Biomedical subjects
Publications and source records attributed to K Kume.
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Cytocidal and anti-phagocytic effects of the heat-stable hemolytic substance produced in culture supernatant by Haemophilus pleuropneumoniae serotype 2 on porcine pulmonary macrophages (PMP) were investigated in vitro. The substance was partially purified by sucrose density gradient ultracentrifugation, and it seems to be a carbohydrate. The partially purified preparation of the heat-stable carbohydrate with hemolytic activity was used as H. pleuropneumoniae hemolysin throughout this study. Viability of the PMP cells exposed to hemolysin with a hemolytic unit of 32 decreased by about 35% during the first 30-min exposure. The response was hemolysin dose- and exposure time-dependent and was neutralized by the anti-hemolysin antiserum. The PMP cells were found to be the most sensitive to hemolysin among the pulmonary, peritoneal, and peripheral blood macrophages obtained from the same animal. Heat treatment of the hemolytic substance in the culture supernatant at 121 degrees C for 2 h did not significantly (P greater than 0.05) alter the cytocidal and hemolytic activities. Pretreatment of the PMP cells with hemolysin at a sublethal dose significantly (P less than 0.01) reduced their phagocytic abilities. Thus, the heat-stable carbohydrate H. pleuropneumoniae hemolysin appears to possess the potential for both cytocidal and antiphagocytic effects on the PMP cells. Death and impaired phagocytosis of the PMP cells by the hemolysin, if it occurs in vivo, would enhance the chances of survival for an invading H. pleuropneumoniae organism. These findings promote understanding of the pathogenesis of pleuropneumonia caused by H. pleuropneumoniae in swine.
A toxin with dermonecrotic activity (DNT) was purified from sonic extracts of Bordetella bronchiseptica L3 of pig origin at phase I by chromatographic and electrophoretic methods. The purification procedure was one developed for obtaining the Pasteurella multocida DNT from sonic extracts with some modifications. Dermonecrotizing activity of B. bronchiseptica-purified DNT was increased by 600-fold compared with that of the crude extract, and the average yield was about 3%. The toxin was homogeneous, as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and disk isoelectric focusing in polyacrylamide gels. The toxin gave a single band on polyacrylamide disk gel electrophoresis (PAGE) and sodium dodecyl sulfate-SDS PAGE. The molecular weight of the toxin was ca. 190,000 +/- 5,000, as determined by SDS-PAGE. The isoelectric point of the toxin was ca. 6.5 to 6.6. The minimal necrotizing dose of the toxin for guinea pigs was about 2 ng of protein per 0.1 ml, the 50% lethal dose per mouse was about 0.3 micrograms, and the minimal cytotoxic dose for embryonic bovine lung cells was about 2 ng/ml. The toxin was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde. The mildly trypsinized B. bronchiseptica DNT preparation dissociated into two polypeptide chains, with molecular weights of ca. 75,000 +/- 4,000 (fragment 1) and ca. 118,000 +/- 5,000 (fragment 2), after treatment with dithiothreitol-SDS or urea. Upon removal of dithiothreitol and urea from the dissociated DNT preparation, the fragments reassociated, and the DNT that was formed was indistinguishable from the native toxin.
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Serial radionuclide ventriculography was performed using a newly developed "real-time" system, and left ventricular ejection fraction (LVEF), right ventricular ejection fraction (RVEF), stroke volume (SV), and cardiac output (CO) were measured during graded supine exercise in five patients with mitral stenosis (MS), in five patients with chronic obstructive pulmonary disease (COPD) and in five healthy subjects. Simultaneous pulmonary gas exchange analysis permitted determining the anaerobic threshold, which is the point during incremental exercise when lactate begins to accumulate in the blood. LVEF at the anaerobic threshold was not significantly changed in any patient groups and in healthy subjects, but RVEF at the anaerobic threshold was lower in COPD and MS patients as compared with healthy subjects. In MS, SV during exercise was reduced at the anaerobic threshold, but not in COPD or in healthy subjects. In conclusion, reduced working capacity is related to decreased RVEF in both COPD and MS, but the inhibited increase in CO during exercise is also important for the working capacity in MS.
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Lesions induced in chickens by an encapsulated or nonencapsulated strain of Haemophilus paragallinarum were investigated. In terms of lesion severity, major differences in pathogenicity were observed between the encapsulated and nonencapsulated variants. The principal lesion manifested by the encapsulated variant was an acute catarrhal inflammation of the upper respiratory tract, mainly of the nasal cavity and paranasal sinus. Infiltration of a large number of mast cells into the lamina propria of the mucous membrane of the nasal cavity was also characteristic. Numerous organisms were found on the cilia or on the surface of the epithelial cells of the nasal mucosa. Chickens with these histologic lesions had severe clinical signs of coryza, and organisms were recovered in high numbers from the nasal cavity and paranasal sinus. In contrast, chickens that were given the nonencapsulated variant did not have clinical signs of coryza, and the organisms were recovered in low numbers from the inoculated sites; slight histopathologic lesions were observed in the nasal mucosa at postinoculation day 1. Mast cell infiltration in the chickens inoculated with the encapsulated variant indicated that mast cells may be responsible for producing clinical signs of coryza via the activation of pharmacologic mediators. Adherence to and colonization of the encapsulated variant on the nasal mucosa seems to be a first step of the infection.
Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.
Chickens were inoculated intranasally with encapsulated or nonencapsulated strains of Haemophilus paragallinarum. The nasal mucosa of the chickens were examined, using scanning and transmission electron microscopy. In chickens given the encapsulated variant, marked loss of cilia and microvilli, infiltration of leukocytes, and deposition of a mucopurulent substance were seen on the surface of the nasal mucosa; the number of microvillous cells were markedly increased, and infiltration of mast cells into the lamina propria of the mucous membrane was observed. Numerous encapsulated organisms were found near cilia and on microvillous cells, and the capsule surrounding the organisms appeared to mediate attachment of the organisms to the cilia. In contrast, the nasal mucosa of the chickens given the nonencapsulated variants changed little morphologically. A few nonencapsulated organisms were found near cilia. Colonization of the nasal mucosa by encapsulated organisms probably was essential to induce the morphologic changes seen in the nasal mucosa.
A procedure was developed to purify dermonecrotic toxin (DNT) from a sonic extract of a serotype D strain of Pasteurella multocida. Sonic extract containing DNT was applied to a DEAE-Sephacel column and eluted by a linear gradient of NaCl. Upon rechromatographing, fractions with dermonecrotic activity for guinea pigs were applied on a second Sephacel column, and a pooled fraction with the toxic activity was filtered through a Sephadex G-200 column. Pooled fractions with the toxic activity were subjected to polyacrylamide disc gel electrophoresis (PAGE), and the toxic substance was eluted from each sliced gel. Eluted fractions with the toxic activity were rechromatographed on a second Sephadex G-200 column, and a pooled fraction with high dermonecrotic activity was referred to as a purified DNT. The activity of purified DNT was increased by 1,000 times, and the average yield was about 1.8%. The purified DNT was homogeneous as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and thin-layer isoelectric focusing in polyacrylamide gels and gave a single band on PAGE and sodium dodecyl sulfate-PAGE. The molecular weight of the toxin was ca. 160,000 as determined by sodium dodecyl sulfate-PAGE. The isoelectric point of the toxin was ca. 4.7 to 4.8. Amino acid analysis of the purified DNT revealed that the toxin was composed of characteristically high proportions of glutamic acid, aspartic acid, glycine, proline, alanine, and leucine. The minimal necrotizing dose of the toxin was about 1 ng of protein, and the 50% lethal dose per mouse was 0.2 micrograms. The purified DNT was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde.
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