Biomedical subjects

P W Andrew

Publications and source records attributed to P W Andrew.

At least 19 recordsLinked to original sources

PubMed1992

Effect of immunization with Freund's adjuvant and pneumolysin on histologic features of pneumococcal infection in the rat lung in vivo.

Immunization with Freund's adjuvant and pneumolysin and stimulation with Freund's adjuvant alone both reduced the severity of the pneumonia caused by injections of bacteria into the apical lobe bronchi of rats. Neither protocol influenced the incidence of pneumococcal bacteremia. Illness sufficiently severe to require sacrifice was delayed from 2.8 days in nonimmunized animals to 5.7 days in those immunized with Freund's adjuvant and pneumolysin (P < 0.05) and 4.5 days in those stimulated with Freund's adjuvant alone (P, not significant).

Adjuvants, Immunologic↗

PubMed1992

The interaction of Streptococcus pneumoniae with intact human respiratory mucosa in vitro.

The interaction of Streptococcus pneumoniae with human ciliated upper respiratory mucosa was studied in an agar-embedded organ culture of nasal turbinate tissue, which only exposed the intact epithelial surface and its secretion. The ciliary beat frequency, measured along the edge of the organ culture, was slowed by 13% in the presence of S. pneumoniae after 16 h (p less than 0.05) compared with the control, and by 24% after 24 h (p less than 0.01). Light microscopy showed bacteria in a thickened gelatinous layer, which obscured the surface of the organ culture. Transmission and scanning electron microscopy confirmed the association of bacteria with the gelatinous layer above an epithelial surface which showed only minor changes compared to uninfected control organ cultures. Contact between bacteria and normal or damaged epithelial cells was not seen. S. pneumoniae in organ culture developed projections from their surface, which were not present after broth culture. S. pneumoniae interactions with epithelial-derived secretions, the formation of a thickened gelatinous layer, and the effects of bacterial toxins on ciliary motility, may be important during colonization of the respiratory tract.

Cilia↗

PubMed1991

Guinea-pig alveolar macrophage killing of Mycobacterium tuberculosis, in vitro, does not require hydrogen peroxide or hydroxyl radical.

Alveolar macrophages from the lungs of guinea-pigs that had been vaccinated, boosted and then intravenously challenged with Mycobacterium bovis BCG, killed both a hydrogen-peroxide-resistant and a hydrogen-peroxide-sensitive strain of M. tuberculosis, in vitro. Pretreatment of the alveolar macrophages, in vitro, with catalase or mannitol, agents which remove hydrogen peroxide and the hydroxyl radical, respectively, did not decrease the extent of killing of either strain. In contrast, pretreatment of alveolar macrophages with catalase, reversed the inhibition of growth of Listeria monocytogenes.

Animals↗

PubMed1991

Interferon gamma fails to activate human monocyte-derived macrophages to kill or inhibit the replication of a non-pathogenic mycobacterial species.

The ability of interferon gamma (gamma) to activate human macrophages to kill mycobacteria was investigated using a mycobacterial species that does not cause disease in man. Although interferon activated human macrophage activity against other intracellular parasites, Toxoplasma gondii and Listeria monocytogenes, it failed to activate human monocyte-derived macrophages to kill not only Mycobacterium tuberculosis but also the non-pathogenic species, Mycobacterium phlei.

Analysis of Variance↗

PubMed1991

Guinea-pig alveolar macrophages kill Mycobacterium tuberculosis in vitro, but killing is independent of susceptibility to hydrogen peroxide or triggering of the respiratory burst.

Alveolar macrophages from the lungs of guinea-pigs that had been vaccinated, boosted and then intravenously challenged with Mycobacterium microti or Mycobacterium bovis BCG killed tubercle bacilli phagocytosed in vitro. The killing was modest, about 40% of phagocytosed bacilli were killed in a day, but alveolar macrophages from animals that had been vaccinated and boosted but had not received the intravenous challenge did not kill bacilli. Different strains of tubercle bacilli had different degrees of susceptibility to these activated macrophages but there was no correlation between killing by macrophages and mycobacterial susceptibility to killing by hydrogen peroxide. The different strains of tubercle bacilli triggered peroxide release from these macrophages but there was no correlation with susceptibility to killing by macrophages or with virulence in the guinea-pig. However, phagocytic uptake of these strains by the activated macrophages was inversely correlated with virulence, and uptake by activated macrophages was less than uptake by normal macrophages.

Animals↗

PubMed1991

A DNA primer/probe system for the rapid and sensitive detection of Mycobacterium tuberculosis-complex pathogens.

A 1.5 kb EcoRI-BamHI restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis-complex organisms. Primers were designed from the terminal sequences of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and probe will prove a useful tool for the early diagnosis of tuberculous infections.

Base Sequence↗

PubMed1991

Effect of growth temperature on virulence of strains of Listeria monocytogenes in the mouse: evidence for a dose dependence.

Growth of Listeria monocytogenes at 4 degrees C significantly increased its virulence for mice by the intravenous route and the effect was dose-dependent. Virulence was apparent only at a dose of about or above 10(4) viable listerias. At slightly lower doses of about 10(3), no such effect was observed. Growth at 4 degrees C did not increase the virulence of the strains for mice by oral-gastric challenge when given at doses of approximately 10(10).

Animals↗

PubMed1991

Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute-phase protein.

Pneumolysin, a membrane-damaging toxin, is known to activate the classical complement pathway. We have shown that 1 microgram ml-1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute-phase proteins, including human C-reactive protein (CRP). Site-directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.

Amino Acid Sequence↗

PubMed1991

Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae.

Pneumolysin is a thiol-activated, membrane-damaging, multifunctional toxin and a known virulence factor of Streptococcus pneumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against pneumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site-directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C-reactive protein, a human acute-phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.

Amino Acid Sequence↗

PubMed1991

Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide.

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

Amino Acid Sequence↗

PubMed1991

Streptococcus pneumoniae produces at least two distinct enzymes with neuraminidase activity: cloning and expression of a second neuraminidase gene in Escherichia coli.

A gene from Streptococcus pneumoniae was cloned in lambda EMBL301 and then expressed in Escherichia coli, which cleaved the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid. The cloned gene therefore encodes an enzyme with neuraminidase activity. On the basis of restriction mapping and DNA hybridization studies, this gene could be distinguished from another pneumococcal neuraminidase gene cloned previously (A. M. Berry, J. C. Paton, E. M. Glare, D. Hansman, and D. E. A. Catcheside, Gene 71:299-305, 1988). Both neuraminidase genes were found in each of five isolates, covering at least three serotypes, of pneumococci tested.

Blotting, Southern↗

PubMed1991

Pneumolysin induces the salient histologic features of pneumococcal infection in the rat lung in vivo.

Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement.

Animals↗

PubMed1991

Activation of mouse peritoneal macrophages by maintenance in serum-free medium.

Normal mouse peritoneal macrophages maintained in a serum-free medium for 48-72 h and then stimulated with phorbol myristate acetate, zymosan or bacteria, released large amounts of hydrogen peroxide. Opsonized zymosan and bacteria stimulated greater release than their unopsonized counterparts. Enhanced peroxide production was not a consequence of increased uptake of particles. Addition of serum to the serum-free medium abolished activation. The addition of interferon-gamma to the serum-free medium enhanced the effect of the serum-free treatment of macrophages from C3H/HeJ mice but abolished the effect of serum free treatment of macrophages from CFLP mice. The results are discussed in terms of negative regulation of receptor-oxidase linkage by serum.

Animals↗

PubMed1990

The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte derived macrophages.

Diphenylene iodonium is an inhibitor of the respiratory burst-generating NADPH oxidase of phagocytes. The effect of this compound on human monocyte-derived macrophages and its usefulness in exploring the antimicrobial mechanisms of phagocytes was examined. 1 microM diphenylene iodonium inhibited hydrogen peroxide production by human macrophages and the activity of these cells against Toxoplasma gondii. At this concentration macrophage degranulation was unaffected.

Acetylglucosaminidase↗

PubMed1990

The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro.

Streptococcus pneumoniae culture filtrates and pneumolysin both slow human ciliary beating and damage respiratory epithelium in vitro. A polyclonal pneumolysin antibody bound to sepharose beads removed pneumolysin from culture filtrates and showed that pneumolysin alone was responsible for the effects on epithelium. In a 48-h organ culture pneumolysin caused ciliary slowing and epithelial disruption in a dose-dependent manner down to 5 ng/ml. Comparison of the ciliary slowing activity and pneumolysin concentration in filtrates in a continuous broth culture showed a maximal effect at 16 h (pneumolysin 7.5 micrograms/ml). Later the activity decreased while the pneumolysin concentration increased (8.8 micrograms/ml). This loss of activity was prevented by neutralisation of the acid pH of the culture medium. Eight different culture filtrates produced significant (P less than 0.05) ciliary slowing which correlated (r = 0.95) with simultaneously measured haemolytic (pneumolysin) activity. Substitution of tryptophan (position 433) by phenylalanine reduced the haemolytic and ciliary slowing activity of pneumolysin, but did not affect its ability to activate complement. There was no correlation between the ciliary slowing produced by the culture filtrate and that produced by the autolysate of a particular strain, nor between ciliary slowing and the extent of autolysis or the serotype of the strain.

Antibodies, Bacterial↗

PubMed1990

Development of an optimized system for electroporation of Listeria species.

Electroporation was used to facilitate transformation of Listeria species with plasmid DNA. Optimal conditions for transformation of L. monocytogenes were a field strength of 8.5 kV/cm, 200 Ohms resistance, 25 microF capacitor with a time constant of 5 ms. With these conditions, 3.9 x 10(6) transformants/micrograms DNA were obtained. Under the same conditions, L. innocua and L. ivanovii exhibited a frequency of transformation similar to that of L. monocytogenes but a somewhat lower level was obtained with L. seeligeri.

Cell Membrane Permeability↗

PubMed1979

Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules.

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.

Animals↗

PubMed1978

The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis.

The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.

Amino Acid Sequence↗