SUE4, a novel PIN1-interacting membrane protein, regulates acropetal auxin transport in response to sulfur deficiency.

Sulfur (S) is an essential macronutrient for plants and a signaling molecule in abiotic stress responses. It is known that S availability modulates root system architecture; however, the underlying molecular mechanisms are largely unknown. We previously reported an Arabidopsis gain-of-function mutant sulfate utilization efficiency4 (sue4) that could tolerate S deficiency during germination and early seedling growth with faster primary root elongation. Here, we report that SUE4, a novel plasma membrane-localized protein, interacts with the polar auxin transporter PIN1, resulting in reduced PIN1 protein levels and thus decreasing auxin transport to the root tips, which promotes primary root elongation. Moreover, SUE4 is induced by sulfate deficiency, consistent with its role in root elongation. Further analyses showed that the SUE4-PIN1 interaction decreased PIN1 levels, possibly through 26 S proteasome-mediated degradation. Taken together, our finding of SUE4-mediated root elongation is consistent with root adaptation to highly mobile sulfate in soil, thus revealing a novel component in the adaptive response of roots to S deficiency.

Airborne host-plant manipulation by whiteflies via an inducible blend of plant volatiles.

The whitefly Bemisia tabaci is one of the world's most important invasive crop pests, possibly because it manipulates plant defense signaling. Upon infestation by whiteflies, plants mobilize salicylic acid (SA)-dependent defenses, which mainly target pathogens. In contrast, jasmonic acid (JA)-dependent defenses are gradually suppressed in whitefly-infested plants. The down-regulation of JA defenses make plants more susceptible to insects, including whiteflies. Here, we report that this host-plant manipulation extends to neighboring plants via airborne signals. Plants respond to insect attack with the release of a blend of inducible volatiles. Perception of these volatiles by neighboring plants usually primes them to prepare for an imminent attack. Here, however, we show that whitefly-induced tomato plant volatiles prime SA-dependent defenses and suppress JA-dependent defenses, thus rendering neighboring tomato plants more susceptible to whiteflies. Experiments with volatiles from caterpillar-damaged and pathogen-infected plants, as well as with synthetic volatiles, confirm that whiteflies modify the quality of neighboring plants for their offspring via whitefly-inducible plant volatiles.

Screening of Serum Biomarkers for Distinguishing between Latent and Active Tuberculosis Using Proteome Microarray.

OBJECTIVE: To identify potential serum biomarkers for distinguishing between latent tuberculosis infection (LTBI) and active tuberculosis (TB). METHODS: A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays (ELISA) were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve (ROC) analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability. RESULTS: Microarray results showed that levels of 152 Mycobacterium tuberculosis (Mtb)-antigen- specific IgG were significantly higher in active TB patients than in LTBI patients (P < 0.05), and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031c, Rv1408, and Rv2421c had higher areas under the curve (AUCs) of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability. CONCLUSION: Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.

Interleukin 8 Gene Polymorphisms Are Not Associated with Tuberculosis Susceptibility in the Chinese Population.

Interleukin 8 (IL8) is an important chemokine that elicits host immune response against tuberculosis (TB). However, whether there is an association between IL8 gene polymorphism and TB susceptibility in the Chinese population is unknown. IL8 gene was amplified and sequenced to search for nucleotide polymorphisms among the Chinese population. Four single nucleotide polymorphisms (SNPs) were identified, selected, and analyzed in a cohort of 438 patients with TB and 536 healthy controls. Allelic, genotypic, and haplotypic analysis demonstrated that the distribution of the four IL8 SNPs between patients with TB and healthy controls was not significantly different (P>0.05). The four IL8 SNPs detected in this study were not associated with TB susceptibility in the Chinese population. Secretion of IL8 by peripheral blood cells was greatly stimulated upon exposure to Mycobacterium tuberculosis whole cell extract, but such enhanced secretion was not associated with the IL8 rs4073 alleles.

L-Cysteine inhibits root elongation through auxin/PLETHORA and SCR/SHR pathway in Arabidopsis thaliana.

L-Cysteine plays a prominent role in sulfur metabolism of plants. However, its role in root development is largely unknown. Here, we report that L-cysteine reduces primary root growth in a dosage-dependent manner. Elevating cellular L-cysteine level by exposing Arabidopsis thaliana seedlings to high L-cysteine, buthionine sulphoximine, or O-acetylserine leads to altered auxin maximum in root tips, the expression of quiescent center cell marker as well as the decrease of the auxin carriers PIN1, PIN2, PIN3, and PIN7 of primary roots. We also show that high L-cysteine significantly reduces the protein level of two sets of stem cell specific transcription factors PLETHORA1/2 and SCR/SHR. However, L-cysteine does not downregulate the transcript level of PINs, PLTs, or SCR/SHR, suggesting that an uncharacterized post-transcriptional mechanism may regulate the accumulation of PIN, PLT, and SCR/SHR proteins and auxin transport in the root tips. These results suggest that endogenous L-cysteine level acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1/2 and SCR/SHR. L-Cysteine may serve as a link between sulfate assimilation and auxin in regulating root growth.

Sulfur nutrient availability regulates root elongation by affecting root indole-3-acetic acid levels and the stem cell niche.

Sulfur is an essential macronutrient for plants with numerous biological functions. However, the influence of sulfur nutrient availability on the regulation of root development remains largely unknown. Here, we report the response of Arabidopsis thaliana L. root development and growth to different levels of sulfate, demonstrating that low sulfate levels promote the primary root elongation. By using various reporter lines, we examined in vivo IAA level and distribution, cell division, and root meristem in response to different sulfate levels. Meanwhile the dynamic changes of in vivo cysteine, glutathione, and IAA levels were measured. Root cysteine, glutathione, and IAA levels are positively correlated with external sulfate levels in the physiological range, which eventually affect root system architecture. Low sulfate levels also downregulate the genes involved in auxin biosynthesis and transport, and elevate the accumulation of PLT1 and PLT2. This study suggests that sulfate level affects the primary root elongation by regulating the endogenous auxin level and root stem cell niche maintenance.

Potential biomarkers for evaluating the BCG vaccination response based on humoral immunity.

Background: The current prophylactic tuberculosis vaccine Bacille Calmette-Guérin (BCG), was derived in the 1920s, but the humoral immune responses induced by BCG vaccination have not been fully elucidated to date. In this study, our aim was to reveal the profiles of antibody responses induced by BCG vaccination in adults and identify the potential biomarkers for evaluating the BCG vaccination response. Methods: Proteome microarrays were performed to reveal the serum profiles of antibody responses induced by BCG vaccination in adults. ELISA was used to validate the potential biomarkers in validation cohort (79 healthy controls and 58 BCG-vaccinated subjects). Then combined panel was established by logistic regression analysis based on OD values of potential biomarkers. Results: Multiple antigens elicited stronger serum IgG or IgM antibody responses in BCG vaccinated subjects than healthy subjects at 12 weeks post BCG vaccination; among the antigens, Rv0060, Rv2026c and Rv3379c were further verified using 137 serum samples and presented the moderate performance in assessment of the BCG vaccination response by receiver operating characteristic analysis. Furthermore, a combined panel exhibited an improved AUC of 0.923, and the sensitivity and specificity were 77.59 % and 91.14 %, respectively. In addition, the antibody response against Rv0060, Rv2026c and Rv3379c was related to the clinical background to a certain extent. Conclusions: The novel antigens identified in our study could offer better knowledge towards developing a more efficacious vaccine based on humoral immune responses, and they could be potential biomarkers in assessments of BCG vaccination responses.

Priming of anti-herbivore defense in tomato by arbuscular mycorrhizal fungus and involvement of the jasmonate pathway.

Mycorrhizas play a vital role in soil fertility, plant nutrition, and resistance to environmental stresses. However, mycorrhizal effects on plant resistance to herbivorous insects and the related mechanisms are poorly understood. This study evaluated effects of root colonization of tomato (Solanum lycopersicum Mill.) by arbuscular mycorrhizal fungi (AMF) Glomus mosseae on plant defense responses against a chewing caterpillar Helicoverpa arimigera. Mycorrhizal inoculation negatively affected larval performance. Real time RT-PCR analyses showed that mycorrhizal inoculation itself did not induce transcripts of most genes tested. However, insect feeding on AMF pre-inoculated plants resulted in much stronger defense response induction of four defense-related genes LOXD, AOC, PI-I, and PI-II in the leaves of tomato plants relative to non-inoculated plants. Four tomato genotypes: a wild-type (WT) plant, a jasmonic acid (JA) biosynthesis mutant (spr2), a JA-signaling perception mutant (jai1), and a JA-overexpressing 35S::PS plant were used to determine the role of the JA pathway in AMF-primed defense. Insect feeding on mycorrhizal 35S::PS plants led to higher induction of defense-related genes relative to WT plants. However, insect feeding on mycorrhizal spr2 and jai1 mutant plants did not induce transcripts of these genes. Bioassays showed that mycorrhizal inoculation on spr2 and jai1 mutants did not change plant resistance against H. arimigera. These results indicates that mycorrhizal colonization could prime systemic defense responses in tomato upon herbivore attack, and that the JA pathway is involved in defense priming by AMF.

[Molecular detection of tomato yellow leaf curl virus (TYLCV)].

Tomato yellow leaf curl virus (TYLCV) is currently considered as one of the most devastating viruses in cultivated tomatoes (Solanum lycopersicum) worldwide. We reported here the development of a PCR-based method to quickly detect TYLCV using the primer pairs (TYLCV-F: 5'-ACG CAT GCC TCT AAT CCA GTG TA-3' and TYLCV-R: 5'-CCA ATA AGG CGT AAG CGT GTA GAC-3'), which was designed based on the genome sequence of TYLCV. A TYLCV-specific band of 543 bp was amplified from infected tomato plants. This protocol provides a rapid, reliable, and sensitive tool for molecular detection and identification of TYLCV in the industrial seedling and virus resistance breeding to facilitate safe and sustainable production of tomato.

[Genetic screening and analysis of suppressors of asa1-1 (soa) defective in jasmonate-mediated lateral root formation in Arabidopsis].

It has been shown that jasmonate modulates the lateral root development through crosstalk with auxin in Arabidopsis thaliana. Exogenous application of jasmonate stimulates lateral root formation in wild type but inhibits lateral root formation in asa1-1. Our previous work has demonstrated that the lateral root formation defect of asa1-1 is co-related with jasmonte effect on PIN2 protein levels. To further elucidate the molecular mechanisms underlying jasmonate-mediated reduction of plasma membrane (PM)-resident PIN2 abundance, we have conducted a genetic screen to identify suppressors of asa1-1 (soa), which showed lateral root formation in the presence of jasmonate. Here, we described the basic characterization of soa563 and soa856. We showed that both soa563 and soa856 displayed restored lateral root formation in response to exogenous jasmonate. In addition, jasmonate-induced PIN2:GFP reduction was blocked in these two mutants. Our on-going effort to identify genes defined by these mutants promise to shed new light on the understanding of the molecular mechanisms controlling jasmonate-mediated regulation of PIN2 protein trafficking and turnover.

Tuberculosis relapse is more common than reinfection in Beijing, China.

BACKGROUND: Recurrent tuberculosis (TB) is a major health problem in countries with a high TB burden. It is very necessary to elucidate the situation of recurrent TB in Beijing, capital of China. OBJECTIVE: To determine the proportion of recurrent tuberculosis (TB) cases and to identify relapsed or reinfected cases, as well as risk factors associated with recurrence in Beijing. METHODS: We conducted a retrospective study that included all TB cases in Beijing that were successfully treated from 2013 to 2015. Recurrence due to relapse or reinfection was determined using the variable number of tandem repeats (VNTR) method. Risk factors associated with recurrence were analysed. RESULTS: Tuberculosis recurred in 275 of the 4043 successfully treated TB patients, giving a recurrence rate of 6.8% (275/4043). 190 of the 275 cases were culture positive in both instances, and genotyping results for both episodes were available for 58 of these patients. The cultured isolates from 40 of the 58 recurrent cases (69%) had identical genotypic patterns in both episodes, indicating a relapse. 31% (18/58) had different genotypes, indicating reinfection by a new strain and suggested recent transmission. Those people in the 30-59 age group (p < .001), and those retreated for pulmonary TB (p < .001) were more likely to have TB recurrence. CONCLUSION: Our results indicate that relapse was more common than reinfection in recurrent TB cases in Beijing from 2013 to 2015. Age and retreatment were found to be risk factors for TB recurrence.

[Influences of Mycobacterium tuberculosis on the levels of human acute monocytic leukemia cell line THP-1 apoptosis and death].

OBJECTIVE: To explore the influences of Mycobacterium tuberculosis on the levels of human acute monocytic leukemia cell line THP-1 apoptosis and death. METHODS: Human acute monocytic leukemia cell line THP-1 were infected with Mycobacterium tuberculosis strains H37Ra, H37Rv, or Beijing genotype (BJTB), respectively, to construct the infection models. Cell apoptosis was detected using flow cytometry. The distribution of the apoptotic proteins was detected using immunofluorescent staining assays. The cells late apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining assays. The change of cell death was determined by Tyrpan blue staining assays. RESULTS: THP-1 apoptosis was induced by Mycobacterium tuberculosis strains H37Ra, H37Rv, and BJTB. H37Ra strongly induced THP-1 apoptosis, H37Rv weakly induced THP-1 apoptosis, and BJTB induced THP-1 apoptosis at the lowest level among these three Mycobacterium tuberculosis strains. On the contrary, BJTB strongly induced THP-1 death, H37Rv weakly induced THP-1 death, and H37Ra induced THP-1 death at the lowest level. CONCLUSIONS: Mycobacterial strains with different virulence induce different levels of apoptosis and death of THP-1 cells. Compared with highly virulent strains, attenuated strains induce more apoptosis and less death.

[A study on the haplotype of the solute carrier family 11 member 1 gene in Tibetan patients with pulmonary tuberculosis in China].

OBJECTIVE: To investigate the association of the haplotype of the solute carrier family 11 member 1 (SLC11A1) gene with susceptibility to pulmonary tuberculosis in Tibetans. METHODS: Four polymorphisms of the SLC11A1 gene [5' (GT)n, INT4, D53N, and 3' UTR] were investigated by denaturalization high performance liquid chromatography and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 140 patients (the patient group) and 139 PPD-positive healthy controls (the control group) of the Tibetan nationality from June 2004 until January 2005. The relationship between the haplotype and susceptibility to pulmonary tuberculosis in these patients was studied by chi2 test, and the linkage disequilibrium as well as the haplotype were analyzed by the SHESIS software. RESULTS: The haplotype frequencies of 5'(GT)9/INT4 G, 3'UTR TGTG/D543N G, 3'UTR TGTG del/D543N A were 64.8% (181/280), 76.6% (215/280), 12.0% (34/280) among the patients and 78.1% (217/276), 84.4% (235/276), 6.4% (18/276) among the controls. 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G haplotypes rendered a lower risk (chi2 = 11.026, P<0.01, chi2 = 6.547, P<0.05, respectively), but 3'UTR TGTG del/D543N A haplotype a higher risk (chi2 = 6.547, P<0.05) for tuberculosis. CONCLUSIONS: 5' (GT)9/INT4 G, 3'UTR TGTG/D543N G and 3'UTR TGTG del/D543N A haplotypes of the SLC11A1 gene may be associated with the susceptibility of the Tibetan population to pulmonary tuberculosis.

[Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates by denaturing high-performance liquid chromatography and DNA sequencing].

OBJECTIVE: To determine the rpsL and rrs gene mutation in Mycobacterium tuberculosis (M. tuberculosis) and compare the consistency between the results of denaturing high-performance liquid chromatography (DHPLC) and those of DNA sequencing. METHODS: The values of streptomycin minimum inhibitory concentration (MIC) against 215 M. tuberculosis clinical isolates, 115 being streptomycin-resistant and 100 being susceptible by a routine proportional method, were tested by DHPLC. DNA sequencing was conducted to detect the rpsL and rrs mutation. RESULTS: 98 of the 115 streptomycin-resistant isolates (85.2%) harbored rpsL and/or rrs mutation, 76.5% of which being rpsL mutation (88/115). There was no significant correlation between the MIC values and mutation types. No mutation was found in all the susceptible isolates. There was a complete consistency between the DHPLC results and those of DNA sequencing. CONCLUSION: DHPLC can be regarded as a useful and powerful tool to detect the streptomycin resistance detection in M. tuberculosis.

[Purification of the heparin-binding haemagglutinin adhesin of Mycobacterium tuberculosis and its application in diagnosis of tuberculosis].

OBJECTIVE: To investigate the application of heparin-binding haemagglutinin adhesin (HBHA) in tuberculosis (TB) diagnosis. METHODS: We prepared native HBHA from cultivated Mycobacterium Bovis Calmetta Guerin (BCG) in Suton liquid medium. After BCG grew to the stationary status, native HBHA was acquired by specific CL-6B chromatography column binding heparin. At the same time, we cloned hbhA gene from Mycobacterium tuberculosis into PET-32alpha (+) expression vector. Recombinant HBHA from E. coli was obtained. Based on the native HBHA and recombinant HBHA, we chose 4 groups of pulmonary TB, extra-pulmonary TB, PPD (-) and PPD (+) healthy control with 47 in each group and conducted ELISA from serum for specific HBHA antibody level. At last we calculated the sensitivity and specificity in TB diagnosis by detection of anti-HBHA antibody level. RESULTS: The native HBHA could be diluted and purified with the PBS containing the 375 mmol/L NaCl by specific CL-6B chromatography column binding heparin; There was no significant difference in experimental result based on the natural and recombinant HBHA protein, also no difference between PPD (-) and PPD (+) healthy control groups. Serum antibody level by ELISA could distinguish pulmonary TB and ertra-pulmonary TB (t = 12.224, P< 0.05). The antibody level of the TB groups (pulmonary TB and ertra-pulmonary TB) was higher than the healthy control groups [PPD (+) and PPD (-) healthy control] (t =25.909, P<0.05). CONCLUSION: Both recombinant and native HBHA can be used as immunological diagnosis in TB. It can be used in TB and especially extra-pulmonary TB diagnosis.

[A study on the mechanisms of mycobacterial clearance induced by CpG-oligodeoxynucleotides in mice].

OBJECTIVE: To investigate the possible mechanisms of mycobacterial clearance induced by CpG-oligodeoxynucleotides (CpG-ODN) in mice. METHODS: Eight-week-old female BALB/c mice were treated with intraperitoneal CpG-ODN (30 microg), while the control mice with normal saline. After 2 weeks, the control mice and the CpG-treated mice were infected with Mycobacterium tuberculosis (1 x 10(6) colony-forming units, H(37)Rv strain) through the tail vein. At 3 weeks, 4 weeks and 6 weeks after mycobacterial infection, the lung and the spleen tissues were examined for histopathological changes. Real time-PCR was performed to measure the messenger RNA (mRNA) of interleukin (IL)-12, IL-18, interferon (IFN)-gamma, IL-4, IL-10 and inducible nitric oxide synthase (iNOS) in the tissues. The homogenates of lungs and spleens were cultured, and the colonies were counted after a 4 weeks incubation period at 37 degrees C. RESULTS: At 3 weeks and 4 weeks of mycobacterial infection, CpG-ODN-pretreated mice showed less mycobacterial burden in the lungs and the spleens than that in the control mice [(0 +/- 0) x 10(6) CFU/g vs (32 +/- 11) x 10(6) CFU/g, (0 +/- 0) x 10(6) CFU/g vs (10 +/- 4) x 10(6) CFU/g; (26 +/- 4) x 10(6) CFU/g vs (56 +/- 8) x 10(6) CFU/g, (4 +/- 3) x 10(6) CFU/g vs (27 +/- 8) x 10(6) CFU/g]. At 4 weeks of mycobacterial infection, CpG-ODN-pretreated mice displayed increased levels of IL-18 mRNA, IFN-gamma mRNA, iNOS mRNA [(3.6 +/- 0.5, 1.6 +/- 1.1, 0.32 +/- 0.14) vs (0.20 +/- 0.10, 23.17 +/- 4.72, 16.18 +/- 5.09)], and decreased level of IL-12p40 mRNA (5.66 +/- 0.64 vs 14.54 +/- 1.89), but there was no difference in the levels of IL-4 mRNA and IL-10 mRNA in the lungs between CpG-ODN-pretreated mice and the control mice [(0.30 +/- 0.09 vs 0.26 +/- 0.05), (0.28 +/- 0.05 vs 0.29 +/- 0.08)]. CpG-ODN-pretreated mice displayed increased levels of IL-18 mRNA, IFN-gamma mRNA, iNOS mRNA [(5.54 +/- 1.29 vs 0.79 +/- 0.36), (0.52 +/- 0.07 vs 0.21 +/- 0.06), (9.07 +/- 1.81 vs 5.97 +/- 1.44)], and decreased levels of IL-12p40 mRNA, IL-4 mRNA and IL-10 mRNA [(2.10 +/- 0.27 vs 5.07 +/- 0.39), (0.23 +/- 0.10 vs 1.21 +/- 0.26), (0.10 +/- 0.04 vs 0.57 +/- 0.13)] in the spleens as compared with the control mice. In CpG-ODN-pretreated mice, expression level of IFN-gamma mRNA in the lungs at 6 weeks post-infection was higher than that at 4 weeks post-infection (0.95 +/- 0.27 vs 0.32 +/- 0.14). CONCLUSION: The activation of Toll-like receptor-9 (TLR-9) with CpG-ODN could enhance murine mycobacterial clearance in vivo. TLR-9-induced anti-mycobacterial activity involves increased expression of IL-18, IFN-gamma, and iNOS but decreased expression of IL-4 and IL-10.

[Therapeutic effects and mechanisms of CpG oligodeoxynucleotides in mice infected with Mycobacterium tuberculosis].

OBJECTIVE: To investigate the protective effects and mechanisms of CpG oligodeoxynucleotides (CpG ODN) in mice infected with Mycobacterium tuberculosis. METHODS: Ninety-six female BALB/c mice were randomized into 4 groups, namely CpG ODN treatment (A group), control ODN treatment (B group), infection control (C group) and healthy control (D group) (n = 24 each). A group and B group were treated once with CpG ODN or control ODN (30 microg/mouse) intraperitoneally 2 weeks before infection with Mycobacterium tuberculosis. 1 x 10(6) bacili in a volume of 300 microl saline was injected into the tail vein of mice from A group, B group and C group. Twelve mice from each group were sacrificed at 3 weeks postinfection. Pathologic changes in lung tissues were observed. The expression of Toll-like receptor 9 (TLR9) mRNA and cytokine mRNA was detected by RT-PCR. The numbers of viable bacteria in lung and spleen were counted. The rest 12 mice from each group were monitored for 60 days to observe the mortality. RESULTS: CpG ODN was shown to increase the survival time of mice infected with Mycobacterium tuberculosis. The body weights of mice from A group [(20.37 +/- 1.12) g] were higher than those of B group [(17.50 +/- 0.62) g] and C group [(17.15 +/- 0.97) g, P < 0.01]. The lung weights of mice from A group [(0.25 +/- 0.02) g] were similar to those of B group [(0.27 +/- 0.34) g, P > 0.05], but less than those of C group [(0.28 +/- 0.26) g, P < 0.01]. The spleens of mice from A group [(0.63 +/- 0.37) g] were larger than those of B group [(0.39 +/- 0.05) g] and C group [(0.38 +/- 0.02) g, P < 0.01]. The inflammation in lung tissue of mice from A group was less than that of B group and C group. There was no mycobacterial outgrowth in lungs and spleens of mice from A group. The expression of TLR9 mRNA in lungs and spleens of mice from A group (0.61 +/- 0.29 and 0.72 +/- 0.48) was similar to that in B group (0.58 +/- 0.35 and 0.64 +/- 0.28) and C group (0.60 +/- 0.32 and 0.65 +/- 0.31, P > 0.05), but higher than that in D group (0.11 +/- 0.08 and 0.26 +/- 0.22, P < 0.01). CpG ODN did not affect the expression of TLR9 mRNA. The expression of IFN-gamma mRNA in lungs and spleens of mice from A group (0.44 +/- 0.07 and 0.76 +/- 0.09) was higher than that in B group (0.19 +/- 0.05 and 0.22 +/- 0.05) and C group (0.16 +/- 0.04 and 0.18 +/- 0.08, P < 0.01). The expression of IL-6 mRNA in lungs and spleens of mice from A group (1.56 +/- 0.29 and 8.21 +/- 0.82) was higher than that in B group (0.86 +/- 0.55 and 0.16 +/- 0.09) and C group (0.78 +/- 0.21 and 0.06 +/- 0.04, P < 0.01). The expression of IL-4 mRNA in lungs and spleens of mice from A group (0.18 +/- 0.05 and 0.06 +/- 0.02) was lower than that in B group (0.31 +/- 0.06 and 1.22 +/- 0.01) and C group (0.35 +/- 0.04 and 1.31 +/- 0.31, P < 0.01). The expression of IL-10 mRNA in spleens of mice from A group (0.05 +/- 0.02) was lower than that in B group (0.57 +/- 0.09) and C group (0.65 +/- 0.15, P < 0.01). CONCLUSION: CpG ODN could increase the immunity of mice against tuberculosis through up-regulation of Th1 immunity and down-regulation of Th2 immunity.

[Study on the association of two polymorphisms of the vitamin D receptor (VDR) gene with the susceptibility to pulmonary tuberculosis (PTB) in Chinese Tibetans].

OBJECTIVE: To investigate the relationship between Fok I and Taq I polymorphisms of the VDR gene and the susceptibility of PTB in Chinese Tibetans. METHODS: Two polymorphisms of the VDR gene were typed by PCR-RFLP among 140 patients and 139 healthy controls who had been sensitized to tuberculosis. The relationship among two polymorphisms and the susceptibility of PTB were studied, and cases were grouped according to the genotypes. RESULTS: The genotype FF, Ff, ff were observed in 60, 56 and 24 cases among tuberculosis group while in 70, 60 and 9 cases among control group. A significant increase of VDR genotype ff (mutant homozygotes) was observed in the PTB patients when compared to controls (P < 0.05, OR 3.111, 95% confidence interval 1.343-7.207). The genotype of TT were in 137 cases, Tt in 3 cases among patients while TT in 134 cases , Tt in 5 cases among controls. No significant difference in these genotype frequencies was observed between two groups (P > 0.05, OR 1.704, 95% confidence interval 0.399-7.272). Two polymorphisms didn't show linkage disequilibrium and D' is 0.475. CONCLUSION: The Fok I polymorphism of VDR gene might be associated with PTB in Chinese Tibetan population, but no evidences supported that the Taq I polymorphism were associated with PTB in Chinese Tibetan. The two polymorphisms didn't show linkage disequilibrium.

[Characterization of the katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant and susceptible strain of Mycobacterium tuberculosis by automated DNA sequencing].

OBJECTIVE: To study the characteristics of katG, inhA, ahpC, kasA, and oxyR gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. METHODS: A total of 101 isoniazid-resistant and 43 susceptible strains of Mycobacterium tuberculosis were analyzed by PCR and sequence analysis of their katG, inhA, ahpC, kasA, and oxyR genes. RESULTS: (1) Sequencing of katG from 101 INH-resistant strains showed point mutations, small deletions or insertions in 81 isolates (80.2%), but no complete deletions were identified. The mutations at 16 position were found for the first time. Point mutations at position 315 were found in the genomes of 38.6% (39/101) of isoniazid-resistant strains. Low level isoniazid resistant strains (1 microg/ml) had higher mutation frequency at 315-Ser than high level isoniazid resistant strains (10 microg/ml; chi(2) = 9.31, P < 0.05). Mutations at position 463 were detected in 58 (57.4%) isoniazid-resistant strains. Arg463leu was also present in 23 of 43 susceptible strains. (2) Mutations in inhA genes were identified in 5 isoniazid-resistant isolates (4.9%). None of the susceptible strains contained any mutation in inhA genes. (3) Only 3 isolates in the 101 (2.97%) isoniazid-resistant clinical isolates had mutations in ahpC genes. No mutations were identified in the ahpC genes in 43 isoniazid-susceptible isolates. (4) Mutations in kasA genes were present in 17 of 101 (16.8%) isoniazid-resistant isolates. However, G312S was also present in 3 of 43 susceptible strains. (5) None of the isoniazid-resistant strains and susceptible isolates contained oxyR gene mutation. (6) Taken together, 91 of 101 (90%) isoniazid-resistant strains had mutations in katG, inhA, ahpC, and kasA genes which were associated with drug resistance. CONCLUSION: These studies provide further evidence supporting the association between katG, inhA, ahpC, and kasA gene mutations and INH resistance in Mycobacterium tuberculosis, while other mechanisms of INH resistant may exist.