Aggregation and opsonization of type A but not type B Hemophilus influenzae by surfactant protein A.

The ability of surfactant protein A (SP-A) to aggregate and opsonize type a and b Hemophilus influenzae was investigated. Type a, but not type b, was aggregated by SP-A. Aggregation was maximal at 24 micrograms SP-A/ml and was Ca(2+)-dependent. Aggregation of type a was inhibited by D-glucosyl-BSA but not by high concentrations of monosaccharides (D-mannose, D-galactose, D-glucose, or L-fucose) or by sialic acid, purified type a capsular polysaccharide, or type IV collagen. In Western blots, 125I-labeled SP-A bound to the major outer membrane protein (putatively P2) of type a hemophilus by a Ca(2+)-dependent mechanism. This binding was competitively inhibited by excess unlabeled SP-A. 125I-labeled SP-A also bound to the major membrane protein of type b, but at less than 5% of the level observed for type a. SP-A did not bind to lipooligosaccharides of either type a or type b. SP-A increased association of type a, but not type b, hemophilus with alveolar macrophages. After opsonization with SP-A, type a hemophilus were killed by alveolar macrophages, as indicated by bactericidal assays and the release of soluble, radiolabeled products from leukocytes. It is concluded that SP-A aggregated and opsonized type a hemophilus, but not type b, possibly because SP-A bound to the P2 outer membrane protein of type a to a greater extent.

Diagnosis of equine influenza by the polymerase chain reaction.

Influenza A is a common respiratory infection of horses, and rapid diagnosis is important for its detection and control. Sensitive detection of influenza currently requires viral culture and is not always feasible. The polymerase chain reaction (PCR) was used to detect DNA produced by reverse transcription of equine influenza in stored nasal secretions, vaccines, and allantoic fluids. Primers directed at a target of 212 bp on conserved segment 7 (matrix gene) of human influenza A/Bangkok/1/79(H3N2) produced amplification products of appropriate size with influenza A/Equine/Prague/1/56 (H7N7), A/Equine/Miami/63 (H3N8), A/Equine/Kentucky/79 (H3N8), and A/Equine/Kentucky/2/91 (H3N8) in infected frozen allantoic fluids and in frozen extracts of nasal swabs of 2 horses with naturally acquired influenza. The products bound a 32P-labeled hybridization probe to an inner region of the target. Control samples, including nasal secretions from a horse infected with herpesvirus, were negative. In a prospective study, 2 ponies inhaled aerosols of influenza A/Equine/Kentucky/2/91 (H3N8), and thereafter supernatants of nasal swabs in transport medium were obtained daily for 10 days for culture and PCR. Amplification products were evaluated by size and binding of a 32P-labeled probe and also by dot-blotting and binding of a biotin-labeled probe. Culture detected influenza more consistently than did PCR in the first 2 days of infection, but PCR detected virus more often later in infection. Gels were the most sensitive, but radiometric and biotin-labeled probes gave specific results and were consistently positive from days 3-6. PCR is suitable for detection of equine influenza in clinical samples.

Comparison of the opsonic activity of human surfactant protein A for Staphylococcus aureus and Streptococcus pneumoniae with rabbit and human macrophages.

Surfactant protein A (SP-A) is a glycosylated apoprotein that may facilitate bacterial phagocytosis and contribute to early bacterial clearance in the lung. The effect of SP-A on attachment (or ingestion) of Staphylococcus aureus and type 25 pneumococci to rabbit alveolar macrophages and human monocyte-derived macrophages was studied. SP-A bound to S. aureus and type 25 pneumococci in a calcium-dependent manner. Bacteria-associated SP-A significantly increased attachment of S. aureus, but not pneumococci, to macrophages. Increased association of SP-A-coated S. aureus with macrophages appeared to consist mainly of attachment without ingestion, as determined by bactericidal tests and release of tritiated bacterial digestion products from macrophages. Preincubation of macrophages with SP-A did not increase attachment or ingestion of S. aureus or type 25 pneumococci, with or without the addition of immune opsonins. SP-A acts as a ligand to facilitate attachment of S. aureus to macrophages but has no effect on S. pneumoniae.

Detection of influenza A and B in respiratory secretions with the polymerase chain reaction.

Influenza A and B are RNA-containing viruses that frequently infect humans. Currently, sensitive detection of these viruses requires fresh respiratory secretions and special facilities for culture. To facilitate diagnosis of influenza, the polymerase chain reaction (PCR) was used in the present studies to detect DNA produced by reverse transcription of influenzal RNA in vaccines, tissue culture fluids, and stored respiratory secretions. Primers were directed at targets on the highly conserved segment 7 (matrix gene) of influenza A (212-bp product) and B (365-bp product). The primers were completely type specific. Critical variables in the assay were the concentration of pleotropic salts used during preparation of samples, the use of carrier RNA and RNase inhibitors during sample preparation, and the use of optimum K+ and Mg2+ levels at each step. Studies of 33 patients with symptoms of viral respiratory infection whose nasal washes had been cultured and frozen for up to 1 year before assay showed that PCR provided type-specific detection of influenza with a sensitivity comparable to that of culture of the fresh secretions. The assay offers a powerful test for detection of devitalized influenza viruses and may be useful in both diagnostic work and epidemiological studies of influenza.

Mechanism of killing of pneumococci by lysozyme.

Lysozyme is abundant in respiratory secretions and may play a role in lung host defenses. Mechanisms by which lysozyme killed Streptococcus pneumoniae, an important respiratory pathogen, were studied. Lysozyme caused optical clearing of pneumococcal suspensions and released fragments containing [3H]choline from their cell walls. Electron micrographs revealed wide-spread cell wall destruction and bacteriolysis. Breakdown of the cell wall appeared to be mediated mostly by the major pneumococcal autolysin, N-acetylmuramoyl-L-alanine amidase, because it was blocked by phosphorylcholine, a specific inhibitor of amidase, or by substitution of ethanolamine for choline in the cell wall. Blockade of amidase did not greatly increase survival of lysozyme-treated pneumococci on blood agar. Pneumococci in which amidase was blocked appeared intact immediately after treatment with lysozyme, but when they were reincubated at 37 degrees C in fresh culture medium they swelled and lysed. Thus, widespread triggering of the major pneumococcal autolysin is not essential for the bactericidal effect of lysozyme.

Spinal epidural abscess: an unusual complication of a duodenal ulcer.

A 50-year-old man had several months of progressively worse low-back pain associated with constitutional symptoms and a history of retroperitoneal tumor and bleeding duodenal ulcer. Initial evaluation suggested a lumbar spine tumor, but myelography confirmed the presence of an epidural abscess. Further evaluation revealed a duodeno-spinal fistula at the site of the previous duodenal ulcer, which proved to be the cause of the abscess.

Species variation in the mechanism of killing of inhaled pneumococci.

Detailed studies in murine models show inhaled staphylococci are killed mainly by alveolar macrophages. Recently, using histology and lung lavage to determine the site of killing of inhaled 59Fe-labeled pneumococci in rats, we found unexpectedly that most of the organisms were killed extracellularly. In the present studies we compared clearance of inhaled 59Fe-pneumococci in rats, guinea pigs, and rabbits to determine if extracellular killing of pneumococci is species-dependent. Absolute clearance rates were measured using Andersen plates. lung lavage and differential centrifugation were used to measure leukocyte-associated and free 59Fe-pneumococci and nonsedimentable 59Fe. Clearance was rapid in all species but was fastest in rabbits, which killed pneumococci almost as quickly as they were deposited (p less than 0.025 versus rats and guinea pigs). At 1-1/2 hours after pneumococcal deposition in rats, when clearance had reached 92%, alveolar macrophages contained only 31% of the total 59Fe while 56% was in the nonsedimentable, extracellular fraction. At 1-1/2 hours in guinea pigs, when clearance was 96% complete, macrophages contained 51% of the 59Fe and 34% was nonsedimentable (p less than 0.002 versus rats). In rabbits at 1-1/2 hours, macrophages had 94% of the 59Fe and only 4% was nonsedimentable (p less than 0.001 versus the other species). In no species were opsonic antibodies detected in sera or concentrated lavage. In vitro, rabbit and guinea pig alveolar macrophages killed pneumococci opsonized with both specific antibody and fresh serum, while rat macrophages had little activity (p less than 0.001). We conclude that the role of alveolar macrophages in killing inhaled pneumococci varies in different species.

Organ-specific disposition of group B streptococci in piglets: evidence for a direct interaction with target cells in the pulmonary circulation.

Despite the serious pulmonary manifestations of early onset group B streptococcal (GBS) sepsis, it is not known whether the organism distributes into lung tissue and whether adverse pulmonary hemodynamic abnormalities relate to an interaction between the organism and target cells in the pulmonary vascular bed. Accordingly, this study evaluated the distribution and fate of GBS in the lung, liver, and spleen of anesthetized infant piglets and in isolated, salt solution-perfused piglet lung preparations. GBS were radiolabeled with 111Indium-oxine and infused at a dose of 10(8) organisms/kg/min for 15 min into anesthetized piglets ranging in age from 5-10 d. Forty-five min after termination of the infusion, animals were killed and specimens of lung, liver, spleen, and blood were excised and the relative deposition and viability of GBS were determined. Most of the recovered bacteria were detected in the lung (53.2 +/- 3.9%) followed by the liver (41.4 +/- 2.0%) and spleen (2.2 +/- 0.38%). GBS detected in the blood was estimated to be only 3.2 +/- 1.0% of the infused dose. Viability of GBS was least in the lung (21.4 +/- 2.6%) relative to the liver (45.7 +/- 11.2%) and spleen (83.4 +/- 19.5%). After a 60-min GBS infusion, transmission electron microscopy localized the organism within pulmonary intravascular macrophages in the lung; there was no evidence for bacterial interaction with either neutrophils or endothelial cells. In the liver, GBS was found exclusively in Kupffer cells. In isolated piglet lungs perfused at a constant flow rate with blood-free physiologic salt solution, GBS (10(6) to 10(8) organisms/mL) provoked concentration-dependent increases in pulmonary vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen radical-dependent bacterial killing and pulmonary hypertension in piglets infected with group B streptococci.

The mechanism by which bacteria are cleared by the pulmonary circulation and the relation of this process to development of hemodynamic abnormalities are not understood. This study tested the hypotheses that clearance of Group B Streptococcus (GBS) during transit through the pulmonary circulation of infant piglets is related to oxygen radical-dependent bacterial killing and that killing of the organism is linked to development of pulmonary hypertension. GBS were radiolabeled with 111In and infused intravenously for 15 min (10(8) organisms/kg/min) into infant piglets ranging in age from 5 to 14 days. Lung specimens were excised at termination of the GBS infusion or 45 min thereafter, and both the relative deposition and viability of the bacteria were determined. The percentage of infused GBS recovered in lung tissue did not differ between the two time points (26 +/- 7% versus 29 +/- 8%), but the relative viability at termination of the infusion, 50 +/- 11%, was reduced to 19 +/- 4% within 45 min. Treatment with an oxygen radical scavenger, dimethylthiourea (DMTU), failed to influence the pulmonary deposition of GBS but significantly increased viability of the organism from 21.4 +/- 2.6 to 33.3 +/- 5.3%. As expected, GBS infusion was accompanied by pulmonary hypertension and arterial hypoxemia; DMTU attenuated these responses by 52 and 78%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein lipase: a source of free fatty acids in bronchoalveolar lining fluid.

Long-chain free fatty acids (FFAs) in pulmonary bronchoalveolar lavage fluid (BALF) are antimicrobial agents that may participate in lung defenses. FFAs may also participate in synthetic and metabolic activities of bronchoalveolar lining cells. In evaluating the origins of FFAs, we found that rat triglyceride lipase activity was readily detectable in rat BALF. This activity appeared to be caused mainly by lipoprotein lipase (LPL), because it was inhibited by protamine, a high salt concentration, or specific anti-LPL antibody. LPL activity was detected in BALF from guinea pigs, humans, and rabbits, but rats had significantly more LPL activity than the other species. LPL activity in rat BALF was enhanced by heat-inactivated serum, but LPL-mediated hydrolysis of triglycerides in BALF proceeded at 37 degrees C in vitro even without serum. The possibility that BALF contained an intrinsic LPL activating factor(s) was suggested by the fact that concentrated, heat-inactivated lavage was 85% as effective as heat-inactivated serum in enhancing the LPL activity of fresh BALF. Macrophages are the likely source of LPL in BALF, and we confirmed that rat resident alveolar macrophages produce LPL in culture in a time-dependent fashion. It was concluded that FFAs in BALF were produced by the hydrolysis of triglycerides by LPL.

Pneumococcal pneumonia.

Pneumococci remain the most common cause of community-acquired pneumonia, and there are still important questions concerning the pathogenesis, management, and prevention of this disease. Infection begins by aspiration of pneumococci from the oropharynx. Alveolar macrophages, granulocytes, and extra-cellular factors, including opsonins, are necessary for control of bacterial proliferation and cure of the infection. Clinically, pneumococcal pneumonia often presents with sudden onset of productive cough, fever, and a rigor, but symptoms may be muted in the young, elderly, or debilitated. About one-fourth of patients have a positive blood culture. Examination of sputum by Gram's stain and culture can provide useful information, but are not definitive. Tests for soluble pneumococcal antigen or the direct quellung reaction on sputum have not proved helpful. Pneumococci isolated from blood and spinal fluid should be tested for penicillin sensitivity routinely. Penicillin G and erythromycin are the mainstays of specific treatment, and rapid subjective improvement on narrow-spectrum therapy is an important point in diagnosis. The mortality rate continues to be about 18%, and prevention by vaccination remains a highly desirable goal.

Impaired antipneumococcal activity of bronchoalveolar lining material of neonatal rats.

Pulmonary clearance of inhaled pneumococci is markedly impaired in neonatal rats compared with that in adult rats. To determine whether this impairment is due to a deficiency of extracellular bactericidal factors, the antipneumococcal activity of free fatty acids (FFA) in lung surfactant and the levels of lysozyme and transferrin in lavage fluids were quantified. Surfactant from adult rats averaged 68 U of antipneumococcal activity per g (dry weight) of lung, compared with less than 0.25 U for rats less than 1 week old (P less than 0.001). The kinds of FFA in surfactant of neonatal and adult rats were essentially identical, and the antipneumococcal activity of highly purified FFA from surfactant of neonatal and adult rats was also the same. However, the quantity of FFA in surfactant varied significantly with age, and rats less than 3 weeks old had much lower levels of surfactant FFA than did adults (P less than 0.001). In addition, lavage fluids from neonatal rats inhibited the antipneumococcal activity of surfactant FFA more than lavage fluids from adults did (P less than 0.02). This inhibitory activity did not appear to be due to protein binding. Lavage fluids from neonates showed an age-related deficiency of lysozyme (P less than 0.001), but lysozyme appeared to play no role in pneumococcal killing by the surfactant fraction of lavage fluids in vitro. Transferrin levels in lavage fluids were similar for neonates and adults. It was concluded that lung surfactant from neonatal rats was deficient in antipneumococcal activity, due mostly to low levels of FFA and to a lesser degree to increased levels of inhibitor(s) in lavage fluids.

Role of leukocytes in lung defenses.

Alveolar macrophages are the primary cells that protect the lung against inhaled or aspirated microbes. They kill microorganisms intracellularly by both oxidative and non-oxidative mechanisms. Alveolar macrophages secrete antimicrobial factors, including lysozyme, peptides, and transferrin, which are found in the bronchoalveolar lining fluid and may kill microbes extracellularly. Macrophage secretory products help initiate a controlled inflammatory response and a limited influx of granulocytes, which appears to be important for complete elimination of many bacterial pathogens from the lung.

Enzyme-linked immunosorbent assay of core antigens for clinical diagnosis of influenza.

A solid-phase enzyme-linked immunosorbent assay (ELISA) with monoclonal secondary antibodies was used to detect matrix protein and nucleoprotein of influenza A. The sensitivity of the ELISA for highly purified A/Brazil nucleoprotein and matrix protein was 0.05 and 1.0 ng, respectively. Nasal washes from 10 of 20 adult subjects with culture-proven, naturally acquired infection caused by A/Brazil/11/78-like influenza virus were positive in the test, and 2 of 13 subjects with rhinovirus infection were falsely positive. To determine if ELISA results could be improved, nasal washes were obtained from 21 adult volunteers who had been inoculated intranasally with wild-type A/Korea/1/82 (five subjects) or A/Korea recombinants with matrix protein or RNA-2 protein of A/Ann Arbor/6/60 (16 subjects), and the nasal washes were processed by a variety of methods. Prompt addition of sodium azide to the nasal washes to limit bacterial growth, avoidance of freezing, and the use of an antiproteolytic agent all failed to improve ELISA results noticeably. Under the best conditions, ELISA was positive in only 12 of the 21 experimentally infected subjects and in 1 of 15 uninfected controls. Positive ELISA results in experimentally infected subjects correlated significantly with the titer of live virus in the nasal washes (r = +0.506; P less than 0.001). Detection of gradient-purified whole influenza virus or isolated core antigen in ELISA was inhibited by prior incubation with nasal washes, and the inhibitory activity was only partly decreased by heat treatment of the secretions. At present, the use of ELISA for detection of influenza antigens in respiratory secretions is not sufficiently sensitive or specific for routine laboratory diagnosis of influenza.

Extracellular killing of inhaled pneumococci in rats.

Early clearance of inhaled Staphylococcus aureus is believed to be caused by phagocytosis by alveolar macrophages. In murine models inhaled pneumococci are cleared even more rapidly than S. aureus. Conventional opsonins appear to play no role in this clearance, and recently it has been shown that murine alveolar lining material contains free fatty acids and other soluble factors that are directly bactericidal for pneumococci. To determine whether non-phagocytic factors are involved in pneumococcal clearance, we compared the site of killing of inhaled pneumococci and S. aureus in rats using histologic methods and bronchoalveolar lavage. Spontaneous lysis of pneumococci was prevented by use of autolysin-defective pneumococci or by substitution of ethanolamine for choline in the cell wall. Histologic studies showed that the percent of inhaled staphylococci associated with alveolar macrophages always exceeded the percent of staphylococci cleared, whereas there was little association of pneumococci with macrophages during clearance. Analysis of the intracellular or extracellular location of iron 59 in bronchoalveolar lavage fluid of rats that had inhaled aerosols of 59Fe-labeled bacteria suggested that staphylococci were killed predominantly in macrophages and pneumococci in the extracellular space. When 59Fe-labeled pneumococci or staphylococci were ingested and killed by macrophages in vitro, the 59Fe remained with the macrophages, suggesting that the extracellular location of 59Fe during pneumococcal killing in vivo was not caused by rapid turnover of 59Fe in macrophages. Studies of the site of killing of inhaled type 25 pneumococci labeled exclusively in the cell wall with carbon 14-ethanolamine confirmed the results obtained with 59Fe-labeled pneumococci. Thus, early killing of inhaled pneumococci, unlike staphylococci, appears to take place outside of macrophages.

Impaired pulmonary clearance of pneumococci in neonatal rats.

Lung infections are a common cause of morbidity and mortality in neonates. To evaluate neonatal lung defenses against pneumococci, we challenged rats with aerosols of encapsulated pneumococci in an airborne infection apparatus. Whereas adult rats cleared greater than 95% of inhaled type 1 or type 25 pneumococci within 4 h, pneumococci proliferated in the lungs of newborn rats and reached 200-600% of the baseline value by 4 h and 1000-1700% by 24 h. As neonatal rats matured, their ability to clear inhaled pneumococci improved, but compared with adults some impairment in clearance was present until approximately 4 wk of age. Newborn rats had significantly fewer resident alveolar macrophages per g of lung tissue than did adults (p less than 0.001). Although the number of resident macrophages increased with time, a significant deficit in alveolar macrophages persisted for the first 3 wk of life (p less than 0.01). Aerosols of pneumococci caused an influx of granulocytes into the lungs of adult rats within 4 h, compared with 24 h for neonatal rats. Even at 24 h after pneumococcal challenge, newborn rats had significantly fewer granulocytes per g of lung tissue (p less than 0.05) than did adults, although 7-day-old rats had reached an adult level by this time. Significant (p less than 0.05) increases in granulocyte chemotactic activity were observed in lavage fluids of adult, but not newborn, rats after pneumococcal challenge. Thus, impaired clearance of pneumococcal aerosols by neonatal rats was associated with an age-dependent deficiency in numbers of resident alveolar macrophages and impaired generation of chemotactic activity and recruitment of granulocytes to the lung.

Different reactivity of rat alveolar and peritoneal macrophages with EAMC and anti-CR3 monoclonal antibodies.

Resident alveolar macrophages of murine species are known to bind complement-coated bacteria and erythrocytes (EAMC, EAMC3b and EAMiC3b) significantly less well than do resident peritoneal macrophages. Because of the potential importance of C3 receptors of resident alveolar macrophages in pulmonary host defenses, we studied the expression of iC3b (CR3) receptors on rat resident alveolar and peritoneal macrophages with a sensitive cytofluorometric method, employing mouse anti-human CR3 monoclonal antibodies. One monoclonal to CR3, designated M42, labelled rat alveolar macrophages as well as it labelled human alveolar macrophages (40.6% versus 45.3%, respectively), whereas rat peritoneal macrophages labelled significantly less well (13.6%-17.4%) (P less than 0.05). A second antihuman CR3 monoclonal, M44, labelled low, but essentially identical percentages of rat alveolar and peritoneal macrophages (21.2% and 23.3%, respectively). Binding of M42 and M44 to rat macrophages appeared to be specific for the CR3 receptor based on cytofluorometric analysis and on the fact that the monoclonals could inhibit the attachment of EAMC to rat macrophages. We conclude that there is specific binding of mouse anti-human CR3 anti-bodies to conserved epitopes of receptors on rat macrophages, and that CR3 expression on resident rat alveolar macrophages is greater than is indicated by attachment of complement-coated particles.

Rôle of surfactant free fatty acids in antimicrobial defenses.

Previous studies of the mechanisms of killing of inhaled bacteria have been confined to the demonstration that alveolar macrophages phagocytose and kill inhaled staphylococcus intracellularly. We have found recently th at the surfactant-containing fraction of rat bronchoalveolar lavage fluid is bactericidal for pneumococci and some other gram-positive bacteria, excluding staphylococci. In studies reviewed herein, we show that these antibacterial factors in rat surfactant are long-chain free fatty acids (FFA). Polyunsaturated FFA appear to be particularly active. Because inhaled pneumococci are cleared very rapidly in vivo in the absence of conventional opsonins, we speculate that FFA may have a rôle in pneumococcal killing. All species tested to date, including humans, dogs, and guinea pigs have detectable FFA in their surfactant, although the level of FFA in these species is lower than in rats. Human and guinea pig surfactant, in fact, have too little FFA in their pulmonary surfactant to give detectable antipneumococcal activity in vitro. Nonetheless, inhaled pneumococci are killed rapidly by guinea pigs, suggesting that the level of FFA in bronchoalveolar lavage is not a good indication of the amount of FFA on alveolar surfaces, or, alternatively that FFA may not play a rôle in pneumococcal clearance in vivo. We have recently completed histological studies which demonstrate that inhaled pneumococci are, in fact, killed extracellularly in rats. This observation adds credence to the concept that mechanisms exist in the alveoli for extracellular killing of some bacteria and indicates that further studies of FFA in this process are worthwhile.