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J D Coonrod

Publications and source records attributed to J D Coonrod.

At least 37 records · Page 2Linked to original sources
PubMed1987

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.

Administration, Inhalation↗
PubMed1986

Electroelution for purification of influenza A matrix protein for use in immunoassay.

A new preparative method for isolation of matrix protein from type A influenza virus was developed. Commercially available whole virus or split virus vaccines were lysed, and the soluble proteins separated by electrophoresis on polyacrylamide gel. The matrix protein was located on the gel by precipitation with KCl, and recovered by electroelution. The method was technically simple and required little direct supervision during the two-step recovery process. Yields of A matrix were consistently high, averaging 68.1% in five trials with A/Brazil/X-71. The method was also successful with other A viruses, although not with influenza B virus. Isolated A matrix had less than 0.5% contamination by hemagglutinin or nucleoprotein, as determined by immunoblotting and ELISA. Matrix protein was immunoreactive in Western blots and was detectable in concentrations as low as 1 ng/ml with ELISA. The isolated matrix provided a suitable standard for detection of matrix protein in nasal washes from patients with influenza A virus infection, and could also be used to detect anti-matrix antibodies, including monoclonal antibodies in tissue culture supernatants. The advantages of electroelution for separation of matrix protein compared to other methods were its technical simplicity, applicability to formalin-fixed influenza virus in commercially available vaccines, its consistently high yield, and its very high level of purification.

Antibodies, Monoclonal↗
PubMed1986

Effect of rat surfactant lipids on complement and Fc receptors of macrophages.

Murine resident alveolar macrophages have very low numbers of complement receptors detectable by rosetting with erythrocyte complexes. These macrophages are relatively inactive in tests of complement- or antibody-mediated bacterial phagocytosis in vitro. It is not known whether these characteristics are intrinsic or are environmentally modulated. We found previously that rat bronchoalveolar lavage fluid or isolated rat surfactant lipid causes a unique and marked reduction in the ability of peritoneal macrophages to form rosettes with immunoglobulin G or complement containing erythrocyte complexes in vitro. In this study the antirosetting activity of rat surfactant was found to be due to its neutral lipid component and, specifically, to the free fatty acid (FFA) fraction of the neutral lipids. Rat surfactant contained a higher level of FFA than has been reported for canine, guinea pig, or human surfactant. Studies with pure FFA showed that activity in blocking macrophage Fc and complement receptors correlated with increasing unsaturation and chain length. A mixture of eight commercially available FFAs, representing the most abundant FFA in rat surfactant and consisting mostly of saturated FFA, had much less effect on rat macrophage receptors than the naturally occurring FFA mixture. These findings suggest that long-chain-unsaturated FFAs in rat surfactant are the most important for the antireceptor activity. The antireceptor activity of rat surfactant or FFA on peritoneal macrophage receptors in vitro and known intrinsic properties of murine alveolar macrophages could not be precisely correlated, suggesting that impaired binding of immune complexes by murine alveolar macrophages and a high level of FFAs in rat surfactant may be independent rather than causally related phenomena.

Animals↗
PubMed1986

The role of extracellular bactericidal factors in pulmonary host defense.

Extracellular killing provides an attractive hypothesis to explain the rapid alveolar killing of inhaled bacterial pathogens in the absence of conventional opsonins for phagocytosis. Some evidence of extracellular killing of inhaled pneumococci has been obtained using histologic studies and bronchoalveolar lavage. Although studies of the antimicrobial activity of lung lavage fluid in vitro have given variable results, a variety of antimicrobial factors have been detected in lung lavage fluids. Studies of lysozyme, peptides, iron binding proteins, free fatty acids and other factors that are found free in lung lavage fluid indicate that some of these factors could be a part of extracellular pulmonary host defenses. However, their precise role is not known. A survey of mechanisms of extracellular killing shows that granulocytes, monocytes, macrophages, and T lymphocytes all have the capacity to kill extracellularly in vitro in some circumstances. It remains to be determined which of these diverse mechanisms operate within the lung and how they function in relationship to other host defenses.

Animals↗
PubMed1984

Etiological diagnosis of influenza A virus by enzymatic radioimmunoassay.

An enzymatic radioimmunoassay for influenza A virus was developed by using polystyrene beads coated with rabbit immunoglobulin G to capture viral hemagglutinins (H1 and H3). Captured hemagglutinin was detected with goat immunoglobulin G followed by affinity-purified rabbit anti-goat immunoglobulin G labeled with alkaline phosphatase. [3H]AMP was added to quantify alkaline phosphatase activity, and free [3H]adenosine was measured with a scintillation counter. The assay detected as little as 0.1 ng of purified hemagglutinin. It was specific for hemagglutinin subtype and, depending on the source of the goat immunoglobulin G used, detected either H1 or H3. There was no reaction with neuraminidase or core antigens of influenza strain WSN-33. The clinical efficacy of the assay was evaluated with sequential nasal washes from 33 patients with naturally acquired H1N1 influenza. In the first 3 days of infection, the assay was consistently less sensitive than the viral culture, although detectable antigen persisted in secretions longer than did the infectious virus. Testing of multiple samples greatly increased the number of individuals in whom an etiological diagnosis could be made by immunoassay (81% of patients were positive for viral antigens at some point in their illness), and such testing was necessary to achieve the sensitivity of a single culture. Mean antigen levels were highest in nasal washes with the highest titers of infectious virus.

Animals↗
PubMed1984

Characterization of the extracellular bactericidal factors of rat alveolar lining material.

The surfactant fraction (55,000-g pellet) of leukocyte-free rat bronchoalveolar lavage fluid contains factors that rapidly kill and lyse pneumococci. These factors were purified and identified biochemically by using a quantitative bactericidal test to monitor fractionation procedures. 91% of the antipneumococcal activity of rat surfactant was recovered in chloroform after extraction of rat surfactant with chloroform-methanol (Bligh-Dyer procedure). After chromatography on silicic acid with chloroform, acetone, and methanol, all detectable antibacterial activity (approximately 80% of the initial activity) eluted with the neutral lipids in chloroform. When rechromatographed on silicic acid with hexane, hexane-chloroform, and chloroform, the antibacterial activity eluted with FFA. Thin-layer chromatography (TLC) established that the antibacterial activity was confined to the FFA fraction. Gas-liquid chromatography showed that the fatty acid fraction contained a mixture of long-chain FFA (C12 to C22) of which 66.7% were saturated and 32.4% were unsaturated. The quantity of TLC-purified FFA needed to kill 50% of 10(8) pneumococci under standardized conditions (one bactericidal unit) was 10.6 +/- 0.5 micrograms. Purified FFA acted as detergents, causing release of [3H]choline from pneumococcal cell walls and increased bacterial cell membrane permeability, evidenced by rapid unloading of 3-O-[3H]methyl-D-glucose. FFA acting as detergents appear to account for the bactericidal and bacteriolytic activity of rat pulmonary surfactant for pneumococci.

Animals↗
PubMed1983

Urine as an antigen reservoir for diagnosis of infectious diseases.

Soluble or particulate microbial antigens are excreted in the urine in many systemic infectious processes. The ease with which urine antigens can be concentrated has facilitated their detection by immunologic methods. The group and type-specific bacterial polysaccharides are among the best studied examples of urinary excretion of microbial antigens. These polysaccharides are often present in the urine as low molecular weight fragments (70,000 daltons or less) and in some instances may represent degradation products of the native polysaccharides. Urine polysaccharides are sufficiently immunoreactive to be detectable by simple precipitin and agglutination techniques in a large percentage of patients with infections due to certain pyogenic bacteria including Haemophilus influenzae and group B streptococci. Both polysaccharide and protein antigens have been detected in the urine by immunologic methods in numerous other infections including parasitic, viral, and spirochetal diseases. Detection of a thermostable antigen in the urine of patients with Legionnaires' disease by radio- and enzyme-linked immunoassays represents an important recent advance. The exact role of immunologic tests for etiologic diagnosis in infectious diseases is not established, but will undoubtedly be influenced by developments such as monoclonal antibody technology and better availability of standardized immunologic reagents.

Animals↗
PubMed1983

Detection and partial characterization of antibacterial factor(s) in alveolar lining material of rats.

Intracellular killing of Staphylococcus aureus by alveolar macrophages is known to be enhanced by exposure to alveolar lining material. Because this material may have a role in pulmonary host defenses, we have studied its effect on pneumococci and other nonstaphylococcal organisms. Alveolar lining material from rats caused rapid killing and lysis of pneumococci. The antipneumococcal activity was localized to the surfactant-containing fraction of the fluid and was not affected by trypsin. Phospholipid extracts of the surfactant fraction or purified lamellar bodies killed pneumococci. Lysis of pneumococci by the surfactant fraction appeared to be mediated by a detergent-like activation of pneumococcal autolysin, in that bacteriolysis was prevented by substitution of ethanolamine for choline in pneumococcal cell walls, and a pneumococcal transformant that lacked autolysin was not lysed. The surfactant fraction readily killed pneumococci containing ethanolamine or the autolysin-defective transformant, and studies with tritiated methyl-D-glucose loading and release showed that killing was associated with increased bacterial cell membrane permeability. Bactericidal activity (without lysis) was observed with several nonpneumococcal gram-positive bacteria, including Streptococcus viridans, unspeciated respiratory streptococci, Streptococcus pyogenes, Streptococcus bovis, and Bacillus species. Purified diacylphospholipids had no antibacterial activity, however, a lysophospholipid, palmitoyl lysophosphatidylcholine, had many properties resembling the surfactant-containing fraction of lavage, including autolysin-mediated pneumococcal lysis, altered cell membrane permeability, and antibacterial activity against several gram-positive bacteria.

Animals↗
PubMed1983

Effect of rat alveolar lining material on macrophage receptors.

Resident alveolar macrophages of many rat strains lack detectable C receptors and cannot carry out C-mediated bacterial attachment and phagocytosis. It has not been established whether this lack of functional receptors is innate or is acquired by contact with the alveolar microenvironment. In the present studies, peritoneal macrophages treated with lavage under conditions designed to maintain viability and adherence to glass showed loss of detectability of C and Fc receptors. Depending on the concentration of lavage and the time allowed for recovery, this effect appeared to be reversible. Continued incubation of peritoneal macrophages with lavage fluid led to loss of adherence to glass and cell death. Lavage-treated 51Cr-labeled macrophages rapidly released radiolabel. Although transmission electron micrographs of lavage-treated peritoneal macrophages appeared normal, micrographs prepared with freeze-fracture techniques revealed clumping and asymmetric distribution of the intramembrane particles. Similar membrane abnormalities were present in untreated resident alveolar macrophages. The anti-receptor effect of rat lavage was not species-specific and lung lavage fluid from dogs, rabbits, and mice affected receptor detectability on rat macrophages. The anti-receptor factor in rat lavage was localized to the surfactant-containing fraction and was stable on heating at 60 degrees C. It was resistant to trypsin and partitioned into chloroform in the Bligh-Dyer extraction procedure, suggesting that it was a lipid. Purified diacyl phospholipids had no effect on macrophage receptors, but purified lysophospholipids mimicked many of the effects of surfactant. We conclude that lipids in the alveolar lining material affect alveolar macrophage membranes and receptor function.

Animals↗
PubMed1982

Early clearance of pneumococci from the lungs of decomplemented rats.

Pneumococcal types which exhibited varying degrees of interaction with the complement system in vitro wee aerosolized into normal and decomplemented rats, and the rate of killing of pneumococci was monitored by serial cultures of whole lung homogenates. The clearance of pneumococci from the alveoli did not correlate with the ability of the pneumococci to be opsonized by complement in vitro. Similarly, rats depleted of complement in the serum and the bronchoalveolar lavage fluid maintained their ability to rapidly inactivate aerosolized pneumococci. These results indicate that the early phase of pneumococcal killing in the alveoli does not require complement and suggest that in the nonimmune host, phagocytosis by alveolar macrophages can be achieved without complement-dependent opsonization or, alternatively, that extracellular factors rapidly inactivate inhaled pneumococci.

Animals↗
PubMed1982

Comparative role of complement in pneumococcal and staphylococcal pneumonia.

To evaluate the role of complement in pneumococcal and staphylococcal pneumonia, we decomplemented rats with cobra venom factor and inoculated them intratracheally with Staphylococcus aureus or type 25 pneumococci. S. aureus produced a patchy bronchopneumonia in normal Sprague-Dawley or Lewis rats, and decomplementation did not increase the severity of staphylococcal infection in either rat strain as judged by quantitative cultures of the lungs and blood at 6, 24, and 48 h after inoculation. In contrast, decomplementation markedly increased the severity of pneumonia caused by type 25 pneumococci in Sprague-Dawley and Lewis rats. In Sprague-Dawley rats, decomplementation significantly increased the number of bacteria in the lungs at 3, 6, and 24 h of infection. Bacteremia developed early in decomplemented Sprague-Dawley rats, but the higher pulmonary bacterial counts did not appear to be caused by bacteremic seeding of the lungs. Decomplemented Sprague-Dawley rats inoculated intravenously with pneumococci failed to develop the very high levels of bacteria in the lungs that were observed when the rats were inoculated intratracheally. Moreover, decomplemented Lewis rats inoculated intratracheally with pneumococci developed significantly increased numbers of pneumococci in the lungs early in infection (3 and 6 h) when they had no detectable bacteremia. These data indicate that in murine models complement plays a major protective role against type 25 pneumococci in the lung, whereas complement is not important to host defense in staphylococcal pneumonia.

Animals↗
PubMed1982

Complement receptors of rat macrophages.

Some species, including rabbits, guinea pigs, and humans, have complement receptors on resident alveolar macrophages, whereas Swiss mice lack detectable receptors on these macrophages. Using complement-coated sheep erythrocytes or bacteria, we were unable to detect complement receptors (CR1, CR2, or CR3) on resident alveolar macrophages of male Sprague-Dawley rats. Nonelicited peritoneal macrophages from Sprague-Dawley rats had CR1 and CR3 receptors and were able to bind bacteria opsonized with complement. Resident alveolar macrophages of Long-Evans and Fisher rats lacked detectable complement receptors, but CR1 and CR3 receptors were detected on the alveolar macrophages of Lewis-Wistar rats. The reason for the lack of complement receptors on murine macrophages is not known, but studies of this phenomenon may prove useful in elucidating the role of macrophage complement receptors in the pulmonary inflammatory response.

Animals↗
PubMed1981

Pulmonary opsonins in Klebsiella pneumoniae pneumonia in rats.

Klebsiella pneumoniae was inoculated intrabronchially into rats, and bronchoalveolar lavage fluid and sera were obtained during the ensuring pneumonia. Klebsiellae recovered by lavage were not maximally coated with C3, as judged by studies with fluorescent antibody, whereas the organisms could be coated fully with C3 by a brief incubation in rat serum. The levels of C1 and C3 in lavage fluid obtained during infection were only a small fraction of the levels in the serum, and klebsiellae were not opsonized during incubation with concentrated lavage fluid. Systemic decomplementation did not affect the severity of K. pneumoniae pneumonia, as judged by the measurement of lung weight or by the numbers of klebsiellae in the lungs, but decomplemented rats had a larger number of klebsiellae in the blood at 24 h of infection than did controls. There were fewer klebsiellae in the lungs 4 h after inoculation of preopsonized organisms than after inoculation of organisms which were incubated in control (heat-inactivated) sera. These studies indicate that the concentration of complement and heat-labile opsonins within the alveoli is lower than that in the systemic circulation and is not adequate for effective opsonization of klebsiellae.

Animals↗
PubMed1980

Evaluation of a hemolytic assay of the alternative complement pathway in human serum.

Alternative pathway activity of human serum was titrated by use of unsensitized rabbit erythrocytes (RE). Under the conditions of the assay, the von Krogh equation could be used to relate the proportion of RE lysed to the level of alternative pathway activity. The use of a 50% hemolytic endpoint provided maximum sensitivity in the assay. The 50% hemolytic endpoint could be calculated from a single measurement in the region of 20% to 80% lysis or RE. Factor B was required for lysis of RE in the test, but neither C2 nor C8 was limiting under the conditions of the assay. Alternative pathway activities of three sera with abnormal IgG levels were in the normal range, but normal serum absorbed with RE at 0 C before testing had diminished lytic activity with the test. Lysis of RE in acute-phase sera of 16 patients who had bacteremic pneumococcal pneumonia was significantly below normal (P < 0.01). Results with Re lysis in these patients correlated well with levels of Factor B that were measured immunochemically and with consumption of whole complement by zymosan.

Animals↗
PubMed1980

Fulminant wound infection with Aeromonas hydrophila.

Aeromonas hydrophila was isolated from a water-soaked traumatic wound of a previously healthy 16-year-old boy. A progressive necrotizing infection developed despite administration of antibiotics. Aeromonas should be considered in wound infections which are water-related.

Adolescent↗
PubMed1980

Comparison of histological and immunological techniques for detection of Pneumocystis carinii in rat bronchial lavage fluid.

We compared histological and immunological techniques in the early diagnosis of Pneumocystis carinii pneumonia in bronchial lavage fluid of steroid-treated rats. The rats were sacrificed weekly and lavage fluids were: (i) examined with cresyl echt violet and Giemsa stains; (ii) examined for P. carinii antigens by indirect fluorescent-antibody, counterimmunoelectrophoresis, and double-diffusion techniques, using high-titer spectific antisera to P. carinii raised in rabbits. P. carinii was detected in lavage fluid by cresyl echt violet at 2 weeks of steroids and persisted even with steroid tapering; the intensity of the infection in lavage fluid closely paralleled that in the lungs. P. carinii was not detected in lavage by Giemsa stain until 4 weeks and disappeared from the fluids with steroid tapering. P. carinii was detected by indirect fluorescent antibody as early as 1 week of steroids, and the results correlated well with those of cresyl echt violet. P. carinii antigens were not detected in lavage fluids or serum by counterimmunoelectrophoresis or double-diffusion techniques. Although precipitin lines sometimes occurred, they were nonspecific. In this model, cresyl echt violet and indirect fluorescent antibody were the preferred techniques for the early diagnosis of P. carinii infection in bronchial lavage fluid.

Animals↗