T细胞、NKT和NK细胞在结核病诊断中的意义探讨

目的探讨CD3+T细胞、CD3+CD16+CD56+N KT细胞及CD3-CD16+CD56+N K细胞在肺结核患者外周血的表达状态及其临床意义。方法采用流式细胞仪分析123例肺结核患者(73例初治肺结核和50例复治肺结核)及83例健康者(43例结核菌素皮试阳性和40例结核菌素皮试阴性)外周血中T细胞、NKT和NK细胞的表达率。结果初、复治肺结核患者的NKT细胞表达率明显高于健康者(P0.01),但初治肺结核患者的NK细胞、T细胞的表达率与健康者相比差异无统计学意义(P0.05)。T细胞表达率在不同程度的初治肺结核患者中差异有统计学意义(P0.01),疾病越重,表达越高。NKT、NK细胞差异无统计学意义(P0.05)。复治肺结核患者的T细胞表达率显著高于初治肺结核患者(P0.01),但NK细胞、NKT细胞的表达率在2组间差异无统计学意义(P0.05)。结论T细胞、NKT细胞是结核病细胞免疫的重要组成部分。对NKT细胞只在活动性肺结核患者才会呈现高表达,测定外周血NKT细胞可能成为辅助诊断活动性肺结核的手段之一。     

Cytokine responses induced by Mycobacterium tuberculosis in patients with HIV-1 infection and tuberculosis.

OBJECTIVE: Tuberculosis (TB) is an important opportunistic infection in HIV patients. Immune responses to Mycobacterium tuberculosis in HIV/TB patients were evaluated. METHODS: Fifteen patients with HIV/TB, ten with HIV, four with TB, and five controls were enrolled. Peripheral blood mononuclear cells were isolated and stimulated with mycobacterial antigen (PPD). Interferon (IFN)-gamma and TNF-alpha in culture supernatants were measured by ELISA. RESULTS: IFN-gamma and TNF-alpha production after PPD stimulation was markedly decreased in HIV patients, but not in HIV/TB patients. In HIV patients with a CD4 cell count of less than 200/mm3, IFN-gamma and TNF-alpha production after PPD stimulation was higher in HIV/TB patients than in HIV patients. Cytokine responses to M. tuberculosis reconstituted after highly active antiretroviral therapy (HAART) and were prominent in HIV/TB patients. CONCLUSIONS: Cytokine responses to M. tuberculosis were retained in HIV-infected patients with tuberculosis, even in patients with a CD4 cell count of less than 200/mm3, and reconstituted after HAART.     

Role of NK,NKT cells and macrophages in liver transplantation

Liver transplantation has become the treatment of choice for acute or chronic liver disease. Because the liver acts as an innate immunity-dominant organ, there are immunological differences between the liver and other organs. The specific features of hepatic natural killer(NK), NKT and Kupffer cells and their role in the mechanism of liver transplant rejection, tolerance and hepatic ischemia-reperfusion injury are discussed in this review.     

Cytokine profiles in immunocompetent persons infected with Mycobacterium avium complex

To evaluate the immunologic factors that contribute to protection against Mycobacterium avium complex (MAC), cytokine production by peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus-negative persons with pulmonary MAC (MAC patients) and healthy control subjects with a delayed hypersensitivity skin test response to M. avium sensitin (MAS-positive control subjects) was measured. In MAC patients, mycobacterium-stimulated PBMC produced higher concentrations of interleukin (IL)-10 but lower concentrations of interferon (IFN)-gamma, IL-12, and tumor necrosis factor (TNF)-alpha, compared with PBMC from MAS-positive control subjects. Immunolabeling for intracellular IL-10 revealed that this cytokine was produced by both monocytes and T cells. Alveolar macrophages produced TNF-alpha and IL-10 in response to MAC, which suggests that these cytokines are produced in the lungs of patients with pulmonary disease caused by this pathogen. Our findings suggest that IFN-gamma, TNF-alpha, and IL-12 contribute to protection against MAC, whereas IL-10 is immunosuppressive.     

Human natural killer T cells (NKT), NK and T cells in pulmonary tuberculosis: potential indicators for disease activity and prognosis

One third of the world's population is infected with Mycobacterium tuberculosis (MTB). However, active disease can develop only in a small percentage, when the immunity is weakened. The acquired immune response to MTB is primarily mediated by T cells. Natural killer (NK) cells play a central role in innate immunity to microbial pathogens. Human NKT cells have characteristics of both T and NK cells and also exhibit antimycobacterial activity. This work aimed to enumerate T, NK and NKT cells in active pulmonary TB compared with healthy controls and to study the correlation between these cells with different factors affecting prognosis of pulmonary TB as disease severity, complications or associated diseases, antitubrculosis chemotherapy, and age & gender. Of the 22 active tuberculosis patients examined, 17 were recent cases and 5 recurrent. Healthy controls were divided into 14 individuals with detectable reaction to purified protein derivative (PPD+) and 14 individuals without detectable reaction to PPD-. The percentages of T, NK and NKT cells in erythrocyte-lysed whole blood samples were determined using flowcytometry. The percentage of NKT cells was significantly higher among the recently diagnosed MTB cases as compared with both PPD+ (P < 0.01) and PPD- (P < 0.01) healthy controls, while no significant difference could be found in the percentages of T or NK cells among these groups. However, comparing recurrent cases with recently diagnosed cases showed a significant difference only in the percentage of T cells (P < 0.01). There was also a significant difference in the percentage of T cells according to severity of disease (P < 0.01) and in the association of diabetes mellitus (P < 0.01). Age, gender and treatment with antituberculosis chemotherapy had no effect on the percentages of T, NK or NKT cells. It is concluded that T and NKT cells play an important role in immunity against TB. In active pulmonary tuberculosis, increased T cell count points to severity of the disease, while their reduced count predicts bad prognosis. Human NKT cell count is a marker of disease activity. Enumeration of these cells in peripheral blood can be used as a non-invasive prognostic indicator for patients with active pulmonary TB.     

Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis.

Mycobacterium smegmatis is typically used as a bacterial host for cloning and expressing single genes or genomic libraries of the human pathogen Mycobacterium tuberculosis. To study virulence of M. tuberculosis, we set out to ask the question, whether a genomic library derived from M. tuberculosis H37Rv confers virulence to the non-virulent M. smegmatis. A representative library from the M. tuberculosis H37Rv genome was generated and transformed into wild-type M. smegmatis. Mice were challenged with recombinant clones by intravenous, aerogenic and intranasal infection. We were unable to detect either growth or persistence of recombinant clones in tissues of infected mice; instead, the infection was cleared. Since the concern that virulent traits might be transferred, bio-safety regulations often require the handling of these experiments at bio-safety Level 3. However, we failed to find any evidence that the M. tuberculosis library confers virulence when expressed in M. smegmatis. We suggest that the results, presented here, should fundamentally alter the containment requirements for similar experiments in the future.     

NK cells in patients during complex therapy of destructive pulmonary tuberculosis

Functional activity of peripheral blood NK-cells was studied in 85 patients aged 18 to 72 years with active destructive tuberculosis of the lungs. There was detected a significant decrease in the activity of the NK-cells them. The most pronounced decrease was observed in the patients over 50 years. The changes in the functional activity of the NK-cells during chemotherapy correlated with the time course of the tuberculosis process. The results of the study provided the use of the NK-cell activity index in addition to the other indices in estimation of the patients immunity status and control of the treatment efficacy. For correction of immunodeficiency detected by the NK-cell activity early use of immunostimulating agents in the combined therapy of the patients with destructive tuberculosis of the lungs was required.     

Genetic polymorphism in Mycobacterium tuberculosis isolates from patients with chronic multidrug-resistant tuberculosis

Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem because treatment is complicated, cure rates are well below those for drug-susceptible tuberculosis (TB), and patients may remain infectious for months or years, despite receiving the best available therapy. To gain a better understanding of MDR-TB, we characterized serial isolates recovered from 13 human immunodeficiency virus-negative patients with MDR-TB, by use of IS6110 restriction fragment-length polymorphism analysis, spacer oligonucleotide genotyping (i.e., "spoligotyping"), and sequencing of rpoB, katG, mabA-inhA (including promoter), pncA, embB, rpsL, rrs, and gyrA. For all 13 patients, chronic MDR-TB was caused by a single strain of Mycobacterium tuberculosis; 8 (62%) of the 13 strains identified as the cause of MDR-TB belonged to the W-Beijing family. The sputum-derived isolates of 4 (31%) of the 13 patients had acquired additional drug-resistance mutations during the study. In these 4 patients, heterogeneous populations of bacilli with different resistance mutations, as well as mixtures of drug-susceptible and drug-resistant genotypes, were observed. This genetic heterogeneity may require treatment targeted at both drug-resistant and drug-susceptible phenotypes.     

Cytokine production in rhesus monkeys infected with Plasmodium coatneyi.

Plasmodium coatneyi infection in rhesus monkeys has been used as a model for studying human malaria. Cytokine production in this model, however, has so far not been examined. In this study, four rhesus monkeys were infected with P. coatneyi, with another four animals serving as uninfected controls. Blood samples were taken for the determination of daily parasitemia, and cytokine and prostaglandin E2 (PGE2) levels at days 0, 3, 5, 7, and 10. All inoculated animals became infected, with synchronized appearance of ring-stage parasites. Infected monkeys had increased plasma levels of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha) during the late stage of the infection. They also had increased production of ciliary neurotrophic factor. In conjunction with the production of proinflammatory cytokines, infected monkeys also had gradual increases in the production of PGE2. A continued definition of the P. coatneyi/rhesus monkey animal model should be useful for the elucidation of the immunopathogenesis of human malaria.     

Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.     

Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.     

结核分枝杆菌感染对BALB/c小鼠B细胞发育分化的影响

目的 探讨结核分枝杆菌感染对小鼠B淋巴细胞分化发育的影响。方法 建立Mtb感染BALB/c小鼠模型,在感染早期(4周)及晚期(8周)分别取小鼠骨髓细胞及脾脏细胞,或经培养后,荧光抗体染色,用流式细胞术对感染早期及晚期小鼠的B细胞表型进行分析,设生理盐水处理的小鼠作为对照。结果 Mtb感染组与对照组比较,pro-B细胞(CD45R⁺CD43⁺)(4周:t=2.886,P<0.05)、未成熟B细胞(CD45R⁺IgM⁺IgD^-)(4周:t=4.760,P<0.05)减少,骨髓成熟B细胞(CD45R⁺IgM^+/-IgD⁺)(4周:t=-3.485,P<0.05;8周:t=-2.594,P<0.05)与脾脏成熟B细胞(CD45R⁺IgMlowIgDhi)(4周:t=-2.275,P<0.05;8周:t=-2.97,P<0.05)增多;小鼠脾脏B细胞活化分子CD69表达升高(4周:t=-2.271,P<0.05;8周:t=-2.052,P<0.05);小鼠脾脏记忆性B细胞 (CD45R⁺CD27⁺IgD^+/-)升高(4周:t=-4.203,P<0.05;8周:t=-5.280,P<0.05)。结论 Mtb感染能影响B细胞的分化发育,促使B细胞向成熟细胞分化,并能促使B细胞活化,使机体更有利于抗结核的感染。     

Quantitative bacillary response to treatment in Mycobacterium tuberculosis infected and M. africanum infected adults with pulmonary tuberculosis.

Data regarding possible differences in microbiological response to therapy of disease caused by Mycobacterium tuberculosis and M. africanum are limited. Presenting clinical characteristics and sputum bacillary load during standard short-course chemotherapy in patients with newly-diagnosed pulmonary tuberculosis due to M. tuberculosis (n = 7) and M. africanum (n = 6) were compared. Changes in sputum bacillary load were measured using quantitative acid-fast bacilli smears, colony forming unit assay, and time until positive culture in the BACTEC radiometric system. Presentation and response to short course chemotherapy were comparable between patients infected with M. tuberculosis and those infected with M. africanum.