聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

Stimulation of breathing by activation of pulmonary peripheral afferents in rabbits

Respiratoryresponse to selective activation of vagal afferents in the peripheralairways was investigated in anesthetized, open-chest, and artificiallyventilated rabbits. Phrenic activity was used as an index of centralrespiratory drive before and after injection of hypertonic saline(8.1%, 0.1 ml) into the periphery of the lung to stimulate theafferents. The amplitude of "integrated" phrenic activity andphrenic burst rate increased by 19 ± 3.4 and 53.7 ± 12.7%(n = 23;P < 0.001),respectively. The response peaked at 5.5 ± 1.6 s andreturned to the baseline at 7 min (median) after the injection. Themagnitude of the response was positively related to the concentrationof injected NaCl. The response could not be elicited by injection ofnormal saline and was abolished by vagotomy. Because artificialventilation caused phrenic activity to be entrained with theventilator, respiratory drive was further assessed after the ventilatorwas stopped. Again, neural hyperpnea and tachypnea were observed.Because activation of a small fraction of the pulmonary peripheralafferents resulted in vigorous stimulation of respiratory drive, wespeculate that initiation of this reflex may contribute to hyperpneaand tachypnea under both physiological and pathophysiologicalconditions.

     

Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-gamma in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242-250, Ag85b199-207, early secreted antigenic target 6 (ESAT-6)28-36, 19-kDa Ag88-97, or the HLA-DR-presented ESAT-6(1-20) epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-6(1-20) could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.     

Experimental pulmonary candidiasis in modified rabbits

Pulmonary lesions induced by an intratracheal inoculation of Candida albicans into rabbits in untreated control, bovine serum albumin (BSA)-sensitized and C. albicans-sensitized groups were examined immunohistochemically to clarify the localization of IgG, IgM and C3. In the control group no inflammatory cells were immunoreactive for IgG and only a few macrophages for IgM and C3, whereas in the BSA- and C. albicans-sensitized groups there were a small number of IgG-positive polymorphonuclear leukocytes and IgM- and C3-positive macrophages in the lesions, the latter group being more prominent. Furthermore, epithelioid granulomatous lesions at the late stage in the C. albicans-sensitized animals showed scattered epithelioid cells containing IgG as well as abundant IgG- and IgM-positive plasma cells. These immunohistochemical results were considered to support the estimation that immune complexes contributed to the modification of fungal lesions in the C. albicans-sensitized hosts, although non-immunological defense mechanisms seemed to be more important in the elimination of the fungus.