Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria.

Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response.

BCGitis detected by 18F-FDG PET/CT after treatment of bladder urothelial carcinoma / BCGitis detectada por 18F-FDG PET/TC después de un tratamiento de carcinoma urotelial de vejiga

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Damage Effects on Bacille Calmette-Guérin by Low-Frequency, Low-Intensity Ultrasound: A Pilot Study.

OBJECTIVES: To perform an in vitro experimental study of the possible damage effects on Bacille Calmette-Guérin (BCG) by low-frequency (42-kHz) ultrasound (US) irradiation at low spatially and temporally averaged intensities and different exposure times. METHODS: A 2-mL BCG suspension was added to the wells of a 24-well cell culture plate. Then the samples were randomly divided into 4 groups, each group including 3 wells, with group 1 as a control group and groups 2, 3, and 4, as US treatment groups. The samples for groups 2, 3, and 4 were irradiated with US at 0.13 W/cm(2) for 5 minutes, 0.13 W/cm(2) for 15 minutes, and 1.53 W/cm(2) for 15 minutes, respectively. After irradiation, the temperature, ratio of damage, and structure of the bacteria were examined. The cavitation effect of the device was detected by the passive cavitation detection method. RESULTS: After US irradiation at the different doses (intensity and exposure time), no significant temperature change was found in all sample suspensions. The ratio of bacterial damage tested by flow cytometry and the optical density of the suspensions as assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method showed that the US-irradiated groups were significantly different from the control group. The BCG damage ratio reached 28% at the intensity of 1.53 W/cm(2). Transmission electron microscopic results showed that the bacterial structure of BCG could be destroyed by low-frequency, low-intensity US. CONCLUSIONS: Low-frequency, low-intensity US can cause acute injury to BCG, and the degree of injury is closely correlated with the US dose applied.

γ Irradiated Mycobacteria Enhance Survival in Bladder Tumor Bearing Mice Although Less Efficaciously than Live Mycobacteria.

PURPOSE: γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent. MATERIALS AND METHODS: The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer. RESULTS: Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin. CONCLUSIONS: Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.

Cryopreservation of Mycobacterium tuberculosis complex cells.

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

UV-induced inactivation rates for airborne Mycobacterium bovis BCG.

Engineering ultraviolet irradiation systems as a control against infectious airborne diseases requires a knowledge of intrinsic ultraviolet (UV) inactivation rates of airborne bacteria. Ultraviolet inactivation rates for airborne Mycobacterium bovis bacillus Calmette-Guérin (BCG) were determined at 50% and 95% relative humidity (RH) in a 0.8 m3 bioaerosol reactor. Ultraviolet inactivation response of waterborne M. bovis BCG pure cultures was also determined. At 50% RH the airborne UV inactivation rates observed were two times greater than those observed in saturated air (RH = 95%), and rates at 95% RH were similar to those observed in otherwise identical cultures suspended in water. Intrinsic UV inactivation rates for M. bovis BCG were statistically similar to rates observed for Mycobacterium parafortuitum at 50% and 95% RH, indicating that M. parafortuitum is a valid surrogate for studying airborne UV responses of M. bovis BCG and Mycobacterium tuberculosis. Results also confirm that UV inactivation responses for bacteria suspended in water cannot be used to estimate UV dose response in unsaturated air.

Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.

Influence of relative humidity on particle size and UV sensitivity of Serratia marcescens and Mycobacterium bovis BCG aerosols.

SETTING: A study of Serratia marcescens and BCG aerosols. OBJECTIVE: To evaluate the effect of relative humidity (RH) on (1) the particle size and (2) sensitivity of 254nm germicidal ultraviolet (UV) irradiation. METHODS: We built a RH controlled experimental chamber into which bacteria were aerosolized, exposed to varying amounts of UV irradiance over measured time periods, and quantitatively evaluated for viability. Aerosolized Serratia marcescens and bacille Calmette-Guérin (BCG) were subject to UV doses ranging from 57-829 microW. sec/cm(2), and sampled with a six-stage Andersen culture plate impactor at RHs ranging from 25-95%. RESULTS: Percent survival for both organisms was inversely related to UV dose. Serratia marcescens was more susceptible to UV than BCG under all conditions. More than 95% of the bacterial aerosol particles were 1.1-4.7 microm in aerodynamic diameter, and particles sizes increased from low (25-36%) to high (85-95%) RH. The count median diameter ranged from 1.9-2.6 microm for Serratia marcescens and from 2.2-2.7 microm for BCG as RH increased. For both Serratia marcescens and BCG, resistance to UV increased as RH increased. The UV resistance of both Serratia marcescens and BCG aerosols dramatically increased at RH higher than 85%. CONCLUSIONS: Our results indicate that differences in UV dose, kinds of microorganisms, airborne particle size and RH affect UV susceptibility.

Effect of local ultraviolet irradiation on infections of mice with Candida albicans, Mycobacterium bovis BCG, and Schistosoma mansoni.

In this study, we investigated whether mice given ultraviolet (UV)-B (280-320 nm) radiation in doses sufficient to alter cutaneous immune cells and impair the induction of contact hypersensitivity would also have impaired resistance to infectious agents administered at the site of UV irradiation. C3H mice were exposed to 400 J/m2 UVR from FS40 sunlamps on four consecutive days. Immediately after the last UV treatment, groups of mice were injected subcutaneously with Candida albicans, injected intradermally (ID) with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or infected percutaneously with Schistosoma mansoni in UV-irradiated skin. The induction of the delayed hypersensitivity response to C. albicans and BCG, as assessed by footpad swelling, was unaffected by UV irradiation. However, the number of viable mycobacteria recovered from the lymphoid organs of BCG-infected mice was increased significantly in the UV-irradiated animals for a period of more than 2 months. Low-dose UV irradiation of the skin at the site of infection did not influence the number of S. mansoni parasites recoverable from the internal organs of mice that had been infected with cercariae percutaneously 6 weeks earlier. We conclude that the ability of UV radiation to impair the development of cell-mediated immunity to antigens introduced in a UV-irradiated site is not universal and depends on the particular antigen administered. We hypothesize that the involvement of epidermal Langerhans cells as the primary antigen-presenting cells in the induction of cell-mediated immunity may be the critical factor in determining whether a particular immune response will be affected by local UV irradiation.

[Consistency in viabilities of mycobacteria detected by FDA/EB staining and colony forming units on the medium].

We have previously reported that the staining of mycobacteria with fluorescein diacetate (FDA) and ethidium bromide (EB) detects viable bacteria and distinguish them from heat-killed bacteria. Whether this method can be applied to clinical specimens has been an important question. In the present experiment, we treated mycobacteria with either UV-irradiation, 70% ethanol, 0.2% benzalconium chloride, 5% saponated cresol solution, or 5% phenol for 24 hours, and stained with FDA/EB to evaluate the effect of sterilization. We also stained samples of sputum from a tuberculosis patient with FDA/EB after treatment with 4% NaOH for 30 min. and neutralized with 1N H2SO4. All the samples were determined for the colony forming units on 1% Ogawa egg media. A good correlation was observed between the results of FDA/EB staining and colony formation. We believe that FDA/EB staining is useful method to detect viable mycobacteria in clinical samples.

Cellular mechanisms of genetically controlled host resistance to Mycobacterium bovis (BCG).

Multiplication of Mycobacterium bovis (BCG) in the reticuloendothelial tissues of the mouse is controlled by the Chromosome 1 locus (Bcg), which exists in two allelic forms, resistant (Bcgr) and susceptible (Bcgs). We have investigated the phenotypic expression of this locus at the cellular level in vivo. No significant differences were observed in the early clearance of BCG from the bloodstream and peritoneal cavity, nor in the uptake of the infectious inoculum in spleen and liver of the mice of Bcgr and Bcgs strains. Inflammatory response to an i.p. injection of live BCG, with respect to both the cell populations involved and the kinetics of their appearance, was comparable in Bcgr and Bcgs mice. A kinetic study of BCG infection in congenitally athymic mice that carried the "nude" mutation on Bcgr (AKR/J) or Bcgs (BALB/c) background showed that the functional absence of T lymphocytes did not influence the expression of the Bcg gene. The population expressing the Bcg gene seems to be a mature cell of the mononuclear phagocyte lineage: it was resistant to a 950-rad dose of x-irradiation; it was susceptible to a prolonged exposure to silica; and, as demonstrated in radiation chimeras, it originated from bone marrow-derived precursors.

Inactivation of Mycobacterium bovis in meat products.

The time-temperature combinations necessary to destroy Mycobacterium bovis in meat products were determined. In any given time, M. bovis was destroyed at temperatures 6 to 7 degrees C (ca. 12 degrees F) lower than those necessary for destruction of members of the Mycobacterium avium-Mycobacterium intracellulare complex. Hence, any processing heat adequate to kill M. avium-M. intracellulare-complex organisms will provide a very large safety factor with respect to M. bovis. Benzalkonium chloride treatment of wiener specimens for cultural examination effectively destroyed the normal flora of wiener emulsion without reducing the numbers of M. bovis. Treatment with a phenolic disinfectant followed by formaldehyde vapor was effective in disinfecting equipment contaminated with meat emulsion containing M. bovis.

[Delayed local reaction (DLR) after BCG vaccination in mice. I.--Description (author's transl]. / La réaction locale granulomateuse après injection sous-cutanée de BCG chez la souris. I--Description.

Mice injected subcutaneously in the hind footpad with BCG vaccine subsequently develop a local reaction. This local reaction could be divided into three stages: a first characterized as an acute inflammatory reaction, a second as an active granulome formation, and a third as a chronic long lasting inflammatory reaction. The second stage is mainly formed by the appearance of a typical histiolymphocytic granuloma. The intensity of the delayed local reaction (DLR) is measured by the swelling of the injected footpad. This reaction does not occur after injection with irradiated (gamma or UV) BCG in normal mice, nor after injection of living BCG in Nude mice, indicating a T-cell immune response which develops during the in vivo multiplication of this intracellular bacteria. When the lymphoproliferation response in draining node was measured, a direct correlation was found with the intensity of the DLR, and the 125IUdR incorporation into DNA after injection of living BCG or after an irradiated BCG inoculation. The intensity and the rapidity of the onset of this DLR depend upon the dose of viable BCG inoculated in the mouse. When the DLR reached its peak, a linear dose relationship was observed in C57Bl/6 and in NCS mice.

Ultraviolet susceptibility of BCG and virulent tubercle bacilli.

To test the effectiveness of irradiating the upper air of a room with ultraviolet light at reducing the concentration of airborne tubercle bacilli, the susceptibility to the germicidal effects of ultraviolet light, Z, was determined for various mycobacteria. Virulent tubercle bacilli and bacille Calmette-Guérin (BCG) were equally susceptible to ultraviolet radiation, whereas Mycobacterium phlei had 10 times their resistance (Z, approximately one-tenth that for M. tuberculosis). The effectiveness against BCG of upper air ultraviolet irradiation in a room was tested directly by nebulizing BCG into the air of the room and monitoring its rate of disappearance. With one 17-watt fixture operating, the rate of disappearance increased 6-fold; with 2 fixtures operating (46 watts total), the rate of disappearance increased 9-fold. This implies that under steady-state conditions, the concentrations of airborne organisms with ultraviolet light(s) on would have been one-sixth and one-ninth, respectively. The increase in rate of decay of the airborne organisms using 1 fixture was equivalent to 10 air changes per hour, whereas that using 2 fixtures was approximately 25 air changes per hour (range: 18 to 33 air changes per hour). These increments are less than those reported previously for Serratia marcescens, because the Z value for BCG is approximately one-seventh that for serratia. These findings with BCG are believed to be directly applicable to virulent tubercle bacilli.