them completely neutralized the toxin in vitro. Affinity chromatography of crude culture supernatant on a column prepared with one of the neutralizing mAbs resulted in the isolation of biologically active toxin.

Cloning and expression of a 30 kDa surface antigen of Pasteurella haemolytica.

Pasteurella haemolytica biotype A serotype 1 is the principal etiologic agent of bovine pneumonic pasteurellosis. A clear understanding of the pathogenesis of this disease and the mechanisms of resistance to it has been limited by a lack of information on the important antigens of the organisms. Using recombinant DNA techniques we have cloned a segment of DNA from P. haemolytica A1 that encodes three proteins of 28, 30, and 32 kDa. Two of these proteins, 30 and 28 kDa, react strongly on a Western blot with a bovine serum raised against live cells of P. haemolytica A1. The gene for the 30 kDa protein was localized to a 3.1 kbp EcoRI fragment, and expression of the 30 kDa protein was found to be independent of an E. coli promoter. The 30 kDa protein comigrated with a 30 kDa P. haemolytica protein that was susceptible to radioiodination and presumably exposed on the bacterial cell surface. The other principal radiolabeled P. haemolytica proteins were 100, 45, and 15 kDa. Antibodies against the 30 kDa protein, isolated from E. coli carrying the recombinant plasmid, recognized 30 kDa and 15 kDa proteins in P. haemolytica serotypes 1-15 and caused agglutination of whole P. haemolytica A1 cells. Cattle vaccinated with live P. haemolytica, P. haemolytica outer membrane proteins, or the cloned 30 kDa protein developed antibodies to the cloned 30 kDa protein as detected by Western blotting and densitometry. Sera were obtained from cattle vaccinated with live or killed P. haemolytica or saline and challenged with P. haemolytica. Those sera were evaluated for antibody responses to the cloned 30 kDa protein. High antibody responses to the 30 kDa protein significantly correlated (P less than 0.01) with resistance to challenge. From these studies it is concluded that the 30 kDa protein represents a surface antigen of P. haemolytica A1 that may be important in inducing immunity to P. haemolytica.

Resistance to host immune defense mechanisms afforded by capsular material of Pasteurella haemolytica, serotype 1.

Selected serum-mediated host immune defense mechanisms against Pasteurella haemolytica were studied using encapsulated and decapsulated organisms. When the capsular material was removed from P. haemolytica, it became more susceptible to serum agglutination, complement-mediated serum killing, and phagocytosis by polymorphonuclear leukocytes. When encapsulated organisms were used, phagocytosis was enhanced by antibodies to capsular material produced by vaccination of calves with any of three P. haemolytica vaccines. The serum bactericidal activity, however, was not facilitated by increased levels of anticapsular antibody in vaccinated cattle. By contrast, when decapsulated organisms were used, vaccination enhanced both the bactericidal and opsonizing capacities of sera from the calves. These studies indicate that capsular material should be considered a principal virulence factor for P. haemolytica.

Comparison of the toxic and antigenic properties of single bovine isolates of Pasteurella haemolytica representing five serotypes and an untypable strain.

Single strains of 5 different P. haemolytica serotypes (1, 2, 5, 6 and 9) and an untypable strain were compared in an attempt to detect differences which might be related to virulence. All but the untypable strain caused extensive lesions when injected into the lungs of healthy cattle. Each strain was found to be encapsulated and to be toxic in vitro for bovine leukocytes. Each strain also produced leukotoxin in vitro. The toxins varied, however, in total toxic activity and in the kinetics of leukotoxin production. Vaccination of cattle with each of the serotype strains elicited antibodies to organism somatic antigens and, to various degrees, the production of leukotoxin-neutralizing antibodies which showed no strain specificity in cross-neutralization studies. Although each of the serotype strains appeared to be a potential bovine pathogen, subtle differences were observed which may explain the importance of Serotype 1 strains in bovine pneumonic pasteurellosis.