Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease最新文献

Setting: Alveolar macrophages (AM), a heterogeneous cell population, play a critical role in eliminating mycobacterial infections in collaboration with lymphocytes. Patients with diabetes mellitus (DM) show increased susceptibility to pulmonary tuberculosis (TB) infection. It is still uncertain whether there is a defect in T cell or AM activation in patients with DM against TB infection.

Objective: To study the difference in activation status of AM and T cells between patients with TB+DM and TB alone.

Method: The heterogeneity of AM from 14 patients with TB+DM, 9 with TB alone, 10 normal subjects and 8 DM alone patients, was studied using Percoll density fractionation. The intracellular H₂O₂production of AM before and after stimulation with phorbol myristate acetate (PMA) or F-Met-Leu-Phen (FMLP) was assayed by loading cells with 2′,7′-dichlorofluorescin (DCFH) and analyzed by flow cytometry. Lymphocytes subsets (CD3, CD4, CD8) and their activation status (CD25) in bronchoalveolar lavage were also measured.

Results: The proportion of the least dense AM (<1.030g/ml) and the magnitude of DCFH oxidation of AM was higher in TB patients than in normal subjects, regardless of DM. Patients with TB+DM had a significantly lower proportion of the least density AM fraction than TB alone patients, regardless of disease extent. Among TB patients, the proportion of the least dense AM was inversely correlated with the bacterial load on sputum and the disease extent on chest radiograph. Stimulation of AM with PMA or FMLP induced an increase in the hypodense AM subpopulations and enhanced intracellular H₂O₂generation in patients with TB+DM and to a similar extent in normal subjects, but not in patients with TB alone. There was no significant difference in CD3 numbers, CD4/CD8 ratio, and CD25+ cells between patients with TB alone and TB+DM. The activation status of AM or T lymphocytes from DM alone patients was not significantly different from those from normal subjects.

Conclusion: Hypodense subpopulations of AM increase in active TB patients and are related to the disease severity as well as activation status of AM. AM in TB patients complicated with DM was less activated, and may be contributory to the susceptibility to mycobacterial infection.

The original Mycobacterium bovis Bacillus Calmette Gué rin vaccine strain has developed into several different substrains which have been used for production of BCG vaccines throughout the world since 1921. Based on the latest genetic and antigenic knowledge, as well as the early literature reports on BCG vaccination, we are able to fit the different pieces of the BCG puzzle together and outline the origin of the different substrains of M. bovis BCG. The BCG vaccine substrains analysed demonstrate two distinct patterns, with an abrupt change consisting of a loss of several genes and altered biochemical characteristics in strains originating from Institut Pasteur after 1927. Further evidence from the literature is provided that a change occurred in virulence of the BCG parent strain at Institut Pasteur in the late 1920s. Based on this information a genealogical tree is proposed and discussed.

Setting: Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients.

Objective: To elucidate the role of HLA-DR genes/gene products on lymphocyte responses toMycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease.

Design: Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects.

Results: Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 μg/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects.

Conclusion: The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response againstM. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects.

Setting: The proportions and absolute cell count of γ/δ T-lymphocytes in the peripheral blood of patients with pulmonary tuberculosis (PTB) remains controversial. Since PTB is an infectious airway disease, bronchoalveolar T-lymphocytes should be a better indicator of local immune T-cell reaction after TB infection than peripheral blood T-lymphocytes.

Objective: To quantitate the absolute cell count and proportions of γ/δ T-lymphocytes in the bronchoalveolar lavage fluid (BALF) of patients with active PTB.

Design: Bronchoalveolar lavage (BAL) and analysis of lymphocytes in the BALF was performed in 25 patients with active PTB and 16 normal controls. All of the patients were negative for HIV infection and none was immunocompromized. BALF and blood were prepared for cell differential count and flow cytometry analysis using monoclonal antibodies CD3, CD4, CD8, CD25, HLA-DR and γ/δ as well as α/β T-lymphocyte receptors.

Results: The number of cells per volume of recovered BALF was significantly higher in the patients with active PTB than in normal controls. BALF from active PTB patients also showed increased percentage of lymphocytes and neutrophils. The absolute number of total lymphocytes, CD3⁺lymphocytes and CD3⁺γ/δ T-lymphocytes were significantly higher in the BALF, but not in the blood, of patients with TB, however, the proportions of CD3⁺γ/δ T-lymphocytes in BALF of patients with TB was comparable to that of normal controls. γ/δ T-lymphocytes in the BALF rarely expressed CD4, CD25, and HLA-DR in both groups.

Conclusion: These results suggest that γ/δ T-lymphocytes are not the major subpopulation of CD3⁺lymphocytes in the BALF that react to mycobacterial infection in the patients with clinically established active TB.

In this study we examined the mechanisms of resistance to rifampin (RMP) and isoniazid (lNH) in 352 clinical isolates of Mycobacterium tuberculosis from Sevilla, Spain, using three different molecular methods: 1) PCR-single strand polymorphism analysis; 2) the commercial system Inno-LiPA RTB for RMP resistance; and 3) sequence analysis. Resistance to RMP was found in 21 strains, where the following mutations in the rpoB gene were detected: Ser₅₃₁→ Leu (n =14 strains); His₅₂₆→ Asp (n =3), Asn₅₁₈→ Ser (n =1), Gln₅₁₃→ Leu (n =1) and a nine nucleotide deletion (n =1). Resistance to INH occurred in 29 strains, with mutations observed in: a) kat G gene: Ser₃₁₅→ Thr (n =12), lle₃₀₄→ Val (n =1), and a partial deletion (n =4); b) regulatory region of the inh A gene: nucleotide substitution C209T (n =3). No mutation was found in the ahp C promoter.

Mycobacterium smegmati 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, biosafety regulations often require the handling of these experiments at Biosafety Level 3. However, we failed to find any evidence that theM. 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.

Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many industrial countries. The diagnosis of tuberculosis depends primarily on identification of mycobacteria and on clinicoradiological evidence of the disease. Compared to other diagnostic methods, serological tests are faster and do not necessarily require samples that contain the tubercle bacilli. We have evaluated a modified version of a commercially available enzyme immunoassay test to detect the presence of circulating anti-mycobacterial IgG and IgM antibodies in tuberculosis patients. The sensitivity and the specificity of the test reaches 87% and 95% respectively.

In conclusion, the modified Anda-TB enzyme immunoassay test offers a good and reliable test for diagnosis of tuberculosis in suspected cases of active pulmonary tuberculosis.

Setting: A deer model has been developed to study protection produced with BCG vaccination, against infection and the development of pathology, following experimental intratonsilar infection with virulent Mycobacterium bovis.

Objective: To determine how the dose of vaccine, the route of vaccination, the viability of the vaccine and exposure to glucocorticoids at the time of vaccination, may affect the protective efficacy of BCG vaccines.

Design: Deer were vaccinated with BCG and later challenged with virulent M. bovis via the tonsilar route. Protection against infection and development of disease was evaluated at necropsy six months after challenge withM. bovis , by histological examination and microbial culture.

Results: Significant protection against infection and disease were obtained following boosting with two low doses (5 × 10⁴cfu) or moderate doses (5 × 10⁷cfu) of live (freshly cultured and lyophilized) BCG. Inferior levels of protection were obtained with high dose (5 × 10⁸cfu) of live BCG. Similar levels of protection were found with vaccines given subcutaneously or via the tonsilar route. Killed vaccine in a mineral-oil adjuvant did not evoke protective immunity and treatment with dexamethasone prior to vaccination with live BCG ablated its efficacy. Protection against infection did not correlate with skin test delayed type hypersensitivity (DTH) or lymphocyte transformation to tuberculin.

Conclusions: Two doses of live BCG gave significant protection against experimental infection and disease caused by virulent M. bovis. Single dose vaccine protected against disease but not infection. Vaccines administered at a dosage which did not evoke DTH, provided protection against tuberculosis infection and disease.

The development of novel vaccines for use in the prevention and immunotherapy of tuberculosis is an area of intense interest for scientific researchers, public health agencies and pharmaceutical manufacturers. Development of effective anti-tuberculosis vaccines for use in specific target populations will require close cooperation among several different international organizations including agencies responsible for evaluating the safety and effectiveness of new biologics for human use. In this review, the major issues that are addressed by regulatory agencies to ensure that vaccines are pure, potent, safe, and effective are discussed. It is hoped that the comments provided here will help accelerate the development of new effective vaccines for the prevention and treatment of tuberculosis.