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Biomedical subjects

P Hambleton

Publications and source records attributed to P Hambleton.

At least 37 records · Page 2Linked to original sources
PubMed1989

Pharmacological investigation of acute cellular accumulation in immunological air pouch inflammation.

Immunologically-mediated cellular accumulation was measured 24 hours after antigenic challenge using a rat subcutaneous air pouch model. This response was inhibited by treatment with prednisolone, colchicine, anti-thymocyte serum and systemic antigen. In contrast, administration of a range of other pharmacological and clinically active agents had little effect. The profile of inhibitory activity suggested that this response was mainly due to delayed type hypersensitivity with little anaphylactic or Arthus-type component.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1989

Studies on carrageenin air pouch inflammation in the rat.

Inflammation was induced in the 6-day subcutaneous air pouch of the rat by injection of carrageenin. The model was characterized in terms of exudate volume, leucocyte accumulation, granuloma, vascular permeability and protein clearance up to 7 days after injection of carrageenin. From days 2-3 rapid and reproducible changes in these responses were observed which indicated a change from polymorphonuclear (PMN) leucocyte-dominated to mononuclear (MN) leucocyte-dominated inflammation. A second injection of carrageenin on day 3 gave increases in exudate formation and PMN accumulation on day 4. Administration of carrageenin mixed with 3 day inflammatory exudate gave an increased exudate volume and decreased leucocyte accumulation at 6 h. Reduction of 6-h cellular accumulation by use of a lower dose of carrageenin or a I-day air pouch gave complete inhibition of exudate formation on day 3. In contrast, inhibition of the 6-h cell response with prednisolone had no effect on the 3-day response. Daily treatment with indomethacin gave increased PMN accumulation on day 3. Similar treatment with prednisolone additionally reduced exudate volume. Treatment on day 2 with prednisolone gave similar effects whereas indomethacin, BW755C and protease inhibitors had no effect. Administration of colchicine at this time gave inhibition of exudate volume on day 3 whereas complement depletion gave increases in volume and PMNs.

Animals↗
PubMed1988

Clostridium botulinum toxins: nature and preparation for clinical use.

C. botulinum neurotoxins are acutely toxic materials and act by inhibiting release of the neurotransmitter acetylcholine. The specific nature of this inhibition is discussed and the preparation and purification of Type A toxin specifically for clinical use is described.

Acetylcholine↗
PubMed1988

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system.

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.

Animals↗
PubMed1988

Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus.

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.

Animals↗
PubMed1988

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.

Chromatography, Affinity↗
PubMed1988

Studies on immunological air pouch inflammation in the rat.

The development of a novel immune-based model of inflammation in the subcutaneous air pouch of the rat is described. Rats were sensitized by injection with methylated bovine serum albumin emulsified with Freund's complete adjuvant and challenged 14 days later by injection of heat-aggregated antigen in saline into a 6-day subcutaneous air pouch. Time course studies revealed a peak polymorph cell infiltration at 24 h with no exudate formation, followed by a separate mononuclear cell response, rapid exudate formation, and granuloma formation over the 2- to 7-day period. Separate peaks of increased vascular permeability were observed in the acute and chronic phases of the response. This reaction was considered to be an example of delayed-type hypersensitivity based on its extended time course, the characteristic time course of oedema formation in foot-pad-challenged rats, and the failure of serum from sensitized rats to elicit a reversed passive Arthus-type reaction. The sensitization schedule was also shown to enhance the acute inflammatory response to carrageenan. In addition, the 'flare-up' reaction to secondary antigenic challenge was investigated in 7-day postchallenge lesions.

Adjuvants, Immunologic↗
PubMed1987

A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles.

1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.

Botulinum Toxins↗
PubMed1987

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system.

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.

Animals↗
PubMed1986

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes.

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.

Animals↗
PubMed1986

Scaled-up production and purification of Clostridium perfringens type A enterotoxin.

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38.8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.

Animals↗
PubMed1986

In vitro assays for botulinum toxin and antitoxins.

Clostridium botulinum produces several powerful neuroparalytic toxins which, although rare in food-poisoning instances, are generally fatal. A considerable amount of effort has therefore been made by the food industry to ensure that food treatment processes adequate to prevent growth and toxin production by Cl. botulinum. Laboratory mice and guinea-pigs are presently used extensively both for the assay of botulinum toxins and for the development and assessment of vaccines used to protect laboratory workers. An amplified ELISA, using a monoclonal antibody, has been developed for botulinum type A toxin with a sensitivity similar to that of the mouse acute toxicity test. The immunoassay has been found to be applicable to the detection of toxin in foodstuffs and could replace the currently used mouse bioassay in many laboratories. Immunoassays have also been developed for the detection of antibodies to botulinum toxins. A preliminary study has shown that antibody titres to botulinum types A and B toxins in sera taken from immunised personnel, as measured by ELISA, showed limited correlation with those measured by the toxin neutralisation test in mice. A more extensive study should determine whether the latter test can be replaced by the ELISA.

Animal Testing Alternatives↗
PubMed1985

Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity.

Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.

Amino Acid Sequence↗
PubMed1985

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.

Animals↗
PubMed1985

Clinical chemical responses to experimental airborne legionellosis in the guinea-pig.

Legionella pneumophila infection of guinea-pigs by the aerosol route with either of two strains, one (serogroup I) giving an acute the other (serogroup 3) giving a protracted illness, induced a pyrexia and similar pneumonic lesions. With both strains there was a bacteraemia with early decreases in serum iron and zinc and increases in serum copper concentrations. Marked changes in other serum components were evident only in those animals which had protracted illness (serogroup 3-infected animals). These included transient increases in aminotransferase, creatine kinase and sorbitol dehydrogenase activities and triglyceride levels, together with gradual decreases in alkaline phosphatase and leucine aminopeptidase activities. Serum lysozyme activity and acute-phase protein synthesis increased, as did the ratio of phenylalanine to tyrosine. The findings confirm the relevance of the aerosol-infected guinea-pig model for the investigation of the disease processes and evaluation of therapeutic measures for use in man.

Acute-Phase Proteins↗
PubMed1984

Anthrax: the disease in relation to vaccines.

The authors trace the origins and history of anthrax and anthrax vaccines. They describe the aetiology and pathogenesis of the disease and the variety of symptoms which result from infection. The authors relate the early work performed by Pasteur, the development of existing vaccines and the efficacy of these vaccines, and predict the type of non-living vaccines which may be used to combat anthrax in the future.

Animals↗