Mycobacterium Vaccae Research Articles

Extracellular 3β-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D

Extracellular 3β-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3β-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3β-hydroxy function followed by Δ 5→Δ 4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 ± 4 kDa. The purified SO obeyed Michaelis–Menten kinetics, double reciprocal plots kinetics revealed K m value towards DHEA 5 × 10 −4 M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.

SRL172 (killed Mycobacterium vaccae) in addition to standard chemotherapy improves quality of life without affecting survival, in patients with advanced non-small-cell lung cancer: phase III results

BackgroundThis open-label, randomised phase III study was designed to further investigate the clinical activity and safety of SRL172 (killed Mycobacterium vaccae suspension) with chemotherapy in the treatment of non-small-cell lung cancer (NSCLC). Patients and methodsPatients were randomised to receive platinum-based chemotherapy, consisting ofup to six cycles of MVP (mitomycin, vinblastine and cisplatin or carboplatin) with (210 patients) or without (209 patients) monthly SRL172. ResultsThere was no statistical difference between the two groups in overall survival (primary efficacy end point) over the course of the study (median overall survival of 223 days versus 225 days; P = 0.65). However, a higher proportion of patients were alive at the end of the 15-week treatment phase in the chemotherapy plus SRL172 group (90%), than in the chemotherapy alone group (83%) (P = 0.061). At the end of the treatment phase, the response rate was 37% in the combined group and 33% in the chemotherapy alone group. Patients in the chemotherapy alone group had greater deterioration in their Global Health Status score (–14.3) than patients in the chemotherapy plus SRL172 group (–6.6) (P = 0.02). ConclusionIn this non-placebo controlled trial, SRL172 when added to standard cancer chemotherapy significantly improved patient quality of life without affecting overall survival times.

Stable carbon isotope fractionation during aerobic biodegradation of chlorinated ethenes.

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.

Mycobacterium vaccae induces a population of pulmonary CD11c+ cells with regulatory potential in allergic mice.

The hygiene hypothesis proposes that common, harmless microorganisms, present throughout our evolutionary history, have helped to develop immunoregulatory mechanisms that prevent inappropriate immune responses by the host. Using a mouse model of allergic pulmonary inflammation, we report that treatment with an ubiquitous saprophytic mycobacterium, Mycobacterium vaccae, significantly reduces allergic inflammation by decreasing type 2 responses such as eosinophilia and IL-4 expression. Rather than observing an increase in type-1 cytokine expression, we found elevated production of IL-10 in the lungs suggesting a role for regulatory T cells. Since induction of these cells may be dependent on APC, we investigated the effects of M. vaccae treatment on pulmonary CD11c+ cells. Increased levels of IL-10, TGF-beta and IFN-alpha mRNA were detected in CD11c+ cells from M. vaccae-treated allergic mice. We propose that M. vaccae-induced CD11c+ cells have a potential regulatory role at the site of inflammation through their secretion of immunomodulatory cytokines.

Effects of Mycobacterium vaccae on toll-like receptor expression

Rationale Toll-like receptors (TLRs), transmembrane proteins expressed on the surface of antigen presenting cells (APCs), contribute to the activation of the innate immune system by recognizing specific pathogen-associated molecular patterns and by directing T cell responses. For example schistosomal lysophosphatidylserine stimulation of dendritic cells (DCs) drives the development of regulatory T cells (Tregs). Since Mycobacterium vaccae has similarly been found to induce Tregs and to improve symptoms of pulmonary allergic inflammation, we investigated its effects on the expression of TLRs to assess their involvement in the bacterium's mode of action. Methods Using real-time PCR, TLRs and cytokine expression were measured from both M. vaccae-stimulated pulmonary CD11c+ APCs from allergic mice and from M. vaccae-stimulated THP-1 cells. We determined the expression of TLR2, -4, -7 and -8 and of CD14, a surface molecule involved in mediating TLR signaling. Results M. vaccae differentially influenced the expression of TLRs both in vitro and in vivo. For example, expression of TLR2 and CD14 was increased in both stimulated THP-1 cells and pulmonary CD11c+ cells. Moreover, TLR2 increased expression in this CD11c+ population correlates with an increased expression of the regulatory cytokines IL-10 and IFN-α. Conclusions These results suggest that stimulation with M. vaccae influences the expression of TLRs on APCs, thereby directing the host's immune responses. It could be speculated that M. vaccae-induced CD11c+ cells have a potential regulatory role through their secretion of immunomodulatory cytokines that are TLRs-dependent.

Intratracheal administration of Mycobacterium vaccae as a long term effect on airway histopathology of chronic asthma in BALB/c mice

Rationale To evaluate whether intratracheal M. vaccae administration may provide a therapeutic effect and long-term protection on airway histopathology of already established chronic asthma in BALB/c mice. Methods BALB/c were divided into four groups. Chronic asthma model was established in groups I, II and III as per protocol. Mice in group IV served as controls. Groups I and II were also treated with 10μl of 10 7CFU of M. vaccae intratracheally (i.t.) (days 42-45-48). Group III did not receive any M. vaccae. Two days after the last i.t. instillation lungs were taken out for histopathologic evaluation from mice in Group I, III and IV. To evaluate long-term efficacy of M. vaccae, mice in Group II were challenged with OVA nebulization three weeks after the last i.t. instillation, and then were sacrificed. Results All of the histopathologic characteristics evaluated in all airways were significantly more in Group III when compared to controls. All of the histopathologic parameters of Group I were significantly less than those of Group III. Comparison of Group II and III, revealed histological parameters were significantly less in Group II. Comparison of Group I and II revealed histological parameters were significantly more in Group I. There was no difference in some of the parameters in comparison of Group I and controls. Most of the histopathologic parameters were not significantly different in Group II when compared with controls. Conclusions M. vaccae administration may provide an effective and long-term therapeutic strategy in treatment of histopathologic changes in murine model of chronic asthma.

Immunotherapy with Mycobacterium vaccae in the treatment of tuberculosis.

All the trials of immunotherapy of tuberculosis with killed Mycobacterium vaccae, published or not, that are known to the authors are reviewed here. Following an introduction giving a brief account of some earlier immunotherapies for tuberculosis, the origins of the concept of immunotherapy with M.vaccae are considered. Progress is traced from the early work with irradiation-killed organisms in leprosy to the study in London of modulation of tuberculin skin-test responses, and the first comparative trials in The Gambia and Kuwait. In the last of these studies, dosages and different preparations were compared. As a result of this subsequent studies have used 109 heat-killed organisms, equivalent to 1mg wet-weight of bacilli, as a standard dose. A series of small trials in Argentina, India, Nigeria, Romania, South Africa and Vietnam have pioneered the way forward, disclosing geographic variability, with South Africa as the only country where almost no effects were recorded. Together the studies have shown that a single dose may not be sufficient. These studies have confirmed the mode of action of M.vaccae to be regulation of cell-mediated immunity with enhancement of Th1 and down-regulation of Th2, and they have shown benefits in faster bacteriological conversion, reduction in ESR, recovery of body weight and resolution of radiological opacities, leading to better recovery from the disease even when given to patients receiving directly observed therapy, short-course (DOTS). Three major randomised, placebo-controlled and partly blinded trials have been carried out in Africa. The first, in South Africa showed no M.vaccae-related effects. The second trial, in Uganda, confirmed the observations made in the earlier studies of faster sputum conversion and better radiological clearance. The third trial, in Zambia and Malawi, showed a trend towards benefits in the treatment of HIV seronegative patients but failed to show beneficial effects in HIV seropositive patients. Studies in patients with multi-drug-resistant tuberculosis have shown that multiple doses of immunotherapy are required in most cases, and that these markedly improve cure-rates for these patients. This is especially so when they are also treated with chemotherapy tailored to the resistance pattern of their infecting organisms. A small study has just commenced in which repeated doses of M.vaccae are being administered to a group of patients who have failed treatment with DOTS-Plus (directly observed therapy with drugs selected on the basis of drug susceptibility profiles). Late in the investigation came publications from China supporting and confirming the data in both drug-sensitive and drug-resistant disease, by the use of multiple injections of their own different preparation of M.vaccae. The trial that is now beginning in Vietnam of 3 doses of M.vaccae in the treatment of newly diagnosed pulmonary tuberculosis, is accompanied by a chemotherapeutic regimen with a shortened continuation phase. If this important study is successful, immunotherapy with killed M.vaccae should be introduced into the treatment regimens for tuberculosis worldwide.

Current and emerging nonsteroidal anti-inflammatory therapies targeting specific mechanisms in asthma and allergy.

Today inhaled corticosteroids (ICS) are regarded as the first-line controller anti-inflammatory treatment in the management of asthma. However, there is an increasing awareness of the risk of long-term adverse effects of ICS and that asthma is not only an organ-specific disease but also a systemic and small airway disease. This thinking has called for systemic treatment alternatives to treat asthma targeting more disease-specific mechanisms without influencing normal physiologic functions. Blocking of disease-specific mediators is a mechanism utilized by anti-leukotrienes and anti-immunoglobulin E treatment, each proven to be effective in both asthma and allergic rhinitis.Different cytokine-modifying strategies have been tested in clinical trials with variable results, some disappointing and some encouraging. Anti-interleukin (IL)-5 monoclonal antibody treatment effectively reduces the number of eosinophils locally in the airways and in peripheral blood in asthmatic patients. Unfortunately, this marked effect on eosinophils was not associated with an improvement in bronchial hyperresponsiveness and/or symptoms. Clinical trials with a recombinant soluble IL-4 receptor have been somewhat more successful at improving asthma control and allowing reduction of ICS therapy in asthma. Treatment with recombinant IL-12 had an effect on bronchial hyperresponsiveness and eosinophilic response, but was associated with unacceptable adverse effects. Other interesting cytokine-modulating treatments include those targeting IL-9, IL-10, IL-12 and IL-13.Immune-modulating treatment with bacterial antigens represents another strategy, originating from the hypothesis that some bacterial infections guide the immune system towards a T helper (Th) type 1 immune response. Mycobacterium vaccae, Bacille Calmette-Guerin (BCG) and immunostimulatory DNA sequences have all been tested in clinical trials, with encouraging results. Future asthma and allergy treatment will probably include not only one but also two or more disease-modifying agents administered to the same patient.

Immunotherapy with mycobacteria.

To summarize and evaluate critically recent progress with mycobacteria as a potential novel disease modifying treatment strategy in asthma. The link between exposure to pathogenic or saprophytic mycobacteria and protection from allergic diseases is still controversial, and recent epidemiological studies, which addressed only exposure to Mycobacterium tuberculosis or bacillus Calmette-Guérin, did not help to clarify this issue. Moreover, the clear efficacy of mycobacterial treatment seen in animal models has not been reproduced in human asthma, and a recent small study testing the hypothesis that heat-killed Mycobacterium vaccae attenuates asthmatic reactions after allergen challenge did not provide convincing results. However, it has been shown that treatment of mice with M. vaccae induces the generation of allergen-specific T regulatory cells capable of suppressing allergen-mediated eosinophilic lung inflammation, suggesting that a general deficiency of T regulatory cell activity might be responsible for the increased prevalence of asthma. This hypothesis is supported by findings that a lack of T regulatory cells, as found in genetic disorders of man and mouse attributable to a mutation of Foxp3, a transcription factor specifically expressed by T regulatory cells, is associated with manifestations of severe atopy and autoimmunity, precisely the spectrum of diseases linked to the hygiene hypothesis. Further studies on the relationship between mycobacteria and atopic disorders are needed, but there is reason to believe that the novel findings and molecular mechanisms associated with mycobacterial infections will further strengthen the currently unproved therapeutic value of immunotherapy with mycobacteria.

Antigen-presenting cells (APC) of mice with chronic graft-versus-host disease (GVHD) cause excessive activation-induced death of T helper cells.

Both experimental and clinical forms of chronic GVHD have unique immunological features. The affected animals/individuals suffer from autoimmune disorders such as systemic lupus erythematosus (SLE), and yet they are unable to mount a self MHC-restricted T cell response to foreign antigens. Pathogenesis of the latter phenomenon was investigated in an experimental model of chronic GVHD. Chronic GVHD was induced in 8-10-week-old (B6xC3H)F1 mice by tail vein injection of 5 x 10(7) spleen cells of C3H parental strain. The recipients, when tested 3 months later, were unable to mount a T helper (Th) cell response to a randomly selected immunogen, a vaccine of l0(8) killed Mycobacterium vaccae. The animals showed evidence of generalized lymphoid hyperplasia, as indicated by GVH index >1.34, and also revealed autoantibodies against erythrocytes and dsDNA, indicating establishment of chronic GVHD. However, mice with chronic GVHD of only 3 weeks duration were able to mount the Th cell response to M. vaccae. Three consecutive immunizations of these mice at 1-week intervals, with the same immunogen, resulted in the mice becoming non-responsive to the antigen. All the three responses tested, namely the DTH, lymphoproliferation and the antibody responses, were adversely affected. The non-responsiveness induced was antigen-specific. Mice receiving two immunizations with M. vaccae responded normally to Salmonella enteritidis. Pulse treatment with cyclosporin A 0.5 mg/mouse by the i.p. route, on days 0, 1, 2, 3 and 4 at the time of immunization with M. vaccae on day 1, prevented emergence of non-responsiveness. Based on this evidence, it was concluded that repeated activation of T cells of mice with chronic GVHD induces non-responsiveness. Extent of clonal loss due to activation-induced cell death (AICD) caused by i.p. injection with a superantigen Staphylococcal enterotoxin B (SEB) was investigated in F1 mice with chronic GVHD. I.p. injection of 25 microg/mouse of SEB induced loss of SEB responding clones in both normal F1 mice and those having chronic GVHD; however, the extent of loss was much greater in the latter. In vitro antigen-specific proliferation of primed splenic T cells of normal F1 mice was observed to be quite poor when antigen was presented by APC of mice with chronic GVHD of 3 weeks duration. Proliferation profiles of T cells of normal F1 mice, in response to stimulation with concanavalin A (Con A) or SEB, were studied, using as APC irradiated spleen cells of normal F1 mice or of F1 mice with chronic GVHD of 3 weeks duration. With Con A and APC of normal F1 mice, peak proliferation was observed at 48 h, which remained at the same level up to 72 h and declined thereafter, possibly due to AICD. With SEB and the normal APC, proliferation progressively peaked at 72 h and declined thereafter. With APC of mice with chronic GVHD, the 48 h proliferative responses of both Con A and SEB were comparable to those caused by APC of normal F1 mice; however, thereafter the responses declined steeply, suggesting greater AICD. Based on these results, it was concluded that APC of mice with chronic GVHD are functionally altered to induce greater AICD.

Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation.

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y(D-mannitol/D-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Mycobacterium vaccae administration during allergen sensitization or challenge suppresses asthmatic features.

The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease. For this reason, bacteria or their components can be used as potential treatment for allergic asthma. We investigated whether heat-killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma. Mice were sensitized with ovalbumin (OVA)/alum on days 0 and 14. Thereafter, mice were challenged on days 35, 39 and 42 by inhalation of either OVA or saline aerosols. M. vaccae-treated mice received an injection with 106, 107 or 108 CFU heat-killed M. vaccae on days 0 and 14 or 107 CFU on days 35 and 39. On day 43, the airway responsiveness of the mice to increasing concentrations of methacholine was assessed, blood was withdrawn to measure serum parameters, and lung lavage was performed to detect cytokines and inflammatory cell number. Treatment of OVA-sensitized mice with 107 CFU M. vaccae either during sensitization or challenge suppresses airway hyper-responsiveness, airway eosinophilia and IL-5 production after OVA challenge. The increases in OVA-specific serum IgE and in IL-4 by respiratory challenges with OVA were only diminished after M. vaccae treatment (107 CFU) during sensitization. Heat-killed M. vaccae prevents allergic and asthmatic manifestations in a mouse model and, more importantly, M. vaccae treatment during challenge suppresses features of asthma, which opens up possibilities for new therapeutic interventions.

P-356 Addition of SRL172 (Mycobacterium vaccae) to standard chemotherapy in small cell lung cancer (SCLC) confers no survival benefit: Results of a randomised multicentre study

P-356 Addition of SRL172 (Mycobacterium vaccae) to standard chemotherapy in small cell lung cancer (SCLC) confers no survival benefit: Results of a randomised multicentre study

O-109 Improved quality of life with the addition of SRL172 (killed mycobacterium vaccae) to standard chemotherapy in patients with advanced non-small cell lung cancer: Phase III results

O-109 Improved quality of life with the addition of SRL172 (killed mycobacterium vaccae) to standard chemotherapy in patients with advanced non-small cell lung cancer: Phase III results

The Slc11a1 (Nramp1) gene controls efficacy of mycobacterial treatment of allergic asthma.

Genes controlling antibacterial resistance may be important in the hygiene hypothesis, which states that lack of bacterial infections during childhood would favor development of allergic disease. We, therefore, studied whether Nramp1 (Slc11a1) alleles, which determine susceptibility (Nramp1(s)) or resistance (Nramp1(r)) to intracellular bacteria, affect the efficacy of heat-killed Mycobacterium vaccae in the treatment of allergic asthma in a mouse model. Treatment of OVA-sensitized Nramp1(s) mice with M. vaccae suppressed airway hyperresponsiveness, airway eosinophilia, Ag-specific IgE, and IL-4 and IL-5 production after OVA aerosol challenge. In contrast, M. vaccae hardly affected these parameters in Nramp1(r) mice. In addition, The Nramp1 gene affected both T cell-mediated responses to M. vaccae in vivo and the level of macrophage activation after stimulation with M. vaccae in vitro. In conclusion, the efficacy of M. vaccae in preventing allergic and asthmatic manifestations in a mouse model is strongly affected by Nramp1 alleles. These findings could have important implications for the future use of mycobacteria and their components in the prevention or treatment of allergic asthma. A new link is described between genes, the environment, and the development of allergy, in which the Nramp1 gene fine tunes the hygiene hypothesis.

Therapeutic treatment with heat-killed Mycobacterium vaccae (SRL172) in a mild and severe mouse model for allergic asthma

The hypothesis that a lack of early childhood bacterial infections would favor the development of allergic disease suggests that bacteria can be used as a potential treatment for allergic asthma. Therefore, in this study, we investigated the therapeutic potential of heat-killed Mycobacterium vaccae in two mouse models of allergic asthma. For this purpose, mice were sensitized i.p. with ovalbumin/alum (severe model) or ovalbumin alone (mild model) and challenged on days 77, 80 and 83 by inhalation of either ovalbumin or saline aerosols. Treatment of mice with M. vaccae (s.c. 10 7 or 10 8 colony-forming units) on days 56 and 63, however, did not reduce airway hyperresponsiveness and eosinophilia, IgE and interleukin-5 production 24 h after ovalbumin challenge in either mouse model. We therefore conclude that treatment of sensitized mice with M. vaccae before allergen exposure is not able to reduce the allergic and asthma-like response in a mild and a severe model of allergic asthma.

Mycobacterium vaccae vaccine* [SRL 172] may attenuate allergen-induced late airway responses

Mycobacterium vaccae vaccine* [SRL 172] may attenuate allergen-induced late airway responses

Impact of Mycobacterium vaccae immunization on lung histopathology in a murine model of chronic asthma.

Therapeutic modalities of asthma have not been proved to be successful in reversing the already established chronic changes of airways. We aimed to determine the impact of heat-killed Mycobacterium vaccae immunization, a potent Th1 stimulant, on chronic changes of asthma. Newborn BALB/c mice were divided into three groups; mice in M. vaccae group received 107 colony-forming units (CFU)/50 micro L of heat-killed M. vaccae subcutaneously on days 3, 14 and 42 before the development of chronic asthma model, whereas mice in control and chronic asthma groups received saline. Subsequently, mice in M. vaccae and chronic asthma groups were administered 10 micro g/100 micro L of ovalbumin (OVA) on days 43, 45, 47, 49, 51, 53 and 55 intraperitoneally, and 20 micro g/10 micro L of OVA on days 83, 86 and 89 intratracheally. Mice in control group received saline on the same days. Comparison of M. vaccae and chronic asthma groups showed statistically significant differences in goblet cell numbers, thickness of basement membrane and subepithelial smooth muscle of small, medium and large airways and epithelial thickness of medium airways. There was no significant difference between the control and M. vaccae groups except for goblet cell numbers of medium and large airways, and epithelial thickness of medium airways. Results of our study suggested that immunization by M. vaccae of newborn mice would prevent some of the chronic changes of airways due to asthma.

Characterization of the initial reactions during the cometabolic oxidation of methyl tert-butyl ether by propane-grown Mycobacterium vaccae JOB5.

The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized. Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1- or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (> or = 80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M. vaccae JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (K(i) = 3.3 to 4.4 microM). Values for K(s) of 1.36 and 1.18 mM and for V(max) of 24.4 and 10.4 nmol min(-1) mg of protein(-1) were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by M. vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by M. vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.

Subcutaneous injection of killed Mycobacterium vaccae (SRP299) soon after birth delays the onset and ameliorates the clinical course of dermatitis in NC/Nga mice

Subcutaneous injection of killed Mycobacterium vaccae (SRP299) soon after birth delays the onset and ameliorates the clinical course of dermatitis in NC/Nga mice