Effect of Mycobacterium phlei F.U.36 on balance of CD4⁺CD25⁺ regulatory T cells and Th17 cells in asthmatic mice / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the effect of early intervention with Mycobacterium phlei F.U.36 injection on the balance of CD4⁺CD25⁺ regulatory T cells and Th17 cells in asthmatic mice, and to investigate the immunomodulatory effect of Mycobacterium phlei F.U.36.</p><p><b>METHODS</b>Thirty female BALB/c mice were randomly divided into three groups: normal control (n=10), asthma model (n=10) and Mycobacterium phlei F.U.36 treatment groups (n=10). A mouse model of asthma was prepared by injection and aerosol inhalation of chicken ovalbumin in the asthma model and Mycobacterium phlei F.U.36 treatment groups, while mice in the normal control group were given normal saline instead. The treatment group was intraperitoneally injected with Mycobacterium phlei F.U.36 (0.57 μg, once every other day) three times in the first two weeks after the first sensitization. All mice were sacrificed at 24 hours after the last challenge. Left lung tissues of these mice were obtained and made into sections for observation of inflammatory changes. The percentages of CD4⁺CD25⁺ regulatory T cells and Th17 cells in CD4⁺ T cells among splenic mononuclear cells were determined by flow cytometry. The levels of interleukin (IL)-10 and IL-17 in serum and bronchoalveolar lavage fluid were measured using ELISA.</p><p><b>RESULTS</b>Compared with the normal control group, the asthma model group had significantly decreased percentages of CD4⁺CD25⁺ regulatory T cells and IL-10 levels (P<0.05) and significantly increased percentages of Th17 cells and IL-17 levels (P<0.05). Compared with the asthma model group, the Mycobacterium phlei F.U.36 treatment group had significantly increased percentages of CD4⁺CD25⁺ regulatory T cells and IL-10 levels (P<0.05) and significantly decreased percentage of Th17 cells and IL-17 levels (P<0.05).</p><p><b>CONCLUSIONS</b>Early intervention with Mycobacterium phlei F.U.36 can promote development of CD4⁺CD25⁺ regulatory T cells and production of IL-10 and inhibit generation of Th17 cells and production of IL-17 in asthmatic mice.</p>

Effects of Mycobacterium phlei F.U.36 on regulatory T cells and TLR4 expression in asthmatic mice / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of early intervention on CD4+CD25+ regulatory T cells and TLR4 expression with Mycobacterium phlei F.U.36 in asthmatic mice.</p><p><b>METHODS</b>Thirty female BALB/c mice were randomly divided into three groups: control, asthma model and Mycobacterium phlei F.U.36 treated asthma groups. Asthma was induced by sensitization and challenges with ovalbumin (OVA) in the later two groups. Mycobacterium phlei F.U.36 was intraperitoneally injected 2 weeks before the first sensitization (0.57 μg/time, once every other day for three times) in the intervention group. After 24 hrs of the last challenge, the mice were sacrificed and the left lung tissues were obtained for the observation of lung pathological changes. Splenic mononuclear cells were isolated. The percentage of CD4+CD25+ regulatory T cells in CD4+ T cells and the mean fluorescence intensity of TLR4 on CD4+ CD25+ T cells were detected by flow cytometry.</p><p><b>RESULTS</b>The percentage of CD4+CD25+ regulatory T cells in the asthma model group was significantly lower than that in the control group (P<0.01), but the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells was not significantly different from the control group. The percentage of CD4+CD25+ regulatory T cells and the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells increased significantly in asthmatic mice receiving Mycobacterium phlei F.U.36 treatment compared with the asthma group (P<0.01).</p><p><b>CONCLUSIONS</b>Early intervention with Mycobacterium phlei F.U.36 can increase TLR4 expression on CD4+CD25+ cells and the number of CD4+CD25+ regulatory T cells, and thus provides therapeutic effects in asthmatic mice.</p>

Mycobacterial infections in human immuno-deficiency virus seropositive patients: role of non-tuberculous mycobacteria.

BACKGROUND: There is high prevalence of tuberculosis in patients with HIV infection; hence the role of non-tuberculous mycobacteria (NTM) in HIV patients has always been undermined. NTM may be responsible for clinical disease in a substantial number of immuno-compromised HIV sero-positive individuals even in a country endemic for Mycobacterium tuberculosis (M. tuberculosis). The study was designed to look for the contribution of NTM to morbidity in HIV seropositive patients. MATERIAL AND METHODS: In a prospective study of ninety-four HIV seropositive individuals presenting with pulmonary or extra-pulmonary symptoms suggestive of mycobacterial infection, appropriate samples were collected and processed. Detailed clinical history was utilized to differentiate colonization or contamination by NTM from true lung disease. RESULTS: Fourteen samples grew mycobacterial species, 8(57.2%) being NTM. The distribution of NTM was--3 M. avium complex, 2 M. fortuitum, 2 M. vaccae, 1 M. phlei. 6 isolates were M. tuberculosis. CONCLUSION: NTM may be responsible for a significant proportion of mycobacterial infections in HIV seropositive individuals. Despite the high endemicity of tuberculosis in developing countries like India, the presence of NTM should be ruled out; especially in immuno-compromised HIV seropositive individuals before instituting anti-tubercular therapy empirically. In addition, non-response of NTM to ATT may be wrongly attributed to multi-drug resistant tuberculosis.

Effect of Mycobacterium phlei F.U.36 suspended liquor on culture and proliferation of dendritic cells derived from human umbilical cord blood in vitro / 中国实验血液学杂志

To investigate the effect of mycobacterium phlei F.U.36 suspended liquor (Utilin"s", U) on the culture and proliferation of dendritic cells (DCs) derived from human umbilical cord blood in vitro, the mononuclear cells (MNCs) were isolated from human umbilical cord blood and cultured with RPMI 1640 in the control group. Test groups consisted of Utilin"s" group (only Utilin"s"), GTI group (GM-CSF, TNF-alpha, IL-4) and GTIU group (GM-CSF, TNF-alpha, IL-4 and Utilin"s"). MNCs in all test groups were cultured with RPMI-1640. The growth of DCs was observed by the light microscopy, the phenotypes of DCs were determined by flow cytometry on the 10th day of culture, and some harvest cells were stained with Wright-Giemsa, then observed and photographed under the oil immersion objective. The results showed that the test groups all displayed some number of typical DCs; both CD1a positive cell rate and HLA-DR positive cell rate of the Utilin"s" group were higher than those of the control; HLA-DR positive cell rate of GTIU group increased most significantly and much higher than that of the GTI group. It is concluded that mycobacterium phlei F.U.36 not only promotes the proliferation of DCs derived from human umbilical cord blood in vitro, but also co-operates with rhGM-CSF, rhTNF-alpha and rhIL-4 in promoting the maturity of DCs.

Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

In-vitro study of mouse peritoneal macrophage response to virulent and avirulent strains of mycobacteria.

An immunological study of pathogenesis of tuberculosis was carried out in BALB-c mice in-vitro. Peritoneal macrophages obtained from BALB-c mice were challenged with virulent (H37Rv) and avirulent (H37Ra, BCG, M. phlei) strains of mycobacteria. Activated peritoneal macrophages showed enlargement, presence of intracellular bacteria and vacuolation. These significant changes in macrophage morphology were clearly evidenced in cells infected with virulent strains of Mycobacterium tuberculosis i.e. H37Rv while being absent in cells infected with avirulent H37Ra, BCG and M. phlei. Virulent mycobacteria (H37Rv) survive the phagocytic action of macrophages by residing inside the vacuoles. The capacity of virulent and avirulent strain to stimulate TNF-alpha production from peritoneal macrophage of BALB-c mice was also examined at different time interval i.e. 1,2,4,6 and 8th day by measuring cytolytic activity of culture supernatant against murine fibroblast cell line. The pattern of highest TNF release was in case of H37Rv and least with M. phlei as measured in culture supernatant after 1,2,4,6 and 8th day.

Nontuberculous mycobacteria in soils of La Pampa province (Argentina)

The presence of nontuberculous mycobacteria (NTM) was investigated in forty soil samples belonging to the four physiographic regions (Eastern, Central, Southern and Western) that constitute La Pampa province. The presence of NTM in 67.5 of these soil samples was determined. The density of mycobacteria ranged 25-4,500 mycobacteria g-1 dry soil (mean = 516 CFU g-1). Significant differences were found in relation to both the investigated regions (p < 0.01) and the soil pH (r = 0.44*) (P = 0.02). The mycobacteria represented less than 0.00001 of the total aerobic bacteria found in the soils. Twenty-seven isolated mycobacteria were classified according to the culture, biochemical, enzymatic characteristics and antibiotic sensitivity. Mycobacterium fortitium was the dominant mycobacterium and was detected in 63 of the positive soils. This species showed ability for living in sandy to sandy loam soils, within a wide pH range (6.5-9.7) and organic matter (4.15-83.63 g kg-1). Two other species were M. phlei (range = 50-4,500 CFU g-1) and M. kansasii (range = 50-500 CFU g-1).

Mycobacterium phlei as an oral immunomodulator with Newcastle disease vaccine.

Experiments were conducted in chickens to understand the effects of oral immunomodulation. Heat inactivated M phlei, a commensal Mycobacterium and a non-specific immunomodulator, was administered orally prior to live Newcastle disease F (ND F) strain vaccination. In experimental birds it lead to an enhanced cell mediated Immune response (CMI) against the vaccine. There was a reduction in the Haemagglutination inhibiting (HI) antibodies. However, it did not affect the protection against a virulent challenge, as the protection percentage was more or less same in vaccinated birds irrespective of the M.phlei administration. M. phlei administration could not enhance the immune response to inactivated ND F vaccine administered orally. The results indicate that M. phlei favours a CMI response to orally administered live ND F vaccine. It may be of potential use in enhancing CMI against vaccines and a cheaper alternative to costlier recombinant cytokines.

Water soluble palmitic acid in media for cultivation of leprosy derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues

Cultivation trials for Mycobacterium leprae resulted in growth of Mycobacterium psychrophilum (L). Media were inoculated with host grown Mycobacterium leprae cells from armadillo tissues, Nu mice foot pads or human lepromata. Cultures were obtained in liquid and on semisolid multifactoria 1 media containing water soluble palmitic acid or its salts. Ammonium thioglycolate and Napalmitate served as carbon and energy sources. The water soluble palmitic acid remained in perfect solution following sterilization in the autoclave, thus easily accessible to the cells. The cyclodextrin-Fe complex served as a siderophore to grow the obtained leprosy derived psychrophilic cells. The leprosy derived cultures and subcultures grew opimally at+10 degrees Celssius but deteriorated rapidly at + 32 degrees Celsius, in the multifactorial media. No growth occurred in 7H9 media. Cultures were not identified for classification.

Polyclonal B-lymphocyte activation of sheep by Mycobacterium phlei against sheep pox virus.

A trypsinized preparation of Mycobacterium phlei, non specific stimulator of immunity (NSI), and Sheep Pox Virus (SPV) were inoculated in different groups of sheep to activate B-lymphocytes and induce SPV neutralizing substance(s). NSI sensitized sheep B-lymphocytes in the presence of NSI or lymphokine elaborated SPV neutralizing substance(s). The SPV sensitized B-lymphocytes also mediated such neutralizing substance(s). Healthy control sheep B-lymphocytes failed to show any appreciable amount of viral neutralizing substance. However, a significant virus neutralizing substance(s) was detected when healthy sheep B-lymphocytes were cultured in presence of NSI antigen along with lymphokines.

Enhanced production of antibody with specific antigen.

On inoculation of nonspecific stimulator of immunity (NSI), prepared from Mycobacterium phlei (M. phlei), simultaneously along with sheep pox virus (SPV) in sheep, the recipient has exhibited appreciable level of SPV specific antibody as early as on 10th day which reached at peak level on 20th day and remained unaltered on 30th day of postimmunisation as evinced by serum neutralisation test (SNT), enzyme linked immunosorbant assay (ELISA) indirect, fluorescent antibody technique (FAT) indirect, counter immunoelectrophoresis (CIEP) and finally by virulent SPV challenge. On the contrary, sheep, when immunised with SPV only could not produce appreciable level of antibody on 10th day but did so on 20th day of inoculation. SPV and NSI immunised sheep produced enhanced protection against virulent SPV challenge in comparison with sheep immunised with SPV only. Healthy control sheep, however, could not resist challenge.

Induction of cell mediated immune response by nonspecific stimulator of immunity against antigenically unrelated oncogenic virus (Marek's disease virus).

An enzyme treated preparation of Mycobacterium phlei (NSI), induced strong cell mediated immune response (CMIR) against specific as well as against nonspecific oncogenic Marek's disease (MDV) in birds, as evinced by Lymphocyte migration inhibition, (LMIT) lymphocyte transformation test (LT) and Lymphokine (Lymphocyte migration inhibition factor) LyIF assay. Maximum CMIR could be observed towards third week post inoculation. All the three tests exhibited a positive correlation. Such phenomenon of CMIR induction by NSI, nonspecifically to unrelated viral/cancerous diseases (MD) in birds generates hopes for immunoprevention of these maladies by utilizing such phenomenon.

Nonspecific immunoprotection against Rous sarcoma tumor in chicks.

An enzyme treated preparation of saprophytic Mycobacterium phlei, referred as NSI, when administered intramuscularly has been found to protect the chicks against Rous Sarcoma Virus induced tumor. A protection level of 35.4%, 24.1% and 21.2% were observed when challenged on 10th, 20th and 30th day post NSI inoculation. The tumor growth inhibitory-activity of NSI was significant (P less than 0.01). Both, systemic and intralesional administration of NSI exhibited significant tumorostatic activity (P less than 0.05). NSI stimulated the cell mediated immune response to specific as well as to nonspecific Rous sarcoma antigen. These studies indicated the immunopreventive activity of NSI against Rous sarcoma tumor which had an immunogenic basis.

Metabolic studies on mycobacteria. III. Demonstration of key enzymes of TCA cycle in M. leprae.

Cell free extracts of armadillo derived M. leprae, M. phlei, M. smegmatis and normal armadillo liver were analysed for the two key enzymes of TCA cycle. Aconitase activity was assayed in the presence of inhibitor fluorocitrate and it was observed that cell free extracts from cultivable mycobacteria as well as aramadillo derived M. leprae had this enzyme activity and 66-82% of this activity was inhibited by 0.1 mM fluorocitrate. 74% of M. leprae derived enzyme activity was inhibited by fluorocitrate in contrast with armadillo derived enzyme which was only 29% inhibited by fluorocitrate. PAGE separation of cell free extracts and staining for Isocitrate dehydrogenase (ICD) activity showed that an additional bond of ICD activity was demonstrable in the cell free extracts of armadillo derived M. leprae and this was NADP dependent. The mobility (ef) of this band of activity was in the same range as ICD from cultivable mycobacteria and much lower than ICD from normal armadillo liver. From this study and from the previously reported work, it is concluded that like other mycobacteria TCA cycle is operative in M. leprae.

Isoenzyme patterns of mycobacteria. I. Factors influencing LDH isoenzymes of mycobacteria.

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.