Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

[A guinea pig model of latent Mycobacterium tuberculosis H37Rv infection].

OBJECTIVE: To establish the guinea pig model of latent Mycobacterium tuberculosis (MTB) H37Rv infection, and to study the multiplication dynamics of MTB in vivo, and the relationship between latent MTB infection and PPD skin test. METHODS: Sixty-two guinea pigs were randomly divided into the model group (n = 42) and the control group (n = 20), and the model group was subdivided into a 4 weeks group (n = 12), an 8 weeks group (n = 21) and a 12 weeks group (n = 9), challenged by 500 CFU H37Rv with restored toxicity. After 2 weeks challenge, the model groups were treated with isoniazid (INH, 10 mg/kg) + pyrazinamidum aldinamide (PZA, 40 mg/kg) for 4 weeks, 8 weeks and 12 weeks respectively. The natural recurrence of tuberculosis was observed in the model 4 weeks group, and the natural and induced recurrence by dexamethasone was observed in the model 8 weeks group and 12 weeks group. PPD skin test, the pathologic changes, and MTB quantity of organs were observed. RESULTS: In the control group, the average MTB quantity of spleen was 3.3 lg CFU after 2 weeks challenge, and the average MTB quantity of spleen and lung in guinea pigs were 4.5 lg CFU and 1.8 lg CFU respectively after 6 weeks challenge, and they reached 5.3 lg CFU and 5.4 lg CFU at 18 weeks respectively. The latent MTB infection of the model 4 weeks group recurred naturally 12 weeks after stopping treatment. The latent MTB infection of the model 8 weeks group recurred naturally and by dexamethasone treatment. The latent MTB infection of the model 12 weeks group did not recur naturally, but dexamethasone induced recurrence. The positive PPD response correlated with recurrence. CONCLUSIONS: A latent MTB infection model was established successfully by H37Rv challenge and treatment with INH and PZA. The latent MTB infection may recur naturally or by induction. The PPD response was related to tuberculosis recurrence.

[The correlation between dendritic cell-specific ICAM-3 grabbing nonintegrin expression and Mycobacterium tuberculosis in humans].

OBJECTIVE: To investigate the correlation between dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expression and Mycobacterium tuberculosis in humans. METHODS: The peripheral blood mononuclear cells were obtained respectively from 25 patients with tuberculosis, 25 patients with pneumonia and 25 healthy individuals, and were cultured in medium with GM-CSF and IL-4. Five days later, expression of CD(11c), CD(86), HLA-DR and DC-SIGN were detected by flow cytometry. The expression of DC-SIGN mRNA was detected by RT-PCR. The levels of IL-12 and IL-10 in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Proliferation induction of T lymphocytes dendritic cells (DCs) was measured by mixed lymphocyte reaction (MLR). RESULTS: The expression of CD(86) on DCs in the tuberculosis patients [(72 +/- 11)%] was significantly lower than those in the other 2 groups [(87 +/- 16)%, (92 +/- 6)%] (F = 11.97, P < 0.01). In patients with tuberculosis, the expression of DC-SIGN on DCs [(85 +/- 8)%] was significantly higher than the other 2 groups [(60 +/- 28)%, (62 +/- 13)%] (F = 8.27, P < 0.01), and so was the DC-SIGN mRNA level (F = 3.99, P < 0.05). The levels of IL-10 secreted by DCs in the tuberculosis patients [(98 +/- 31) ng/L] were significantly higher than those in the other 2 groups [(74 +/- 38) ng/L and (66 +/- 27) ng/L] (F = 4.19, P < 0.05). MLR showed lower potency of proliferation induction of T lymphocytes in the tuberculosis patients (1858 +/- 628) than the other 2 groups (3066 +/- 1389), (3383 +/- 1163) (F = 7.92, P < 0.01). Expression of CD(11c) and HLA-DR on DCs and IL-12 levels were not significantly different among the 3 groups (P > 0.05). CONCLUSION: The decreased DC maturity, elevated IL-10 secretion by DCs, decreased proliferation induction of T lymphocytes and impaired immune response may be due to overexpression of DC-SIGN in patients with tuberculosis.

[The effect of interventional therapy in multimodality treatment on multi-drug resistant pulmonary tuberculosis].

OBJECTIVE: To evaluate the effect of interventional therapy with antituberculous drug instillation to the lesions in the treatment of multi-drug resistant pulmonary tuberculosis (MDR-PTB) on conventional therapy. METHODS: Sixty-one cases of MDR TB were included from January 2001 to October 2002 in five hospitals. Pasiniazide, rifapentine levofloxacin, ethambutol, ethionamide, amikacin and clarithromycin were used as the basic chemotherapy regimen. In addition, M. vaccac and interventional therapy were used, and chemotherapy was continued for a total of 18 months. RESULTS: The sputum negative conversion rate (including sputum smear and culture) was 50.8% (31/61) after 3 months of interventional therapy. The rate increased to 83.6% (51/61) after 18 months of therapy. Chest X-ray showed that, the foci were markedly absorbed in 50.8% (31/61), and the effective rate was 93.4% (57/61) after 3 months of therapy. The foci were markedly absorbed in 78.7% (48/61) after 18 months of treatment. The effective rate was 96.7%. The rate of cavity closure was 21.3% (13/61) after 3 months of interventional therapy and it increased to 49.2% (30/61) after 18 months of treatment. The rate of symptom disappearance was 73.2%-94.4%, including fever, hemoptysis and dyspnea. CONCLUSION: For the treatment of MDR-TB, interventional therapy is effective in improving sputum negative conversion, lesion absorption and cavity closure.

Clinical evaluation of the recombinant 38 kDa protein of Mycobacterium tuberculosis.

The diagnosis of tuberculosis remains among public health concerns due to shortcomings of the purified protein derivative (PPD). Recombinant truncated 38 kDa protein (rTPA38) of Mycobacterium tuberculosis was evaluated to screen new tuberculosis-specific tuberculin. 539 patients, 1133 healthy controls, and 55 guinea pigs were recruited to assess their sensitivity and specificity to rTPA38; 221 healthy controls, with negative responses to rTPA38 and PPD, were vaccinated with M. bovis BCG to determine their cross-reactions with M. bovis BCG. The Mantoux technique was adopted to perform skin tests. No difference in the sensitivity of skin tests was detected between rTPA38 and PPD (78.2% vs 83.4%), but there was a significant difference in the specificity of skin tests between rTPA38 and PPD (75.2% vs 47.0%). Compared to PPD, rTPA38 elicited low positive responses for those recruitments vaccinated with M. bovis BCG. The rTPA38 had significant skin reactions in M. tuberculosis-sensitized guinea pigs, and the opposite was true for both M. fortuitum- and M. kansasii-sensitized guinea pigs. These findings indicate that rTPA38 may have potential as a tuberculosis-specific skin test antigen.

[The animal experimental study on rifampicin-dependent Mycobacterium tuberculosis].

OBJECTIVE: To investigate the presence of rifampicin-dependent Mycobacterium tuberculosis strains by use of a guinea pig model of tuberculosis of rifampicin-dependent Mycobacterium tuberculosis. METHODS: Guinea pigs were randomly divided into groups of infection by rifampicin-dependent Mycobacterium tuberculosis strains (1130 strain, 1219 strain, b858 strain), rifampicin-resistant Mycobacterium tuberculosis strain (1290 strain) and ATCC 35810 strain and each group was further divided into an experimental group and a control group. The guinea pigs were challenged with 1130 strain, 1219 strain, b858 strain, 1290 strain and ATCC 35810 strain to establish the tuberculosis model. The experimental groups were treated with rifampicin. The parameters including macroscopic visceral pathological change index, visceral weight index (spleen, lungs and liver), the colony-forming units (CFU) quantity of visceral Mycobacterium tuberculosis culture (spleen, lungs) and tissue pathology of guinea pigs were observed. RESULTS: At the 7th week after challenged with 1130 strain, 1219 strain, 1290 strain and b858 strain, all animals were sacrificed. The macroscopic visceral pathological change indices of the experimental group were 68.7 +/- 13.8, 60.0 +/- 13.5, 70.0 +/- 5.8 and 23.8 +/- 18.9, whereas all those parameters of the control group were 76.2 +/- 18.9, 40.0 +/- 16.8, 63.8 +/- 10.3 and 22.5 +/- 15.5 respectively, and there was no significance between the experimental group and the control group (t = 0.64, 1.85, 0.35 and 0.10, all P > 0.05). The spleen weight indices of experimental group were 0.229 +/- 0.048, 0.256 +/- 0.067, 0.324 +/- 0.054 and 0.199 +/- 0.029, whereas all those parameters of control groups were 0.278 +/- 0.025, 0.216 +/- 0.076, 0.368 +/- 0.033 and 0.213 +/- 0.038 respectively, and there was no significance between the experimental group and the control group (t = 1.75, 0.79, 1.41 and 0.57, all P > 0.05). The CFU quantity of spleen Mycobacterium tuberculosis culture of the experimental group were 4.98 +/- 0.30, 4.68 +/- 1.26, 5.07 +/- 0.47 and 3.85 +/- 0.45, whereas all those parameters of control groups were 4.90 +/- 1.03, 4.79 +/- 0.45, 5.08 +/- 0.55 and 4.23 +/- 0.95 respectively, and there was no significance between the experimental group and the control group (t = 0.11, 0.15, 0.03 and 0.73, all P > 0.05); Moreover, the tissue pathology of both groups was similar. CONCLUSIONS: The tuberculosis model of rifampicin-dependent Mycobacterium tuberculosis strains was similar to the model of rifampicin-resistant Mycobacterium tuberculosis in guinea pigs. Rifampicin-dependency was not evident in this guinea pig tuberculosis model.

[A controlled clinical trial of long course chemotherapy regimens containing rifabutin in the treatment of multi-drug resistant pulmonary tuberculosis].

OBJECTIVE: To evaluate the curative effect and safety of a long course regimen containing Chinese-made rifabutin as compared to the regimen containing rifapentine in the treatment of multi-drug resistant pulmonary tuberculosis. METHOD: During 18 month treatment, 130 patients with multi-drug resistant pulmonary tuberculosis were divided into a treatment group (rifabutin, pasiniazide, levofloxacin, ethambutol, ethionamide, amikacin for 3 months, rifabutin, pasiniazide, levofloxacin, ethambutol, ethionamide for 6 months, rifabutin, pasiniazide, levofloxacin, ethambutol for 9 months), and a control group (rifapentine, pasiniazide, levofloxacin, ethambutol, ethionamide, amikacin for 3 months, rifabutin, pasiniazide, levofloxacin, ethambutol, ethionamide for 6 months, rifabutin, pasiniazide, levofloxacin, ethambutol for 9 months) with proportion 1:1 random, and parallel compared method. RESULTS: After intensive phase, the sputum negative conversion rates (smear negative, culture negative) of the treatment group and the control group were 41.54% (27/65) and 35.94% (23/65), chi(2) = 2.42, P > 0.05, respectively. The remarkable effective rates in chest X-ray of the two groups were all 10.77% (7/65), chi(2) = 0.01, P > 0.05, and the effective rates were 67.69% (44/65) and 56.92% (37/65), chi(2) = 1.44, P > 0.05, respectively. At the end of the treatment, the sputum negative conversion rate (smear negative, culture negative) of the treatment group was 75.0% (48/65), and of the control group was 65.08% (41/65), chi(2) = 1.88, P > 0.05. The remarkable effective rates in chest X-ray of the two groups were 46.15% (30/65) and 44.62% (29/65), chi(2) = 0.02, P > 0.05, and the effective rates were 76.92% (50/65) and 73.85% (48/65), chi(2) = 0.19, P > 0.05, respectively. The cavity closure rates were 23.64% (13/55) and 33.33% (17/51), chi(2) = 0.00, P > 0.05, respectively. CONCLUSION: Regimens containing rifabutin or rifapentine. are very effective in sputum negative conversion rate, lesion absorption and cavity closing for the treatment of multi-drug resistant pulmonary tuberculosis, with good safety and tolerance.

[Study on the sequences of amino-acids and their gene codes of the related proteins of rifampin-dependent Mycobacterium tuberculosis].

OBJECTIVE: To study the sequences of amino-acids and their related genes of rifampin-dependent Mycobacterium tuberculosis. METHODS: The strains of rifampin-dependent, rifampin-resistant and rifampin-sensitive Mycobacterium tuberculosis were evaluated by L-J method. The proteins of 134 strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of amino-acids of the proteins with differential expressions were determined by Q-TOF2 liquid chromatography-electra mist spray-quadrupole -time of flight cascade mass spectrograph (LC-ESI-MS-MS, Q-TOF2). The corresponding gene codes for the proteins were analyzed. RESULTS: The protein SDS-PAGE profile of 41 of 49 (41/49) rifampin-dependent strains showed a high expression of a protein band (molecular weight 43,000), representative of 30%-50% of the total proteins differed from rifampin resistance and the control strains. The protein profile of H(37)Rv standard strains, 28 (28/30) rifampin-sensitive strains and 44 (44/54) rifampin-resistant strains showed no relevant high expression. The protein of high expression was identified as the hypothetical protein Rv0341 of Mycobacterium tuberculosis H(37)Rv with a molecular weight (M) of 43,894, a iso-potential point of 5.25 and a score of 183. No mutation was found in the 1,440 bp encoding sequence in various strains. CONCLUSIONS: The hypothetical protein Rv0341 is related to the rifampin-dependent strains, so it can be regarded as an indicator for rifampin-dependent Mycobacterium tuberculosis. Rifampin upregulated the expression of Rv0341 in rifampin-dependent Mycobacterium tuberculosis. Whether the hypothetical protein Rv0341 is related to virulence and synthesis of the cell wall of Mycobacterium tuberculosis needs further study.

[Differentiation of infection with Mycobacterium tuberculosis by recombinant Mycobacterium tuberculosis 11000 protein].

OBJECTIVE: To study the differentiation effect of recombinant Mycobacterium tuberculosis 11000 protein on infection of Mycobacterium tuberculosis. METHODS: Guinea pigs were immunized with different strains of mycobacterium, and then all guinea pigs were given intradermal injections with recombinant Mycobacterium tuberculosis 11000 protein and purified protein derivative of tuberculin (PPD) or purified protein derived from M. intracellulare (PPD-B). Skin reactions defined with two transverse diameters were read double-blinded after 24 and (or) 48 hours, and the means of the two transverse diameters were counted as the reaction diameters. RESULTS: All guinea pigs immunized with different strains of Mycobacteria responded to PPD or PPD-B with positive skin reactions. The recombinant Mycobacterium tuberculosis 11000 protein elicited positive skin reactions in guinea pigs infected with live Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium kansasii, and the reaction diameters were (14.7 +/- 2.0) mm, (9.3 +/- 3.8) mm, (18.7 +/- 2.4) mm and (14.8 +/- 4.2) mm, respectively. But it failed to elicit positive skin reaction in guinea pigs immunized with killed Mycobacterium tuberculosis, live BCG and other MOTT (mycobacteria other than Mycobacterium tuberculosis). CONCLUSIONS: Recombinant Mycobacterium tuberculosis 11000 protein can differentiate infection with live Mycobacterium tuberculosis from immunization with killed Mycobacterium tuberculosis, live BCG or other MOTT.

[Effects of Mycobacterium smegmatis vaccine on cytokines production and Th1/Th2 responses in mice].

OBJECTIVE: To validate the immunogenicity of Mycobacterium smegmatis and to study the immune modulatory function of Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis by analyzing the effects of the vaccine on immune responses in mice. METHODS: Spleen cells and peritoneal macrophages from BALB/c mice which were randomized into a control group and Mycobacterium smegmatis vaccine groups (low, middle, and high doses) were cultured in vitro. Then the supernatants were collected and the concentrations of IL-2, IL-4, IL-12, and IFN-gamma were analyzed through ELISA. RESULTS: (1) IL-12 produced by the control mice and mice immunized with low, middle, high doses of Mycobacterium smegmatis vaccine was (32.6 +/- 22.7), (58.9 +/- 18.6), (77.3 +/- 38.0), (114.7 +/- 9.9) pg/ml respectively, and the middle and high dose group showed significant difference as compared with the control group (P < 0.05). (2) IL-2 produced by the control mice and mice immunized with low, middle, high dose of Mycobacterium smegmatis vaccine was (5.0 +/- 2.6), (13.4 +/- 9.23), (15.3 +/- 9.7), (22.6 +/- 7.5) pg/ml respectively, and the high dose group showed significant difference when compared with the control group (P < 0.01). (3) When the cells were stimulated with ConA in vitro, IFN-gamma produced by the control mice and mice immunized with middle dose of Mycobacterium smegmatis vaccine was (662 +/- 279) and (807 +/- 163) pg/ml, IL-4 produced by the two groups was (407 +/- 127) and (101 +/- 26) pg/ml, but the differences were not statistically significant (P > 0.05). When the cells were stimulated with Mycobacterium smegmatis-purified protein derivative (PPD) in vitro, IFN-gamma produced by the control mice and mice immunized with middle dose of Mycobacterium smegmatis vaccine was (14.0 +/- 6.31) and (55.3 +/- 32.4) pg/ml, the difference being statistically significant (P < 0.05), but IL-4 produced by the two groups was under the limit of detection. CONCLUSION: Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis showed strong immunogenicity promoted Th1 responses and inhibited Th2 response in mice.

[The in vitro killing of intracellular Mycobacterium smegmatis by Mycobacteriophage].

OBJECTIVE: To study the effect of Mycobacteriophage on the lysis of intracellular Mycobacterium smegmatis. METHODS: Peritoneal macrophages from BALB/C mice were incubated with Mycobacterium smegmatis for 4 h, and the extracellular bacteria were removed. Then the infected macrophages were treated for 2 h with normal saline, or different doses of Mycobacteriophages (2.1 x 10(7) PFU, 2.1 x 10(6) PFU, and 2.1 x 10(5) PFU, respectively), all in a volume of 0.1 ml, and then the extracellular phages and Mycobacterium smegmatis were removed by washing. After incubation for 24 h, the number of viable intracellular bacteria was determined. The intracellular changes after infection of host bacteria by bacteriophages in the macrophages were observed by electron microscopy. RESULTS: The logarithm 10 of viable intracellular bacteria unit was 5.74 +/- 0.18 in the saline group, 4.77 +/- 0.08 in the high dose phage group (P < 0.01), 4.97 +/- 0.17 in the moderate dose phage group (P < 0.01), and 5.33 +/- 0.13 in the low dose phage group (P > 0.05). Electron microscopy confirmed the infection of intracellular bacteria by the bacteriophages and the production of filial bacteriophages. CONCLUSIONS: Mycobacteriophages phagocytosed by macrophages are capable of killing the infected mycobacteria. The result suggests that the use of Mycobacteriophages is a potentially novel strategy in the treatment of intracellular bacterial infection.

[Study on the molecular epidemiology of Stenotrophomonas maltophilia infection in patients on mechanical ventilation].

OBJECTIVE: To investigate a potential outbreak of Stenotrophomonas maltophilia (pma) infection occurred in patients on mechanical ventilation in a respiratory ICU (RICU) and to track the infective origins by antibiotype and pulsed-field gel electrophoresis (PFGE) typing methods. METHODS: (1) Thirteen pma strains were isolated from 9 patients on mechanical ventilation (9 strains), hand swabs of medical staffs in RICU (2 strains) and fiberscope used for intubations and aspiration (2 strains) from December, 2002 to February, 2003. (2) Sixteen strains gathered from different wards during the period of 1997 - 2000 were collected and used as control. (3) Antibiotic susceptibility data of all strains were collected. (4) Homology of the strains was analyzed by the methods of antibiotype and PFGE genotype. RESULTS: Of the 9 pma isolated from patients on mechanical ventilation, eight had identical PFGE genotype. The isolates from two RICU staffs and two fiberscope displayed the same genotype with the eight patients above. Seven of the 9 isolates shared an identical antibiotype. The consistent rate of antibiotype with PFGE genotype was 85% (11/13). There were 11 PFGE genotypes and 9 antibiotypes in 16 strains of the control group, which indicated that they came from different clones. CONCLUSIONS: Eight pma strains of patients in RICU came from the same clone. This result proved that clone transmission occurred in patients on mechanical ventilation. Contaminated fiberscope and hands of staffs may be the infective origin and the route of transmission.

[A study of standardization to the rapid detection of Mycobacterium tuberculosis based on phage amplified biologically assay].

OBJECTIVE: To evaluate the phage amplified biologically (PhaB) assay in the rapid detection of Mycobacteria tuberculosis in samples. METHODS: The conditions of the PhaB assay, including various infection times prior to addition of virucide and the effect of the inactivation agents which could inactive the extracellular phages, were investigated and compared. The sensitivity, specificity and accuracy of PhaB assay were tested when it was used in rapid detection of Mycobacterium tuberculosis. Some agents of the method, including Mycobacteriophage, virucide and help cells (Mycobacterium smegmatis) were investigated at different times when they were preserved at 4 degrees C. RESULTS: (1) The optimal infection time prior to addition of virucide was between 3 and 4 hours. Four percent FAS (ferrous ammonium sulphate) could inactive 1 x 10(9) PFU (plaque-forming unit) in five minutes. (2) The samples were positive when 80 - 200 CFU of Mycobacterium tuberculosis were present. However, the positive rate of non-Tuberculosis Mycobateria (NTM) was varied. All bacteria lived in the respiratory tract were negative. (3) The important agents used in this test showed optical effect when they were preserved at 4 degrees C. CONCLUSIONS: The method based on mycobacteriophage-amplified biologically assay could rapidly detect Mycobacterium tuberculosis, and it was effective, accurate, and simple to perform. It was appropriate for using in developing countries, compared with a variety of molecular techniques.