The Effect of IFN-gamma on the Phagocytosis of Mycobctcterium tuberculosis and Activation of Human Pulmonary Alveolar Macrophage / 결핵

BACKGROUND: IFN-gamma is known to activate mononuclear phagocytes and to mediate host defense mechanism against some intracellular microorganisms, but litfle is known about anti-mycobacterial activity and mechanism of IFN-gamma in human. In this study, we investigated the role of IFN-gamma in the pathogenesis of tuberculosis by observing the effect of IFN-gamma on the phagocytosis of M.tuberculosis(MTB) and on the production of TNF-alpha by human pulmonary alveolar macrophage. METHOD: Pulmonary alveolar macrophage(PAM) were prepared with adhesion purification method from bronchoalveolar lavage fluid obtained from 8 persons without active lung lesion and cultured(1 x 106cells/ml) with MTB(3 x 107 bacteria/ml) with or without IFN-gamma(300U/ml), LPS(0.5ug/ml) and autologous serum(10%). After 2 hours, the percentage of PAM-phagocytosed MTh was counted after AFB staining(modified Kynion method). TNF-alpha production by PAM stimulated by IFN-gamma(300U/ml), MTB(1 x l06bacteria/ml) and LPS(0.5ug/ml) for 24hours was measured in culture supernatant using ELISA method. The degree of phagocytosis of MTh by PAM stimulated with IFN-gamma(300U/ml) and LPS(0.5ug/ml) for 24hours was also investigated. RESULTS: IFN-gamma did not influence the phagocytosis of MTB by PAM(percentage of PAM-phagocytosed MTB: control: 22.1+/- 4.9, IFN-gamma: 20.3+/- 5.3) and did not increase TNF-alpha production by PAM(controfl 21+/-38pg/ml, IFN-gamma : 87+/-106pg/ml), and the degree of phagocytosis of MTh by PAM pre-stimulated with IFN-gamma for 24 hours, was not increased (controL 24.5+/-9.5, IFN-gamma : 23.4+/-10.1). CONCLUSION: IFN-gamma does not influence on the phagocytosis of MTB and TNF-alpha production by PAM.

The Comparison of Methods Processing Cells Recovered by Bronchoalveloar Lavage / 결핵

BACKGROUND: The total and differential cell count of bronchoalveolar lavage(BAL) fluid are useful assessing activity, prognosis and response to therapy in diffuse interstitial lung disease. But controversy exist as to the appropriate method in processing BAL fluid. Therefore we investigated the effect of gauze filtration, centrifugation and different storage time of BAL fluid on the total and differential cell count. METHOD: We obtained BAL fluid from 6 persons with no active lung lesion and divided pooled BAL fluid into several siliconized glass tubes and filtered through 0,1, 2, 4 folds of cotton guaze(pore size:lmm), and compared total cell count using hemocytometer after trypan blue staining and differential cell count after Wright-Giemsa staining of cytocentrifuged preparations. And we also counted total and differential cell count after centrifugation(400g for 30 mm) and various storage time(2hr, 24hr, and 48hr). RESULTS: There was no difference in total and differential cell count according to folds of gauze filtraion. But without gauze filtration, mucus threads that hampered total and differential cell count were found in 2 cases (33%). Centrifugation resulted in loss of total cell count(24+/-18%) without change in differential cell count. There was no change in total cell count after 21w storage but significant cell loss was found after 241w storage time(24hr : 28+/-21%, 48hr: 41+/-24%). However there was no change in differential cell count with 48hr storage time. CONCLUSION: Total and differential cell count of BAL fluid may be best performed after cotton gauze filtration without centrifugation and within 2 hours.

Phagocytosis of Drug-Resistant Mycobacterium Tuberculosis by Peripheral Blood Monocytes / 결핵

BACKGROUND: Phagocytosis is probably the first step for mycobacteria to be virulent in host because virulent strains are more readily phagocytosed by macrophage than attenuated strains. According 13 the traditional concept, multi-drug resistant strains have been regarded as less virulent. However, this concept has been challenged, since recent studies(reported) showed that the degree of virulence and drug-resistance is not related. The purpose of this study is to evaluate whether the phagocytic activity of M. tuberculosis by peripheral blood mononuclear cells(PBMC) is different according to drug-resistance or host factor. To evaluate this, we estimated the difference of phagocytic activity of drug-resistant and drug-sensitive M. tuberculosis and also estimated the phagocytic activity of PBMC from intractable tuberculosis patients and healthy controls. METHODS: PBMC from ten intractable tuberculosis patients and twelve healthy control and three different strains of heat-killed M. tuberculosis, ie, ADS(all drug sensitive), MDR(multi-drug resistant), and ADR(all drug resistant) were used. After incubation of various strains of M. tuberculosis with PBMC, the phagocytic activity was evaluated by estimating proportion of PBMC which have phagocytosed M. tuberculosis. RESULTS: Drug-resistant strains of M. tuberculosis were phagocylosed easily than drug sensitive strains(Percentage of PBMC phagocytosed M. tuberculosis in healthy control : ADS : 32.3α2.9%, ADR : 49.6α3.4%, p=0.0022, Percentage of PBMC phagocytosed M. tuberculosis in intractable tuberculosis patients : ADS : 34.9α3.6%, ADR : 50.7α4.5%), p=0.0069). However, there was no difference in phagocytic activity of PBMC from healthy control and intractable tuberculosis patients. CONCLUSION: Drug-resistant strains of M. tuberculosis were phagocytosed easily than drug sensitive strains and host factors does not seems to influence the phagocytosis of M. tuberculosis.