Martins Marcia Valéria B S, Lima Mônica Cristina B S, Duppre Nadia C, Matos Haroldo J, Spencer John S, Brennan Patrick J, Sarno Euzenir N, Fonseca Leila, Pereira Geraldo M B, Pessolani Maria Cristina V
Leprosy Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.
Tuberculosis (Edinb). 2007 May;87(3):202-11. doi: 10.1016/j.tube.2006.07.006. Epub 2006 Oct 13.
There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome sequence allowing selection of the best candidates for new diagnostics (including new skin tests), and vaccines for leprosy and tuberculosis.
目前尚无可靠方法检测亚临床分枝杆菌感染。近期对多个分枝杆菌基因组进行测序，为开发病原体特异性诊断检测带来了新机遇，这在麻风病和结核病防控方面至关重要。在本研究中，我们应用多参数流式细胞术分析来研究T细胞功能，以确定可衡量既往分枝杆菌暴露情况的免疫标志物。我们比较了5名PPD阳性和5名PPD阴性健康志愿者对PPD（用于测量既往结核分枝杆菌暴露情况的金标准皮肤试验试剂）的体内反应，以及白细胞活化的体外参数。在全血中计数PPD刺激后表达CD4、CD69、皮肤淋巴细胞相关抗原（CLA）和细胞内IFN-γ的外周白细胞，并与PPD刺激的外周血单个核细胞上清液中通过ELISA测量的体内PPD诱导硬结大小和IFN-γ产生水平进行比较。对结核菌素皮肤试验（TST）的反应性与PPD反应性活化（CD69+）和产生IFN-γ的CD4+T细胞频率显著增加相关。检测PPD特异性产生IFN-γ的白细胞仅限于CD4+T细胞，且这些细胞的一个亚群显示表达皮肤归巢分子CLA。对PPD反应的多元线性回归模型显示，皮肤试验硬结与PPD反应性产生IFN-γ的CD4+CD69+T细胞频率之间的相关性最高。我们的数据表明，体外计数抗原特异性产生IFN-γ的CD4+T细胞可为检测潜伏性结核分枝杆菌感染提供一种替代体内结核菌素试验的方法。此外，测量这些免疫参数可有助于筛选由基因组序列定义的新的特异性抗原，从而选择新诊断方法（包括新的皮肤试验）和麻风病及结核病疫苗的最佳候选物。
