Objective: The interferon-γ (IFN-γ) release assays still fail to meet the diagnostic needs of etiology negative tuberculosis (TB) patients. The purpose of this paper is to explore the value of the IL-2-enzyme-linked immunospot (ELISPOT) assay in the auxiliary diagnosis of TB. Methods: We recruited 127 TB patients, 49 non-TB patients with other diseases (NTB), and 198 healthy people, whose peripheral blood mononuclear cells were isolated and stimulated by Mtb-specific antigen CFP-10/ESAT-6 fusion protein to study the changes of T cells secreting IFN-γ and Interleukin-2 (IL-2) by ELISPOT method. Results: The combination of IFN-γ and IL-2 had an area under the curve (AUC) of 0.867 (IFN-γ: 0.835, IL-2: 0.866) in the diagnosis of TB and could increase the sensitivity of IFN-γ detection alone from 79.5% to 86.6%. The cut-off value for IL-2 and IFN-γ was 16. IL-2 could increase the positive diagnostic rates of IFN-γ-ELISPOT (-) in the etiologically positive and negative ATB patients by 9.4% and 7.1%. In the ATB group, the positive rates and the numbers of spot-forming cells (SFC) of IFN-γ and IL-2 showed significant differences compared to the normal control subjects and NTB group (P<0.05). And SFCs of IFN-γ-ELISPOT were significantly higher than that of IL-2-ELISPOT in ATB patients. Before the treatment, the SFC values of the etiologically positive TB patients were higher than those of the etiologically negative patients. However, this situation was totally reversed after anti-TB treatment. After effective treatment, the positive rates and SFC values of IFN-γ and IL-2 in TB patients decreased and exhibited a decreasing and increasing trend in etiologically positive and negative TB patients, respectively. Conclusions: The combination of IL-2 and IFN-γ could improve the diagnostic sensitivity of single-factor detection in TB patients and these two cytokines could be used as effective biomarkers for the treatment monitoring in TB patients.

Background: Verapamil enhances the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs, promotes the macrophage anti-TB ability, and reduces drug resistance, but its mechanism is unclear. Herein, we have investigated the effect of verapamil on cytokine expression in mouse peritoneal macrophages. Methods: Macrophages from mice infected with M. tuberculosis or S. aureus were cultured with verapamil, the cytokines were detected by enzyme-linked immunosorbent assay, and the RNA was measured with quantitative real-time polymerase chain reaction and agarose gel electrophoresis. The intracellular calcium signaling was measured by confocal microscopy. Results: Significantly higher levels of NF-κB, IL-12, TNF-α, and IL-1β were observed after TB infection. The levels of NF-κB and IL-12 increased when verapamil concentration was less than 50 μg/ml, but decreased when verapamil concentration was greater than 50μg/ml. With the increase in verapamil concentration, TNF-α and IL-1β expressed by macrophages decreased. The L-type calcium channel transcription significantly increased in M. tuberculosis rather than S. aureus-infected macrophages. Furthermore, during bacillus Calmette-Guerin (BCG) infection, verapamil stimulated a sharp peak in calcium concentration in macrophages, while calcium concentration increased mildly and decreased smoothly over time in the absence of verapamil. Conclusions: Verapamil enhanced macrophage immunity via the NF-κB pathway, and its effects on cytokine expression may be achieved by its regulation of intracellular calcium signaling.

Abstract Background The aim of the present study is to investigate the clinical value and characteristics of peripheral blood lymphocyte subsets in patients with pulmonary tuberculosis (PTB) using flow cytometry. Methods The absolute counts of T, CD4+T, CD8+T, natural killer (NK), NKT and B lymphocytes in 217 cases of PTB were detected, and the variations in lymphocyte subset counts between different ages and genders and between aetiological detection results and chest radiography results were analysed. Results In 75.3% of the patients with PTB, six subset counts were lower than the normal reference range, and 44% showed lower‐than‐normal CD4+T lymphocyte levels. The counts of T, CD4+T, CD8+T and B lymphocytes were significantly lower in patients aged >60 years, and the NKT cell counts were significantly lower in female patients than in male patients. Among the patients with positive aetiological results, 40.8% had reduced CD8+T counts; these were significantly lower than those in patients with negative aetiological results (P = 0.0295). The cell counts of T, CD4+T, CD8+T and B lymphocytes reduced as lesion lobe numbers increased. The counts of T, CD4+T and CD8+T lymphocytes were significantly higher in the group with lesions affecting one lobe than in the groups with two to three lobes or four to five lobes, and the counts of B lymphocytes were significantly higher in the group with one lobe and the group with two to three lobes than in the group with four to five lobes. The counts of CD4+T and CD8+T lymphocytes were highest in the no cavity group and showed a downward trend with the increase in cavities; the T lymphocyte count was significantly higher in the no cavity group than in the group with five or more cavities (P = 0.014), and the CD8+T lymphocyte count was significantly higher in the no cavity group than in the group with one to two cavities and the group with five or more cavities (P = 0.001 and 0.01, respectively). Conclusions In most patients with tuberculosis, immune function is impaired. The absolute counts of peripheral blood lymphocyte subsets are closely related to the aetiological results and lesion severity in patients with PTB; this could be used as evidence for immune intervention and monitoring curative effects.

结核病是由结核分枝杆菌感染引起的慢性传染性疾病.当结核分枝杆菌侵入机体的菌量和毒力大于机体免疫系统的防御能力时,就会发病;免疫功能低下者更易感染发病.目前淋巴细胞亚群检测成为临床分析结核病患者免疫状况的手段之一,但其诊疗意义尚没有统一的认识.作者通过概述结核病患者外周血中淋巴细胞亚群百分比及绝对计数在结核病诊断和治疗中的应用研究进展,以判断其临床价值,为结核病患者的免疫诊断和免疫治疗提供依据.

Background This research aimed to investigate the clinical value and characteristics of peripheral blood lymphocyte subsets in tuberculosis (TB) patients by flow cytometry. Methods The Absolute counts of T, CD4 + T, CD8 + T, NK, NKT and B lymphocytes were detected in 217 cases of pulmonary TB (PTB), 163 cases of PTB & extra-PTB and 65 cases of extra-PTB patients. We analyzed the change characteristic of subset counts with the clinical parameters in PTB and compared the subsets differences among PTB, PTB with extra-PTB, and extra-PTB patients. Results The absolute counts of six subsets in 75.3% of PTB patients were lower than the normal reference range, and 44% patients showed lower CD4 + lymphocytes. (1) The absolute counts of T, CD4 + T, CD8 + T and B lymphocytes was significantly lower in patients older than 60 years old. (2) The NKT cell counts was significantly lower in female patients than the males; (3) 40.8% of patients with positive etiological results and 49.2% of extra-PTB patients showed lower CD8 + T and NK cell counts below the reference range respectively. (4) The T and CD4 + T lymphocyte counts in PTB with positive IGRA were significantly higher than those with negative IGRA; (5) The T and CD8 + T cell counts in PTB with positive IgG antibody were decreased; (6) The T, CD4 + T, CD8 + T and B cell counts reduced with the increasing lesion lobe numbers, accelerated ESR, complications with anemia and low serum albumin, and NK cells also decreased in patients with anemia and low serum albumin; and the T, CD8 + , and B cells significantly decreased in PTB patients with diabetes, while NKT increased. Conclusions The immune function in most TB patients is impaired and the absolute counts of lymphocyte subsets could be used as the evidence for immune intervention and monitoring the curative effect.

目的 探讨肺结核并发糖尿病(pulmonary tuberculosis complicated with diabetes mellitus,PTB-DM)患者临床特征及淋巴细胞亚群特点.方法 回顾性收集2018年4月1日至2019年5月31日在解放军总医院第八医学中心结核二科住院的符合纳入标准的肺结核患者.根据是否并发糖尿病将其分为两组:PTB-DM组85例,单纯肺结核组(PTB组)96例.对两组患者临床特征包括性别、年龄、治疗史和胸部CT检查结果进行比较分析;对两组患者淋巴细胞亚群和结核分枝杆菌特异性反应T细胞检测结果分别进行比较分析.结果 PTB-DM组男性和女性分别占76.4％(65/85)和23.6％(20/85),PTB组分别占57.3％(55/96)和42.7％(41/96),差异有统计学意义(x2=7.42,P=0.006).PTB-DM组＜25岁、25～60岁和＞60岁的患者分别占2.4％(2/85)、68.2％(58/85)和29.4％(25/85);PTB组则分别占25.0％(24/96)、63.5％(61/96)和11.5％(11/96),差异有统计学意义(x2=23.55,P=0.000).PTB-DM组初治和复治患者分别占74.1％(63/85)和25.9％(22/85),PTB组分别占84.4％(81/96)和15.6％(15/96),差异无统计学意义(x2=2.92,P=0.088).PTB-DM组胸部CT扫描显示无空洞、有空洞、病变范围＜3个肺叶和≥3个肺叶者分别占23.5％(20/85)、76.5％(65/85)、31.8％(27/85)和68.2％(58/85);PTB组分别为53.1％(51/96)、46.9％(45/96)、54.2％(52/96)和45.8％(44/96);PTB-DM组胸部CT有空洞和病变范围≥3个肺叶的患者明显多于PTB组,差异有统计学意义(x2=16.56,P=0.000和x2=9.20,P=0.002).PTB-DM组结核分枝杆菌特异性反应T细胞阳性率为81.3％(65/80),PTB组阳性率为74.2％(66/89),差异无统计学意义(x2=1.22,P=0.270).PTB-DM组总T淋巴细胞绝对计数、CD4+T淋巴细胞绝对计数、CD8+T淋巴细胞绝对计数、自然杀伤细胞(NK细胞)绝对计数、NK样T淋巴细胞绝对计数和总B淋巴细胞绝对计数的中位数(四分位数)值分别为1069.00 (753.50,2372.50)、548.00 (381.00,787.50)、380.00(270.50,574.50)、184.00(111.00,294.50)、60.00(36.00,120.50)和162.00(80.50,244.00)个μl,PTB组则分别为1161.50(858.50,1601.00)、628.00(472.75,860.50)、457.50(286.00,614.75)、191.50(115.75,315.75)、65.50(34.50,119.50)和184.50(112.25,301.00)个/μl,两组间6项指标差异无统计学意义(Z=-1.80,P=0.073;Z=-1.47,P=0.142;Z=1.46,P=0.144;Z=-0.57,P=0.568;Z=-0.09,P=0.931;Z=-1.93,P=0.053).结论 PTB-DM中老年男性较多,病灶范围广泛、严重;PTB是否并发DM对淋巴细胞亚群6项指标检测结果无明显影响.

In this study, the Mycobacterium tuberculosis (MTB) latency-associated antigens Rv2660c, Rv1733c, Rv1813c, Rv2628, Rv2029c, and Rv2659c were compared regarding their immunogenicity and potential therapeutic effects in an MTB reactivation mouse model. Normal mice or MTB reactivation mice were immunized intramuscularly three times at 2-week intervals with saline, plasmid vector pVAX1, Mycobacterium vaccae vaccine (a commercial inactivated vaccine), rv1813c DNA, rv2628 DNA, rv2029c DNA, rv2659c DNA, rv1733c DNA, or rv2660c DNA. The normal mice immunized with rv2628 DNA or rv2659c DNA had low numbers of Th1 cells and a lower ratio of Th1:Th2 immune cells in whole blood (p < 0.05). Compared to the saline group, Tc1 cells in the rv2029c DNA group and Tc1:Tc2 cell ratio in the rv1813c DNA, rv2628 DNA, and rv2029c DNA groups were significantly decreased (p < 0.05). The proportion of Foxp3+CD4+ T cells in the rv2628 DNA and rv2659c DNA groups and the proportion of CD4+CD25+ T cells in the rv2029c DNA group were significantly increased (p < 0.05). The level of anti-Rv1813c-immunoglobulin G (IgG) in the rv1813c DNA group was significantly increased (p < 0.01). The levels of specific IgG, IgG1, and IgG2a in the rv2628 DNA, rv2029c DNA, and rv2659c DNA groups were significantly increased (p < 0.05). Lung colony-forming units in M. vaccae and the six DNA groups decreased to different degrees in the MTB reactivation mouse model, but only the lung colony-forming units in the rv2628 DNA group (4.38 ± 0.70 log10) significantly decreased compared to the vector group (5.90 ± 0.42 log10; p < 0.05). The MTB rv1813c DNA, rv2628 DNA, rv2029c DNA, and rv2659c DNA could elicit a strong humoral immune response and a higher proportion of CD4+CD25+or CD4+Foxp3+ T cells but could not increase the proportions of Th1 and Tc1 cells. These results suggest that latency-associated DNA vaccines, especially rv2628 DNA, had some therapeutic effect on the endogenous resurgence mouse tuberculosis model.

目的 研究结核分枝杆菌(MTB)耐药基因芯片直接检测结核病患者临床标本中利福平和异烟肼耐药相关基因的临床价值.方法 回顾性分析125例通过硝基苯甲酸(PNB)和噻吩-2-羧酸肼(TCH)鉴别培养为MTB的临床标本,其中痰标本90例、支气管灌洗液12例、脓液12例、胸腔积液6例、组织标本3例、腹腔积液和尿液标本各1例,应用绝对浓度法同时进行利福平(RFP)和异烟肼(INH)药物敏感性试验(简称“绝对浓度法药敏试验”),并用基因芯片直接检测临床标本中MTB rpoB、katG和inhA基因型.以绝对浓度法药敏试验为标准,评价基因芯片检测RFP、INH耐药和耐多药(MDR)的敏感度和特异度,并且比较两种检测结果的一致性.结果 在125例结核病患者临床标本中,绝对浓度法药敏试验显示对RFP、INH的耐药率和MDR发生率分别为20.0％(25/125)[其中高耐占88.0％(22/25)、低耐占12.0％(3/25)]、17.6％(22/125)[其中高耐占18.2％(4/22)、低耐占81.80％(18/22)]、16.8％(21/125).初治和复治结核病患者临床标本中MDR-MTB的检出率分别为7.6％ (7/92)和39.4％(13/33).以绝对浓度法药敏试验为标准,应用基因芯片直接检测临床标本中MTB rpoB、katG和inhA基因型,预示对RFP、INH耐药和MDR的敏感度分别为72.0％ (18/25)、63.6％(14/22)和61.9％(13/21),特异度分别为91.0％(91/100)、86.4％(89/103)和89.4％(93/104),两种方法的一致率分别为87.2％(109/125)、82.4％(103/125)和84.8％(106/125).结论 应用耐药基因芯片直接检测结核病患者临床标本中MTB对RFP和INH耐药的相关基因突变具有中等的敏感度和较高的特异度,可快速检出对RFP、INH耐药和MDR-TB患者,为临床早期开展有效化疗提供实验依据.

Relapse of pulmonary tuberculosis (PTB) is associated with a failure of the host immune system to control the invading Mycobacterium tuberculosis. Severe immunodeficiency or immune disorders may be the main reason for TB recurrence. This study aimed to quantify serum inflammatory cytokine and soluble adhesion molecule levels in Re-treated smear-positive PTB patients before and after re-anti-TB drug therapy. Serum samples were collected from 30 healthy controls and 215 Treated active PTB patients at baseline and 2, 4, and 6 months post-re treatment. Levels of 18 serum cytokines and soluble adhesion molecules were measured by a high-throughput Cytometric Bead Array. At baseline, IL-1, IL-2, IL-12P70, and soluble CD62E levels were significantly higher in PTB patients than those in the healthy controls (p < 0.05); IL-4, IL-5, IL-7, IL-8, IL-10, IL-17, IL-21, soluble CD54, MIG, and TGF-beta levels in PTB patients were significantly lower than those in the healthy controls (p < 0.05), of which TGF-beta, IL-7, IL-8, IL-10, soluble CD54, and MIG were most notably (p < 0.0005). After re treatment, IFN-gamma, IL-2, IL-7, and soluble CD54 levels and IL-2/IL-10 and IFN-gamma/IL-10 ratios showed an upward trend during the re-treatment period. They were more sensitive than other cytokines and adhesion molecules and could be effective as serum indicators for re-treatment response. The immune response was imbalance in treated smear-positive PTB patients: Th1 response was elevated, but Th2 and Th17 responses were reduced. Systematic and comprehensive understanding of the cytokine and soluble adhesion molecule profiles provides a theoretical basis for immuno-diagnosis, immunotherapy, and immuno-monitoring of Re-treated PTB patients.

Tuberculosis (TB) is a major global public health problem. Latent TB infection (LTBI) is a major source of active TB. New vaccines to treat LTBI are urgently demanded. In this study, the gene encoding latency-associated antigen Rv3407 of Mycobacterium tuberculosis (MTB) rv3407 DNA vaccine was used to prepare and the immunogenicity and therapeutic effects were evaluated. Normal mice were immunized intramuscularly three times at two-week intervals with sterile water for injection, plasmid vector pVAX1, M. vaccae vaccine, ag85a DNA or rv3407 DNA. TB-infected mice were immunized intramuscularly three times at two-week intervals with phosphate-buffered saline (PBS) and rv3407 DNA. The normal mice immunized with rv3407 DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-gamma) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevated ratio of Th1/Th2 immune cells in whole blood, indicating that a Th1-type immune response was predominant. The levels of anti-Ag85A or anti-Rv3407 IgG antibody were significantly increased in the ag85a DNA and rv3407 DNA groups compared to the sterile water for injection, vector, and M. vaccae groups (p < .0001). Compared with the PBS group, the rv3407 DNA group had pulmonary bacterial loads that were lower by 0.56 log(10) (p < .01)(.) The mice vaccinated with rv3407 DNA developed antigen-specific cellular and humoral responses. The rv3407 DNA is a potential DNA vaccine candidate against TB.

Background: The diagnosis of bacterium-negative pulmonary tuberculosis (TB) and extra-pulmonary TB is challenging clinically. The detection of the anti-TB antibody has an important, auxiliary, clinical diagnostic value. Therefore, TB antibody detection kits should be screened and evaluated, and the reagents with the highest sensitivity and specificity should be chosen and used clinically. Methods: The diagnostic performance of 7 commercially available TB antibody detection kits (kits A, B, C, D, E, F and G) based on the gold immunoassay detection of immunoglobulin (Ig) G or IgM antibodies were simultaneously evaluated and compared in 62 TB cases and 56 non-TB cases in a laboratory. A retrospective analysis including 2549 cases was carried out to assess the clinical diagnosis values of bacteriological examinations and TB antibody tests (kits B and H used in the clinic). Results: The sensitivities of TB antibody kits A, B, C, D, E, F and G in the sera from 62 TB patients were 50.0%, 83.9%, 38.7%, 9.7%, 48.4%, 69.4% and 79.0%, respectively; the sensitivities in the sera from 24 smear-negative TB patients were 29.2%, 79.2%, 29.2%, 12.5%, 29.2%, 54.2% and 79.2%, respectively; the specificities in the sera from 56 non-TB patients were 73.2%, 25.0%, 85.7%, 96.4%, 78.6%, 78.6% and 50.0%, respectively. Of the 2549 clinically diagnosed cases, there were 1752 pulmonary TB cases, 505 extra-pulmonary TB cases, 87 old pulmonary TB cases and 205 nonTB cases. The positive results for smear, culture, TB antibody kit B and kit H in pulmonary TB cases were 39.8% (543/1365), 48.6% (372/765), 45.8% (802/1752) and 25.2% (442/1752), respectively; the results in extra-pulmonary TB cases were 3.4% (6/178), 5.8% (4/69), 35.4% (179/505), and 11.3% (57/505), respectively; the results in old pulmonary TB cases were 0% (0/64), 0% (0/30), 32.2% (28/87), and 9.2% (8/87), respectively; and the results in non-TB cases were 0% (0/121), 0% (0/56), 21.5% (44/205), and 2.4% (5/205), respectively. Of 624 smear-positive and/or culture-positive pulmonary TB cases, the sensitivities of antibody test kits B and H were 53.0% and 36.4%, respectively. Of 901 smear-negative and/or culture-negative pulmonary TB cases, the sensitivities of antibody test kits B and H were 42. 5% and 19.0%, respectively. The positive rate of antibody detection in the bacterium-positive pulmonary TB cases was significantly higher than that in the bacterium-negative pulmonary TB cases (P < 0.05). Conclusions: The colloidal gold-labeled TB antibody IgG detection assay is a simple, rapid and economical method that provides a better clinical auxiliary diagnosis value on TB, especially in smear-negative pulmonary TB and extra-pulmonary TB. The production, quality control, screening and evaluation of antibody detection kits are very important for its clinical application.

There is an urgent need for a more effective vaccine against tuberculosis (TB). Cytotoxic T lymphocytes (CTLs) play a critical role in combating Mycobacterium tuberculosis (M.tb). The identification of novel CTL epitopes is essential for the design of peptide-based vaccines. In this study, we predicted CTL epitope peptides of M.tb antigen Rv2629 restricted by HLA-A2, using bioinformatics methods. The affinity and stability of binding of these peptides with HLA-A2 molecules were detected by flow cytometry. Their ability to induce CTLs generation was determined in peripheral blood mononuclear cells (PBMCs) from healthy uninfected subjects, Latent tuberculosis infection (LTBI) subjects, and TB patients ex vivo. The cytotoxic activity induced by the epitope peptides was tested by lactate dehydrogenase (LDH) release assay. Finally, we found four novel CTL epitope peptides, Rv2629-p190-2L, Rv2629-p190-1Y2L, Rv2629-p274, and Rv2629-p315, which had high-affinity and stability of binding with T2 cells. Their ability of inducing CTLs was highest in PBMCs from TB patients (P < 0.05). In addition, these peptides could induce the CTLs to generate specific cytotoxic activity. They showed higher immunogenicity in TB patients and had the potential to become candidate vaccines for TB therapy.

目的观察缺氧诱导因子（hypoxia inducible factor-1,HIF-1α）、转化生长因子（transforming growth factor-β,TGF-β）在尿路移行上皮癌中的表达,确认2个细胞因子与尿路移行上皮癌进展的相关性。方法收集低级别移行上皮癌30例、高级别移行上皮癌60例（浸润内1/2和外1/2肌层各30例）,采用免疫组化和组织芯片技术观察肿瘤组织中HIF-1α、TGF-β的表达,并与其临床病理行相关性分析。结果 （1）HIF-1α在低级别和高级别癌中均表达,阳性率分别为33%（10/30）和67%（40/60）,差别具有统计学意义（P＜0.05）;TGF-β在低级别和高级别癌中均表达,阳性率分别为30%（9/30）和70%（42/60）,差别具有统计学意义（P＜0.05）;（2）HIF-1α表达水平与组织学类型、浸润深度、淋巴结转移相关（P＜0.05）,与肿瘤体积、脉管内癌栓、TNM分期无关（P＞0.05）;TGF-β表达水平与肿瘤体积、脉管内癌栓、组织学类型密切相关（P＜0.05）,与淋巴结转移、TNM分期、浸润深度无关（P＞0.05）。（3）HIF-1α、TGF-β表达水平在低级别尿路移行上皮癌中无相关性（r=0.2802,P＞0.05）;在高级别尿路移行上皮癌中呈正相关（r=0.7803,P＜0.05）。结论 HIF-1α和TGF-β在移行上皮癌中均存表达,随移行上皮癌恶性级别升高而表达明显增强,提示HIF-1α和TGF-β与移行上皮癌的进展密切相关。

Objectives To clone,express,and purify Rv1813c protein,which is related to latent infection by Mycobacterium tuberculosis,and to analyze its immunological activity.Methods The amino acid sequence of Rv1813c protein was analyzed bioinformatically.Primers were synthesized in accordance with the sequence of the M.tuberculosis H37Rv genome,rv1813c was amplified with PCR and cloned into a pGEM-T vector.The gene from the positive clone was sequenced,and then cloned into a pET30a vector.Expression of the recombinant protein in E.coli BL21 was induced with IPTG,and the protein was then purified using metal chelate chromatography.The titer of antibodies against the recombinant Rv1813c protein was determined in sera from 39 healthy individuals,19 patients with tuberculosis,and 10 patients with a non-tuberculous respiratory disease according to ELISA.Results Rv1813c protein contained transmembrane domains and a signal peptide portion before amino acid 34;the protein may be an extracellular.Gene sequencing indicated that the recombinant plasmid pET30a-Rv1813c had the same sequence as intended.The recombinant protein was expressed in the form of inclusion bodies,and expression peaked 3-4 hours after it was induced with IPTG.The recombinant Rv1813c protein accounted for more than 40％ of all bacterial proteins expressed.The protein was more than 95％ pure.ELISA results indicated that the recombinant Rv1813c protein had a significantly higher level of reactivity to sera from patients with tuberculosis (0.50±0.34) than to sera from healthy individuals (0.23±0.18) and sera from patients with a non-tuberculous respiratory disease (0.30±0.27) (P＜0.05).Conclusion The recombinant Rv1813c protein of M.tuberculosis was successfully prepared and had good immunological activity,laying the foundation for further study of the protein's function and its potential use.

Objective To predict the epitopes of Mycobacterium tuberculosis Rv2657c protein, to understand its immunogenicity Methods The T?cell and B?cell epitopes of Mycobacterium tuberculosis Rv2657c protein were predicted by DNAStar software package. The homology of Rv2657c amino acid sequence with the human protein sequences was prepared using Blast method, then the CTL epitopes were predicted using SYFPEITHI supermotif method, BIMAS quantitative motif method and NetCTL prediction method, and the Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif prediction method. Results The prediction using DNAStar software package showed that Rv2657c protein had 5 B?cell epitopes and 6 T?cell epitopes. The protein had 6 CTL epitopes and there were 38 Th epitopes. Conclusion Rv2657c protein has both B?cell epitopes and T?cell epitopes. It may be a candidate target antigen for the studies of vaccine and diagnosis of tuberculosis.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), is becoming increasingly recognized as an important cause of fatal chronic illnesses in China. In this study, we report an infectious disease among 84 rhesus macaques at a Chinese zoo. Their clinical signs and symptoms were very similar with the manifestations of TB in humans. To determine the potential pathogens of this outbreak, many methods were used. First, tuberculin skin tests showed that none of the monkeys displayed significant skin reactions. Subsequently, the sera were tested for specific antibody IgG; 29 (34.5%) and 39 (46.4%) blood samples tested positive by TB-IgG and TB-DOT, respectively. Radiographic examination showed characteristic imageology changes in 14 (16.7%) monkeys. One individual determined as positive by the above three methods was euthanized, and histopathological analysis demonstrated typical granulomas and caseous necrosis in the lung, liver, spleen, and intestine. Furthermore, the pathogenic mycobacteria were isolated from lung lobe, cultured on acidic Lowenstein-Jensen culture medium, and identified as M. tuberculosis by real-time PCR and DNA sequencing. Nevertheless, the origin of the infection remained unknown. These findings emphasize the need to strengthen the management and training of staff, especially those working at animal shelters.

The Excel data file [FOLT] Figshare, [DOI: 10.6084/m9.figshare.4668148 and https://figshare.com/s/bd46c22986c673579bb6 ] includes all datasets supporting the conclusions of this article: IFN-Îł in spleen lymphocyte culture supernatants, IL-4 in spleen lymphocyte culture supernatants, CD4+ T cell subsets expressing intracellular IFN-Îł or IL-4, CFU in the lungs and spleens.. (XLS 143 kb)

Objective To study the value of recombinant Mycobacterium tuberculosis latent infection related protein Rv1813c in the diagnosis of latent infection of Mycobacterium tuberculosis.Methods 20 LTBI subjects and 79 healthy controls were given chest X-ray examination,PPD skin test,antibody detection,and IGRAs by ELISA with recombinant protein CFP10-ESAT6 and latent infection protein Rv1813c.Results In all the subjects,the positive rates of PPD skin test and antibody test were 50％ and 28％ respectively.After stimulation with Rv1813c,IFN-γ level produced in LTBI was significantly higher than that in healthy controls (P ＜ 0.05),the strong positive group (diameter≥ 15 mm) was lower than that of the weak positive group of TST (5mm ≤diameter ＜ 15mm) though the difference was not statistically significant.After stimulation with CFP10-ESAT6,the IFN-γ level in the strong positive group was higher than the weak positive group (P ＜ 0.05).There was no significant difference between antibody negative and positive groups after stimulation by CFP10-ESAT6 and Rvl813c (P ＞ 0.05).The area under the ROC curve of tuberculosis infection diagnosis was 0.834.While specificity was 80％,sensitivity was 85％.And while specificity was 90％,sensitivity was 45％.Conclusion Combined detection of Rv1813c and CFP10-ESAT6 specific IFN-γvalues is helpful to screen the high risk population of latent infection,which has important application value for diagnosis of LTBI.

Objective To compare the anti-tuberculosis effects of four kinds of genes encoding the antigens in proliferation phase of M.tuberculosis in mouse tuberculosis (TB) model,and provide experimental basis for developing new TB immunotherapy agents.Methods Seventy female BALB/c mice were infected via the tail vein with 6.4× 105 CFUs of M.tuberculosis H37Rv,then randomly divided into 7 groups and treated as follow at the third day after infection:saline,plasmid vector pVAX1,M.vaccae vaccine,ag85a DNA,rv1291c DNA,rv1419 DNA and rv2223c DNA,which were injected intramuscularly 3 times at two-week intervals.The mice were sacrificed at two weeks after the final immunization.The lungs and spleens from the mice were taken and their pathological changes,weights and number of mycobacterial colony were examined.Data were expressed as means and standard deviations.Results The lung histopathological examination showed that the lesions were severe and extensive in saline group,lighter in plasmid vector pVAX1 group than those in saline group,slight and limited in the M.vaccae vaccine,ag85a DNA,rv1291c DNA,rv1419 DNA and rv2223c DNA groups with relatively clear and normal structure of alveoli.Compared with saline group,ag85a DNA group,rv1291c DNA group and rv1419 DNA group reduced the pulmonary bacterial loads respectively by 0.53 lg CFU,0.67 lg CFU and 0.41 lg CFU (x2 =27.07,P＜0.01);spleen bacterial loads respectively by 0.43 lg CFU (t＞3.004,P＜0.05),0.34 lg CFU (t＜3.004,P＞0.05),and 0.41 lg CFU (t＞3.004,P＜0.05);M.vaccae vaccine and rv2223c DNA groups also decreased lung and spleen bacterial loads,but there were not significant differences (t＜3.004,all P＜0.05).Conclusion M.tuberculosis rv1291c and rv1419 genes encoding the antigens in proliferation period had better irnmunotherapeutic effects on TB,which were similar to ag85a DNA in mice.Rv2223c DNA had not obvious immunotherapeutic effect.