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M A Chambers

Publications and source records attributed to M A Chambers.

36 records · Page 2Linked to original sources
PubMed2000

Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M. bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin.

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Animals↗
PubMed1999

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian badger (Meles meles).

The Eurasian badger (Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.

Animals↗
PubMed1998

Some issues in the assessment of clinical practice: a review of the literature.

This paper focuses on some issues in the assessment of clinical practice of particular interest to the author. The assessment of students of nursing in clinical practice is acknowledged as a long-standing and difficult problem. There is little consensus as to what is meant by the term competence when applied to clinical nursing practice, making the assessment of clinical practice a mainly subjective process. The English National Board (1989) has distinguished the term mentor as meaning counsellor and guide, nevertheless the roles of mentor and assessor are frequently confused. It is suggested that nurses are equally accountable for the accurate assessment of student nurses' clinical skills as they are for their own practice. The validity and reliability of tools used to assess clinical practice are difficult to establish, making objective assessment complex at best, and impossible at worst. The assessment of the reflective process has been suggested as one way out of the dilemma, but the ability to think and to write does not necessarily translate into competent clinical practice.

Clinical Competence↗
PubMed1997

Differential responses to challenge with live and dead Mycobacterium bovis Bacillus Calmette-Guérin.

Bacillus Calmette-Guérin (BCG) vaccination has been shown to protect against challenge with virulent Mycobacterium tuberculosis in a range of experimental animal models: in each case, protective efficacy requires vaccination with live bacteria. With the goal of moving to a new generation of safer, nonliving vaccines, efforts have been made to identify the factors that determine the efficacy of live vaccination. We show that injection of live, but not dead, BCG induces localized swelling in the mouse footpad model. Live and dead bacteria induce similar responses during the first week after vaccination as determined by immunohistochemical analysis of the site of injection and of the draining lymph node. The subsequent differential response is characterized by migration of acid-fast bacilli to the draining lymph node in the case of the live vaccine. This is accompanied by an increase in mononuclear cells in the lymph node and by expression of inducible nitric oxide synthase (iNOS) both in the lymph node and at the site of injection. The ability of the bacteria to migrate to the lymph node may be an important element in the efficacy of live BCG vaccination.

Animals↗
PubMed1997

Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and their suppression by an interleukin-10-secreting recombinant.

We have analyzed mycobacterium-induced cytokine secretion in the J774A.1 macrophage-like cell line. Tumor necrosis factor alpha (TNF-alpha) was preferentially induced by live organisms, both slow and rapid growing. Expression of interleukin-10 by a recombinant strain of Mycobacterium smegmatis caused reduced production of TNF-alpha and nitric oxide during the early stages of infection.

Blotting, Western↗
PubMed1996

Effect of bovine herpesvirus-1 on expression of interleukin-2 receptors and effect of interleukin-12 on lymphocyte proliferation.

Expression of the interleukin-2 receptor alpha chain (IL-2R alpha) by peripheral blood mononuclear cells (PBMC) from Holstein calves, both experimentally-infected with bovine herpesvirus-l (BHV-l) and controls, was measured by flow cytometry. Expression of IL-2R alpha was 35 percent and 23 percent higher in infected calves than controls, on days 2 and 3 postinfection (PI), respectively. Concurrent with this increase in IL-2R alpha expression, a significant decrease (P < 0.001) was observed in the PHA-induced proliferative responses of PBMC from infected compared with control calves. In vitro treatment with recombinant human (rhu) IL-12 enhanced PHA-induced proliferative responses of PBMC from both infected and control calves. This rhuIL-12 enhancement of mitogen-induced proliferative responses was significant (P < 0.001) in infected calves on day 2 PI and was sufficient to abrogate the decrease observed due to BHV-1 infection. Since the expression of the beta and gamma chains of IL-2R was not measured it is difficult to speculate as to the status of high affinity receptor expression during BHV-1 infection. However results of the present study suggest that the decrease in proliferative responses observed during infection may not be due to a decrease in IL-2R alpha expression but may possibly be due to a selective down-regulation of signal transduction through IL-2R and/or by modulation of the expression of other cytokines involved in lymphocyte activation and proliferation.

Animals↗
PubMed1996

Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

Animals↗
PubMed1996

Novel indirect fluorescent antibody test for Lyme disease.

An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.

Animals↗
PubMed1995

Effect of stress on interleukin-2 receptor expression by bovine mononuclear leukocytes.

Holstein calves given three consecutive i.m. injections of dexamethasone (DEX) (0.04 mg kg-1) showed lymphopenia and neutrophilia with increased numbers of mature neutrophils on post-injection Days 1 and 2, but these values returned to normal levels by post-injection Day 3. Interleukin-2 receptor (IL-2R) expression by peripheral blood mononuclear cells (PBMC) was evaluated by flow cytometry using a monoclonal antibody specific for bovine IL-2R alpha. Treatment with DEX significantly decreased expression of IL-2R alpha in Concanavalin A (Con A)-activated PBMC on Day 1 (P < 0.02) and on Day 2 (P < 0.1). On Day 3, expression of IL-2R alpha by PBMC was similar in control and DEX-treated calves. This decrease in IL-2R alpha expression correlated with decreased proliferative responses of PBMC to the T-cell mitogens, phytohemagglutinin (PHA) and Con A. Following in vitro treatment with recombinant human (rhu) interleukin-12 (IL-12) Con A-induced proliferative responses of PBMC tended to be higher in both groups. However, the rhu IL-12 induced increase of Con A activated proliferative responses were significantly greater in DEX-treated calves than in control calves. IL-2R alpha expression by PBMC was found to be less in calves transported 800 km in a truck as compared to that in PBMC from controls. These data suggest that stress-induced immunosuppression in calves may involve decreased IL-2R alpha expression and decreased IL-12 production. Serum chemistry results indicated a trend toward higher creatine kinase (CK) levels in DEX-treated calves. This may be due to the lysis of corticosteroid sensitive lymphocytes.

Animals↗
PubMed1994

"Natural" presentation of human papillomavirus type-16 E7 protein to immunocompetent mice results in antigen-specific sensitization or sustained unresponsiveness.

We have used a mouse model that utilizes the exclusively epithelial nature of human papillomavirus (HPV) infections to investigate the in vivo immune response to the E7 protein of human papillomavirus type-16. A keratinocyte cell line expressing E7 protein has been established and grafted onto syngeneic mice using a transplantation technique that permits the reformation of a differentiated epithelium on a granulation tissue bed. In this way viral antigens may be presented to the immune system in a way comparable to natural infection. A delayed-type hypersensitivity (DTH) response was studied post grafting by intradermal challenge with recombinant E7 protein. A significant response to E7 has been demonstrated in this way; however, priming with a low amount of HPV-16 E7 antigen induces immunological unresponsiveness, as measured by a loss of DTH reactivity to the protein, and persistence of keratinocytes expressing E7. Lymphocytes from mice exhibiting DTH reactivity have been shown to proliferate when stimulated with purified recombinant E7 protein in vitro, while immunoperoxidase staining of tissue from the sites of immunologically-induced inflammation has defined the cell infiltrate to be phenotypically characteristic of DTH. The observations reported here have important implications for vaccine strategy.

Animals↗
PubMed1994

Delayed-type hypersensitivity response to human papillomavirus type 16 E6 protein in a mouse model.

A mouse model incorporating the epitheliotropic nature of human papillomavirus (HPV) infections has been used to study an immune response to HPV type 16 (HPV-16) E6 protein in vivo. Using a transplantation technique, a novel immortal keratinocyte cell line expressing the E6 protein has been grafted onto syngeneic mice to re-form a differentiated epithelium overlying a granulation tissue bed. By this approach the presentation of viral antigens to the immune system can be modelled in a way analogous to the natural infection. Here we report a delayed-type hypersensitivity (DTH) reaction in grafted mice challenged intradermally with a recombinant vaccinia virus expressing the HPV-16 E6 protein. The specificity of the response was confirmed by the absence of a DTH reaction to challenge with virus expressing either HPV-16 E7 or L1 protein.

Animals↗
PubMed1994

A randomized, double blind, placebo controlled trial of intravenous loading with S-adenosylmethionine (SAM) followed by oral SAM therapy in patients with knee osteoarthritis.

OBJECTIVE: We evaluated the effectiveness and rapidity of onset of S-adenosylmethionine (SAM), administered as daily intravenous boluses of 400 mg for 5 days, followed by oral tablets, 200 mg thrice daily for 23 days, versus a matching placebo regimen, in the treatment of 81 patients with symptomatic knee osteoarthritis (OA). METHODS: The study was bicentric, randomized, double blinded, and placebo controlled. Patients underwent a 7-day washout of arthritis medications prior to initiation of this study treatment. Major outcome measures were the Stanford Health Assessment Questionnaire disability and pain scales, and supplemental visual analog scales for rest and walking pain. RESULTS: At one site, patients had milder OA, the baseline characteristics of the treatment groups were well matched, and the SAM treated group showed significantly greater reduction in overall pain and rest pain (p < 0.05) than the placebo treated group. At the other site, the patients had more severe OA, randomization yielded markedly different treatment groups, and the response to treatment did not differ between groups. Onset of SAM effect was seen as early as 14 days after the start of treatment. CONCLUSION: SAM may be an effective treatment for some patients with symptomatic knee OA, and merits further study. Intravenous loading before oral maintenance therapy may be advantageous.

Administration, Oral↗
PubMed1990

Bioactivation of 8-methoxypsoralen and irreversible inactivation of cytochrome P-450 in mouse liver microsomes: modification by monoclonal antibodies, inhibition of drug metabolism and distribution of covalent adducts.

The in vitro bioactivation of 8-MOP was studied in liver microsomes of male CD-1 mice. In 10-min incubations with 40 microM [14C]8-MOP, covalent binding (mean +/- S.D.) was 1.8 +/- 0.4, 3.1 +/- 0.6 and 5.4 +/- 0.4 nmol/mg protein, respectively, in microsomes from mice pretreated for 3 days with vehicle, phenobarbital or beta-naphthoflavone (BNF). A monoclonal antibody (MAb 1-7-1), which recognizes isozymes of cytochrome P-450 induced by 3-methylcholanthrene (P1-450 and P3-450), selectively inhibited the metabolism of 8-MOP (-57%) and covalent binding of its metabolites (-40%) in microsomes from mice pretreated with BNF, but had no effect in microsomes of mice pretreated with phenobarbital or vehicle. Monoclonal antibody 2-66-3, which recognizes the major isozymes of rat cytochrome P-450 induced by phenobarbital and unknown isozymes in the mouse, enhanced the covalent binding of 8-MOP metabolites in microsomes of mice pretreated with vehicle (+74%), phenobarbital (+44%) or BNF (+31%) without affecting the disappearance of 8-MOP. Preincubation of liver microsomes from BNF-pretreated mice with 40 microM 8-MOP decreased the activity of 7-ethoxycoumarin de-ethylase in a time-dependent manner. Preincubation with 40 microM 8-MOP for 10 min decreased the Vmax from 3.4 to 1.2 nmol/min/mg protein and increased the Michaelis constant from 46 to 90 microM, thus demonstrating mixed competitive and noncompetitive inhibition of 7-ethoxycoumarin de-ethylase. Cysteine trapped three-fourths of the reactive intermediates of 8-MOP but was ineffective in preventing the irreversible inhibition of 7-ethoxycoumarin de-ethylase activity or the 45% spectral loss of cytochrome P-450. Cysteine was ineffective probably because it did not prevent the irreversible binding of metabolites of 8-MOP to cytochrome P-450. There was no spectral evidence that 8-MOP formed cytochrome P-420 or metabolite-intermediate complexes with cytochrome P-450. These findings support the hypothesis that irreversible inactivation of cytochrome P-450 by 8-MOP is caused by modification of the apoprotein by reactive metabolites.

7-Alkoxycoumarin O-Dealkylase↗
PubMedDate not supplied

Inhibition and induction of theophylline metabolism by 8-methoxypsoralen. In vivo study in rats and humans.

The effects of 8-methoxypsoralen (8-MOP) on the metabolism of theophylline were studied in rats and humans. Rats were randomized into three groups and prepared with iv jugular catheters. Group I (N = 4) received a single ip injection of 27 mg/kg of 8-MOP, group II (N = 5) vehicle (corn oil), and group III (N = 4) 50 mg/kg/day of 8-MOP for 3 days. Rats were subsequently administered 15 mg/kg of theophylline iv, and timed blood samples (0.2 ml) were assayed for theophylline by HPLC. Theophylline clearance (ml/min/kg; mean +/- SD) was 1.7 +/- 0.3, 2.4 +/- 0.5, and 9.5 +/- 1.6 in groups I, II, and III, respectively. The half-life (harmonic mean) from 0.5 to 12 hr was 7.2, 3.6, and 0.8 hr. Urinary excretion of unchanged theophylline (mean +/- SD) from 0 to 24 hr was 60 +/- 10, 41 +/- 6, and 13 +/- 3% of the administered dose. In a crossover study, three healthy, male, nonsmokers received 600 mg of oral theophylline. Urine and plasma were collected for 48 hr. One week later, subjects received 1.2 mg/kg of oral 8-MOP followed in 1 hr by 600 mg of oral theophylline. Mean residence time of theophylline increased from 10.7, 17.2, and 12.2 hr in the control period, to 20.3, 19.0, and 18.4 hr after 8-MOP. The AUC (microgram.hr/ml) of theophylline increased from 204, 213, and 204, to 555, 364, and 432, while clearance (ml/min/kg) decreased from 0.74, 0.57 and 0.63, to 0.27, 0.33, and 0.30, respectively. Urinary excretion of unchanged theophylline from 0 to 48 hr increased from 14, 14, and 15, to 24, 21, and 20%. We conclude that 8-MOP administered acutely is a potent inhibitor of theophylline metabolism and chronically in the rat is a powerful inducer.

Animals↗