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Biomedical subjects

M J Walker

Publications and source records attributed to M J Walker.

At least 55 records · Page 3Linked to original sources
PubMed1998

Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits.

Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis, the causative agent of whooping cough. Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp. We have individually expressed the five PT subunits in Salmonella typhimurium aroA and subsequently expressed a synthetic pertussis toxin operon (pDP16), via the use of a temperature inducible expression system. In S. typhimurium aroA containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed. In S. typhimurium aroA (pDP16) S5 was completely processed, S1 was partially processed, whilst S2, S3 and S4 remained unprocessed. Induction of the synthetic PT operon decreased the in vitro invasiveness of S. typhimurium aroA, compared to vector-only and plasmid-less strains. Following oral immunisation of Balb/c mice with S. typhimurium aroA (pDP16), statistically significant IgG (p < 0.05) specific to PT was detected in the serum of mice. Nonetheless, despite a statistically significant anti-PT serum antibody response in immunised mice, vaccination with S. typhimurium aroA (pDP16) failed to protect mice from virulent challenge.

Administration, Oral↗
PubMed1998

Flow cytometric analysis of cellular changes in mice after intradermal inoculation with a liposome-iscom adjuvanted vaccine.

As it is not known what changes to leucocyte homeostasis are mandatory for effective adjuvant action, the biological relevance of systemic changes elicited by different vaccine formulations can only be interpreted in the context of the immunological outcomes. We used flow cytometry to quantify the changes in leucocyte subsets induced in mice intradermally immunized with SAMA4 (adjuvant group), outer membrane proteins (OMP) purified from Actinobacillus pleuropneumoniae (OMP antigen group), SAMA4 adjuvanted OMP (OMP vaccine group), or phosphate-buffered saline (PBS: control group). This approach allowed direct comparisons to be made between the effects of antigen, adjuvant or antigen-adjuvant complexes on immune effector cell populations. Antigens complexed with the liposome-iscom hybrid adjuvant, SAMA4, generated strong antibody responses and cytotoxic T-cell activity in animals immunized intradermally, reflecting remobilization and recruitment of specific cell populations. Splenomegaly, due to granulocytosis, monocytosis and megakaryocytosis, was most prominent in the OMP vaccine group. Histological examination of spleen sections confirmed that these changes were due primarily to splenic haematopoiesis. Circulating numbers of granulocytes and monocytes increased significantly (P < 0.05) in the blood of the OMP vaccine group, as did granulocyte numbers in the lungs (P < 0.05). No changes in T- and B-cell numbers were detected by flow cytometry in the spleens, lungs or blood over the 28-day period in any treatment group. Thymocyte numbers (predominantly CD4+CD8+ cells) in the OMP vaccine group fell by 95% within 3 days of immunization. Identical cellular responses were obtained when an innocuous antigen, ovalbumin, was complexed with SAMA4 instead of OMP, thus demonstrating that the adjuvant effects of SAMA4 were due to synergistic interaction between antigen and adjuvant and not due to the presence of toxic components. The association of strong adaptive immune responses with such complex changes in leucocyte homeostasis induced by complexing adjuvant and antigen suggested that the changes were important for effective vaccination and were not purely circumstantial.

Adjuvants, Immunologic↗
PubMed1998

Phase II trial of recombinant human interleukin-4 in patients with disseminated malignant melanoma: a Southwest Oncology Group study.

Malignant melanoma is increasing in incidence in this country. Metastatic disease generally responds poorly to most chemotherapy drugs. Immunologic and biologic agents have shown some activity in this disease. Interleukin 4 (IL-4) is a cytokine produced by activated T-lymphocytes with pluripotent activities including growth inhibition of various tumor cell lines in vitro and immune- mediated tumor growth inhibition in in vivo animal tumor models. In this phase II trial, patients with advanced malignant melanoma with no prior systemic therapy for metastatic disease and Southwest Oncology Group performance status 0-1 were treated with recombinant human IL-4 at a dose of 5 micrograms/kg/day by daily subcutaneous injection days 1-28 followed by a 7-day rest period, after which the cycle was repeated. Thirty-six patients were registered to this study. Two patients were ineligible by study criteria. Among the 34 eligible patients, there was 1 complete response, 0 partial responses, 2 stable/no responses, 27 increasing disease/progression, 1 early death, and 3 patients whose assessment was inadequate to determine response. The overall estimated response rate was 3% (1 of 34) with a 95% confidence interval 0.1-15%. The duration of the complete response is 421+ days. Thirty-one of the 34 eligible patients have died. The estimated median survival is 6 months (95% confidence interval 4-9 months). The most common toxicities were elevated liver function tests, nausea/vomiting/diarrhea, malaise/fatigue, edema, headache, myalgias/arthralgias, and fever/chills. Despite promising preclinical growth inhibitory and immunomodulatory effects, IL-4 in this dose and schedule showed only low antitumor activity. Alternative methods and routes of administration or combinations of IL-4 with other cytokines might produce greater antitumor effects.

Adult↗
PubMed1998

Sodium channel-blocking properties of spiradoline, a kappa receptor agonist, are responsible for its antiarrhythmic action in the rat.

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

Animals↗
PubMed1998

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis.

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

Animals↗
PubMed1998

A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress, production of acid phosphatase, and in vivo persistence.

Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.

Acid Phosphatase↗
PubMed1998

Phase II study of carmustine, dacarbazine, cisplatin, and tamoxifen in advanced melanoma: a Southwest Oncology Group study.

PURPOSE: The combination of carmustine (BCNU), dacarbazine (DTIC), cisplatin (DDP), and tamoxifen (Tam) has been reported in small series to provide a response rate of 50%, but with significant myelosuppression and risk of thromboembolic complications. We performed this phase II study to assess the antitumor activity and important toxicities of this combination in the cooperative group setting. PATIENTS AND METHODS: Seventy-nine eligible patients were treated with BCNU 150 mg/m2/d, every 6 weeks, DTIC 220 mg/m2/d on days 1 to 3 every 3 weeks, DDP 25 mg/m2/d on days 1 to 3 every 3 weeks, and Tam 20 mg orally daily throughout treatment. Treatment cycles were repeated every 6 weeks in responding or stable patients for a maximum duration of 1 year. RESULTS: Twelve objective responses were achieved (response rate 15%, 95% confidence interval 8%-25%). Five responses were complete (CR) and seven were partial (PR). The median response duration was 8+ (range, 4-19+) months, (16+ [4-19+] for CR and 8+ [4-11] for PR), and the median survival of the entire group was 9 months. The toxicities were predominantly neutropenia and thrombocytopenia. Four patients developed thromboembolic events. Two patients died while on protocol therapy, one with complications of neutropenia, and the other with disease progression. CONCLUSION: The activity of this regimen is in the range reported for single agents or DTIC plus DDP, and the addition of BCNU and Tam appears to increase toxicity. We do not recommend this combination for routine treatment of advanced melanoma or as the control arm in randomized studies of combination therapy.

Adolescent↗
PubMed1997

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells.

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1-4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37 degrees C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.

Blotting, Southern↗
PubMed1997

Temperature dependent expression of an acid phosphatase by Bordetella bronchiseptica: role in intracellular survival.

Bordetella bronchiseptica has the ability to invade and survive intracellularly. This potential to survive for extended periods within eukaryotic cells might play an important role in the pathogenesis of the infections caused by this microorganism. The bacterial factors involved in this process, however, have not yet been determined. In this study we have identified an acid phosphatase produced by B. bronchiseptica, but not by other Bordetella spp. The expression of this enzyme was demonstrated to be strictly regulated by temperature (optimal expression at 30 degrees C) and seems to be partially repressed by the product of the bvg locus. The enzyme was localized in the cytoplasmic fraction, the optimal activity was observed at pH 5.5, and the apparent molecular mass obtained by zymogram was 40 kDa. To further investigate the pathogenic role of this enzyme, mutants lacking acid phosphatase activity were obtained from both bvg-positive and bvg-negative parental strains using minitransposons. The growth pattern of these recombinant clones in vitro was similar to the parent strains, however, the tested clones exhibited a significant reduction (P < or = 0.05) in their intracellular survival ability. This newly described acid phosphatase from B. bronchiseptica seems to play a role in intracellular survival, and therefore represents a novel pathogenicity factor.

Acid Phosphatase↗
PubMed1997

KC8851, a tedisamil analogue with mixed channel blockade, exhibits antiarrhythmic properties against ischemia- and electrically-induced arrhythmias.

KC8851, a structural analogue of tedisamil, has previously been found to exhibit mixed blockade of K+ and Na+ currents in isolated rat ventricular myocytes. We have now investigated the antiarrhythmic actions of this compound in the anaesthetized rat and isolated rat heart. In the rat, KC8851, at concentrations as low as 0.2 micromol kg(-1) min(-1), widened the QT intervals of the ECG and prolonged the effective refractory period in a dose-dependent manner. Such actions were consistent with blockade of repolarizing K+ currents. At relatively higher doses (above 0.5 micromol kg(-1) min(-1)), KC8851 increased RSh amplitude suggesting blockade of Na+ currents. The compound was found to be effective against occlusion-induced arrhythmias at doses of 0.5 to 2 micromol kg(-1) min(-1). In isolated hearts, the effects of KC8851 on PR and QRS intervals were potentiated by elevated concentrations of K+ and H+. Overall, KC8851 was found to exhibit antiarrhythmic actions consistent with inhibition of both K+ and Na+ currents.

Animals↗
PubMed1997

Expression of a synthetic pertussis toxin operon in Escherichia coli.

Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli beta-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

Amino Acid Sequence↗
PubMed1997

A model of myocardial ischemia for the simultaneous assessment of electrophysiological changes and arrhythmias in intact rabbits.

A method of recording epicardial monophasic action potentials (MAPs) and ischemia-induced arrhythmias following coronary artery ligation in intact rabbits is described. It is expected that this model will have utility in analyzing drug effects and mechanisms of ischemic arrhythmogenesis. Rabbits were found to have two arrhythmic phases following coronary artery occlusion which correspond to phase Ia and Ib arrhythmias in other species. Epicardial MAPs recorded from ischemic tissue allowed electrophysiological effects to be correlated with these phases. Phase Ia arrhythmias occurred within the first 2 min of coronary artery occlusion and were associated with a reduction in the maximum upstroke velocity of MAPs and changes in MAP duration, including the occurrence of alternans in duration. Phase Ib arrhythmias occurred between 8 and 15 min after coronary artery occlusion. These arrhythmias were associated with a decrease in MAP duration and amplitude, alternans in MAP duration as well as conduction block. Coronary artery occlusion reliably induced arrhythmias in rabbits if the left branch of the coronary artery and the left anterior descending artery were occluded. There was a 95% incidence of premature ventricular contractions, 38% of ventricular tachycardia, and 48% of ventricular fibrillation (n = 21). The results of this study show that epicardial MAPs can be used to aid in the characterization of the electrophysiological mechanisms of ischemia-induced arrhythmias in vivo.

Action Potentials↗
PubMed1997

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day-1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.

Animals↗
PubMed1997

Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF).

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a commercially expensive respiratory disease of swine. Salmonella typhimurium SL3261 was used as a live carrier of plasmid pKF1, which encodes a 15-kDa recombinant M. hyopneumoniae protein. This expressed recombinant protein consists of the carboxy-terminal 11 kDa of a 42-kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Rabbit anti-15-kDa serum was able to inhibit the growth of viable M. hyopneumoniae J in vitro. When used as a live oral vaccine, S. typhimurium SL3261(pKF1) induced a significant secretory immunoglobulin A immune response in the lungs of mice orally immunized against the M. hyopneumoniae antigen. Utilization of live oral vaccines expressing potentially protective M. hyopneumoniae proteins, such as the NrdF antigen, which can stimulate a lung mucosal response against surface-accessible proteins may provide a cost-effective alternative to the present control strategies used for porcine enzootic pneumonia.

Administration, Oral↗
PubMed1997

Investigation of the validity and reliability of four objective techniques for measuring forward shoulder posture.

Clinicians often rely on visual inspection and descriptive terms to documents a patient's forward shoulder posture. The purpose of this study was to assess the validity and intrarater reliability of four objective techniques to measure forward shoulder posture. Subjects were 25 males and 24 females. Subjects had a lateral cervical spine radiograph taken, from which the horizontal distance from the C7 spinous process to the anterior tip of the left anterior acromion process was measured. Subjects then proceeded twice through a random order of four measurements: the Baylor square, the double square, the Sahrmann technique, and scapular position. These results were then used to determine the intrarater reliability of each technique. Multiple regression analyses were performed on each measure's mean scores to determine both the correlation with and the predictive value for the radiographic measurement. The intraclass correlation coefficients for intrarater reliability ranged from .89 to .91. The correlation coefficients ranged from -.33 to .77, and the coefficients of determination ranged from .10 to .59 (N = 49). The researchers demonstrated clinical reliability for each technique; however, validity compared with the radiographic measurement could not be established. These techniques may have clinical value in objectively measuring change in a patient's shoulder posture as a result of a treatment program. Before any of these measures could be universally recommended in clinical practice, future research is necessary to establish interrater reliability and assess each technique's ability to detect postural changes over time.

Adult↗
PubMed1997

Phase II trial of topotecan in malignant melanoma.

Topotecan (S-9-dimethyl-10-hydroxycamptothecin hydrochloride SKF 104864-A) is a semisynthetic analog of the alkaloid camptothecin and, similar to the parent compound, a potent inhibitor of topoisomerase I. The cytotoxicity induced by topotecan appears due to interference with the normal breakage reunion reaction of topoisomerase I leading to DNA damage and cell death. Since preclinical studies of topotecan suggested antitumor activity against refractory solid tumors, a phase II trial of the drug was initiated in melanoma patients with recurrent and/or metastatic disease. Topotecan 1.5 mg/m2 was given as a daily 30-min infusion for 5 days and repeated every 21-28 days. Seventeen patients were entered into the treatment program with all evaluable for toxicity but I patient, inevaluable for response. There were no objective responses. Toxicity was predominantly severe myelosuppression, which occurred in 12 of 17 (70%) patients. Topotecan in this dose and schedule is inactive in malignant melanoma.

Adult↗