Mycobacterium africanum elicits an attenuated T cell response to early secreted antigenic target, 6 kDa, in patients with tuberculosis and their household contacts

BACKGROUND: Mycobacterium africanum, a member of the M. tuberculosis complex that is infrequently found outside of western Africa, is the cause of up to half of the tuberculosis cases there. METHODS: We genotyped mycobacterial isolates obtained from a study of patients with tuberculosis and their household contacts and compared T cell responses and tuberculin skin test results by infecting genotype. RESULTS: The T cell response to early secreted antigenic target, 6 kDa (ESAT-6), was attenuated in patients with tuberculosis (odds ratio [OR], 0.41 [95% confidence interval {CI}, 0.19-0.89]; P = .024) and household contacts (OR, 0.56 [95% CI, 0.38-0.83]; P = .004) infected with M. africanum, compared with the response in those infected with M. tuberculosis. In these same groups, responses to culture filtrate protein, 10 kDa (CFP-10), were nonsignificantly attenuated (P = .22 and P = .16, respectively), as were tuberculin skin test results (P = .30 and P = .46, respectively). Sequencing of region of difference 1 of M. africanum revealed that Rv3879c is a pseudogene in M. africanum; however, this finding does not provide an obvious mechanism for the attenuated ESAT-6 response. CONCLUSIONS: This is the first evidence, to our knowledge, that strain differences affect interferon- gamma -based T cell responses. Our findings highlight the need to test new diagnostic candidates against different strains of mycobacteria. Integrating additional immunologic and genomic comparisons of M. tuberculosis and M. africanum into further studies may provide fundamental insights into the interactions between humans and mycobacteria.     

结核分枝杆菌早期分泌抗原靶-6（ESAT-6）对人THP-1单核细胞IL-12产生的影响

目的研究结核分枝杆菌早期分泌抗原靶-6（ESAT-6）对人THP-1单核细胞IL-12产生的影响及其作用的细胞信号传导机制。方法应用亲和层析方法纯化大肠埃希菌表达重组的结核分枝杆菌ESAT-6抗原,采用ELISA法检测不同浓度ESAT-6刺激人THP-1单核细胞IL-12产生的影响,以及对LPS刺激的反应,应用各种细胞信号传导通路抑制剂来探讨ESAT-6诱导单核细胞IL-12产生相关可能的信号通路。结果结核分枝杆菌ESAT-6分泌抗原在2.5～10g/ml浓度范围能依赖性地刺激人THP-1单核细胞产生IL-12（p70 及其亚单位p40）。细胞信号传导通路JNKMAPK的选择性抑制剂SP600125能促进ESAT-6刺激单核细胞IL-12p40产生,而信号通路PKR抑制剂2-AP有显著性抑制其作用。结论ESAT-6抗原诱导人THP-1单核细胞IL-12产生,JNK MAPK及PKR信号通路可能参与了此过程的调控。     

Immune regulatory activities of early secreted antigenic target of 6-kD protein of Mycobacterium tuberculosis and implications for tuberculosis vaccine design

Although ESAT-6 was originally identified as a strong T cell immunogen in short-term culture filtrate of Mtb, and has therefore been a candidate vaccine antigen for many years, recent work has demonstrated that ESAT-6 is also a virulence factor that mediates pathogenicity of Mtb. The studies described in this review suggest that ESAT-6 secreted by Mtb subverts host immunity by manipulating intracellular signaling pathways in macrophages and T cells, which are critical in protection against Mtb. Furthermore, ESAT-6 elicits pro-inflammatory responses that can be detrimental to the host. Understanding the molecular mechanisms through which ESAT-6 inhibits immunity will permit design of ESAT-6-based vaccine constructs that elicit protective immune responses with minimal negative effects.     

Key antigenic determinants in regulation of the immune response.

The immune response to beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23)is characterized by a wave of early help followed by a wave of suppression to a subsequent in vitro challenge with galactosidase-fluorescein. A cyanogen bromide peptide of beta-galactosidase, CB2, mimics the suppression seen with the enzyme. It is time dependent, carrier specific, and anti-theta sensitive; however, this suppression is not preceded by a wave of help. It is possible that CB2 cannot stimulate helpers, and is only able to activate suppressor cells. These data indicate that one small region of an antigen, capable of activating suppressors, can nullify the positive effect induced in helper T cells reactive with other epitopes on beta-galactosidase. Key determinants on macromolecules may in this way be influential in regulating the immune response to the entire antigen molecule.     

Antibody response to four secretory proteins from Mycobacterium tuberculosis and their complex antigen in TB patients.

OBJECTIVE: To evaluate antibody responses to a panel of four protein antigens secreted from Mycobacterium tuberculosis, CFP-21, ESAT-6, MPT-63 and MPT-64, and their complex antigen. DESIGN: Seventy-eight samples of serum and cerebral spinal fluid were tested by ELISA. RESULTS: Serum IgM antibody positive rates of protein antigens CFP-21, ESAT-6, MPT-63 and MPT-64 and their multi-antigen complex were respectively 34.6%, 60.3%, 52.6%, 78.2% and 96.2%; IgG antibody positive rates were respectively 46.2%, 64.1%, 93.6%, 57.7% and 84.6%. The specificity of the antibodies to the four proteins secreted from M. tuberculosis and its mixture antigen was 100%; sensitivity to the multi-antigen complex was higher than for the four single antigens. Among the IgM positive rates, the differences between the multi-antigen complex and CFP-21, ESAT-6, MPT-63 and MPT-64 were significant (P < 0.01); among the IgG positive rates, the differences between the multi-antigen complex and CFP-21, ESAT-6 and MPT-64 were significant (P < 0.01). Although the IgG positive rate for MPT-63 was higher than for the multi-antigen complex, the difference was not significantly different (P > 0.05). CONCLUSION: These findings indicate that the multi-antigen complex composed of CFP-21, ESAT-6, MPT-63 and MPT-64 may be useful in the diagnosis of TB.     

MTB分泌蛋白抗原85B-早期分泌靶抗原6融合蛋白对结核性胸膜炎的辅助诊断价值

目的 研究MTB分泌蛋白抗原85B(Ag85B)-早期分泌靶抗原6(ESAT6)融合蛋白在结核性胸膜炎辅助诊断中的价值。方法 研究对象为2014年1月至2015年1月于河北省胸科医院住院的结核性胸膜炎(结核组)患者45例,非结核性胸膜炎(非结核组)患者26例,以及体检健康人群(健康组)25名。应用流式细胞仪分别检测结核分枝杆菌Ag85B-ESAT6融合蛋白刺激前后结核组、非结核组外周血、胸腔积液及健康组外周血中γ干扰素(IFN-γ)的含量,采用免疫组织化学染色法检测结核组与非结核组患者组织(通过手术或穿刺活检获得的胸膜组织)中CD4 ⁺和CD8 ⁺细胞的表达水平。采用SPSS 13.0软件进行统计学处理,数据符合正态分布的组间比较采用t检验,以P<0.05为差异有统计学意义。 结果 MTB Ag85B-ESAT6融合蛋白刺激前后,结核组外周血中IFN-γ的含量分别为(42.63±10.51)pg/ml和(401.90±72.54)pg/ml;非结核组分别为(38.97±7.08)pg/ml和(40.04±6.80)pg/ml;健康组分别为(39.61±7.28)pg/ml和(39.86±6.97)pg/ml。结核组胸腔积液中IFN-γ的含量分别为(411.91±41.56)pg/ml和(1342.67±167.96)pg/ml;非结核组分别为(47.99±11.49)pg/ml和(48.76±11.25)pg/ml。刺激前结核组胸腔积液中IFN-γ的含量明显高于非结核组,两组比较差异有统计学意义(t=55.194,P=0.000);刺激后结核组外周血单个核细胞产生的IFN-γ含量相对于刺激前及刺激后的其他两组比较,差异均有统计学意义(t=32.879、33.211和33.204,P值均为0.000);刺激后结核组与刺激前及非结核组刺激前后胸腔积液中IFN-γ的含量比较,差异均具有统计学意义(t=36.085、51.478和51.499,P值均为0.000)。免疫组织化学染色结果显示结核组与非结核组患者胸膜组织中的CD4 ⁺和CD8 ⁺积累光密度值(结核组:16349.91±2376.36和10525.77±1164.86;非结核组:1853.64±670.40和1327.15±175.55)差异均具有统计学意义(t=14.381和19.127,P值均为0.000)。 结论 结核分枝杆菌Ag85B-ESAT6融合蛋白刺激外周血和胸腔积液后均明显提高结核特异性IFN-γ的含量,对结核性胸膜炎的辅助诊断有较高的临床应用价值。     

高度重视老年患者肺结核的早期诊断

老年肺结核有逐年增多趋势。本文通过对老年肺结核的临床症状中需要关注的重点问题、胸部影像学检查的价值及合理评价、结核病相关的化验检查及合并症情况等进行综合分析,提示老年肺结患者早期诊断困难,极易误诊、漏诊,临床上应高度重视。     

Cytolytic T cells in the immune response to mycobacterium tuberculosis

Cytolytic T cells (CTL) are of paramount importance in immune defense against tumors and viruses. Work over the past decade has revealed that lysis of infected cells is also involved in protective immunity to bacteria and parasites, including Mycobacterium tuberculosis. Experiments involving gene-deleted mice and the characterization of CTL lines derived from tuberculosis patients suggest an important role of CTL in immunity to tuberculosis. More recently, the identification of an effector pathway of human CTL provided evidence for direct antimicrobial activity of CTL. This pathway involves the combined action of the pore-forming perforin and the antibacterial granulysin, both expressed in the granules of CTL. Granulysin binds to the bacterial cell surface, thereby disrupting the membrane and causing osmotic lysis. The relevance of this pathway for protection against intracellular pathogens is suggested by the expression of high amounts of granulysin in tissue from patients with tuberculoid leprosy, which are able to contain the spread of the bacilli. These findings support the current concept of designing novel vaccination strategies which elicit not only CD4 + T helper cells, but also CD8 + CTL with direct antibacterial activity.     

Apoptosis modulation in mononuclear cells recovered from individuals exposed to Plasmodium falciparum infection

In endemic areas, asymptomatic infection by the malaria parasite Plasmodium falciparum was found associated with elevated percentages of human host's mononuclear cell spontaneous in-vitro apoptosis. In Dielmo, a village where malaria is holoendemic, apoptosis was age-and parasite-dependent. In-vitro exposure of peripheral blood mononuclear cells (PBMC) to the parasite extract induced a marked increase in the mononuclear cell membrane expression of functional CD95 antigen: a 3-h exposure of the mononuclear cells to anti-CD95 antibodies led to a detectable increase in the mean percentage of apoptotic nuclei found in the cultures carried out in the presence of P. falciparum extracts compared to control cultures. IL-2, IL-4, IL-6 and IL-10 promoted the viability of PBMC in cultures while IL-1alpha or IFN-gamma had no obvious impact and TNFalpha gave conflicting results. IL-2 was the most efficient cytokine at rescuing PBMC from cell death and this effect was associated with a strong increase in T cell activation. In contrast, IL-4 and IL-10 had no such effect on T cell activation, hence they acted as survival factors and not through their mitogenic activity. Taken together, these different observations suggested that the levels of in-vitro apoptosis observed were not only associated with parasite infection, but also potentially modulated by the human host through different pathways.     

Production of secretory immunoglobulin A in rat self-filling blind loops. Local secretory immunoglobulin A immune response to luminal bacterial flora

The immunoglobulin A (IgA) response to small intestinal bacteria was studied in rats with self-filling blind loops (SFBLs), surgically constructed in continuity with the intestine or at the end of a Roux-en-Y loop (RY-SFBL) so as to avoid filling with chyme. Total bile salt in the RY-SFBL lumen was much lower (4.35 +/- 0.8 mumol) than in the SFBL lumen (116 +/- 15 mumol), but other parameters, such as the number of anaerobic bacteria and disaccharidase activities were similar. Within 1 wk of establishing the blind loops, they had accumulated at least 14 times as much IgA as found in the normal jejunum. Luminal IgA per milligram mucosal protein was almost as high in the RY-SFBL as in the SFBL, indicating that a significant proportion of the IgA must be nonbiliary and probably mucosal in origin. Oral treatment with lincomycin significantly reduced luminal IgA accumulation in the RY-SFBL. Column chromatography and enzyme-linked immunosorbent assay (ELISA), which employed antirat secretory component antibody, established that the majority of the luminal IgA was nonmonomeric and complexed with secretory component. Centrifugation of luminal contents to separate soluble and particulate bound IgA showed that the RY-SFBL contained a higher proportion of precipitable IgA than either the SFBL or lincomycin-treated RY-SFBL. Immunoglobulin A eluted from the precipitates by KSCN was bound to a greater extent by bacterial sonicates than IgA in the supernatant. For either precipitate or supernatant IgA, the greatest binding was observed when the IgA was obtained from the RY-SFBL. These observations indicate that rat intestinal mucosa rapidly responds to bacterial overgrowth by secreting secretory immunoglobulin A (sIgA) with specificity for luminal bacterial antigens. As the sIgA present within the SFBL is to a certain extent derived from bile, the lower proportion of SFBL sIgA bound by bacterial antigens than of RY-SFBL sIgA suggests that biliary sIgA is less specific for local antigens than the sIgA that is secreted by the local mucosa.     

Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.     

Incipient and subclinical tuberculosis: defining early disease states in the context of host immune response

Latent Mycobacterium tuberculosis infection (LTBI) and active tuberculosis (TB) are 2 ends of a spectrum of states ranging from asymptomatic infection to overt disease. While progressing from LTBI to TB, patients often undergo asymptomatic states with detectable manifestations indicative of disease. Such asymptomatic disease states frequently remain undiagnosed, and their manifestations and duration are mostly dependent on host immune response. Various terms referring to such states are used in the literature, often interchangeably and without explicit definitions. Defining these intermediate states in concrete terms is important for pragmatic reasons, as they might impact upon the diagnostic performance of TB biomarkers and could also present targets for therapeutic interventions. We here propose definitions for 2 commonly used terms, "incipient" and "subclinical" TB, to describe asymptomatic disease states occurring at opposite ends of the host response spectrum. We propose using the term "incipient TB" when referring to early, contained disease in asymptomatic, relatively immunocompetent persons. In contrast, we propose using the term "subclinical TB" to refer to disease in asymptomatic, immunocompromised individuals in whom it is largely associated with loss of effective containment. The rationale for this article is to facilitate the discussion of such early disease states, especially in relation to their impact on TB biomarker discovery and assessment of new diagnostics, and with regard to treatment decisions and ultimately outcome.     

Spectrum of immune response abnormalities in different clinical forms of tuberculosis.

In an attempt to explain the reasons for the development of different clinical forms of tuberculosis in different persons, their immunologic status was compared to their clinical patterns. The spectrum of immunologic abnormalities correlated with the clinical forms. Also, an inverse relationship between cell-mediated and humoral immune responses was observed. Immunologic abnormalities reverted to normal concomitant with clinical improvement on chemotherapy, suggesting that the abnormalities were the result of the illness rather than its cause. Malnutrition could have been the underlying factor for the immunologic deficiencies seen in some patients.     

Reconstitution of immune responses to tuberculosis in patients with HIV infection who receive antiretroviral therapy

STUDY OBJECTIVE:s: To assess the restoration of immune responses to tuberculosis, as manifested by secretion of T-helper type 1 cytokines (interferon [IFN]-gamma, interleukin [IL]-12, and IL-2) and T-helper type 2 cytokines (IL-10), in HIV-positive patients who receive antiretroviral therapy (ART). DESIGN: Prospective cohort study. SETTING: University hospital. PATIENTS: Ten HIV-positive patients, all na?ve to ART and all about to start ART for clinical indications, and 11 healthy, HIV-negative control subjects. INTERVENTIONS: Assessment of T-cell proliferation and cytokine production after administration of ART to patients with HIV infection. MEASUREMENTS AND RESULTS: All patients had a negative tuberculin skin test result at baseline and were anergic. Highly active ART reduced the viral load to very low levels in all patients within a short time after starting therapy. Blood samples were drawn every 2 months after starting therapy, and continued for 1 year while the patients continued to receive ART. There were trends toward increased proliferation of peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium tuberculosis-specific stimuli, but these were delayed until several months of ART had elapsed. Similar trends were noted in relation to the secretion of IFN-gamma. Neither PBMC proliferation nor IFN-gamma secretion reached levels seen in healthy control subjects. No consistent trends in IL-2, IL-10, or IL-12 production were noted. CONCLUSION: ART restores immune responses to M tuberculosis, although this restoration is delayed and does not reach levels seen in healthy, HIV-negative control subjects. These results may explain in part the phenomenon of paradoxic reactions to antituberculosis therapy in patients with HIV infection. A larger study in which patients are followed up for a longer period of time will allow the magnitude and timing of this reconstitution to be more precisely defined.     

Severe immune hemolytic anemia in disseminated tuberculosis with response to antituberculosis therapy

Severe hemolytic anemia in patients with disseminated tuberculosis is exceedingly rare. We report an episode of Coombs'-positive hemolytic anemia in a previously healthy young man with miliary tuberculosis, resulting in a hemoglobin level of 5 g/dL and an undetectable haptoglobin level. The patient responded well to treatment with antituberculosis drugs, and the results of the direct Coombs' test became negative without the need of blood transfusion or steroid therapy.