Angiocentric pulmonary granulomas in tuberculosis

An open-lung biopsy performed in a 15-year-old girl because of left sided pulmonary nodules revealed striking angiocentric necrotising granulomas. No acid fast bacilli (AFB) were cultured or demonstrated in tissue sections, however, the diagnosis of tuberculosis was suggested. Anti-tuberculous therapy resulted in both clinical and radiological cure within 12 months. The differential diagnosis of this type of lesion should include pulmonary tuberculosis as well as the non-infective angiocentric granulomas such as lymphomatoid granulomatosis and has major therapeutic implications.     

Immunopathogenesis of pulmonary granulomas in the guinea pig after infection with Mycobacterium tuberculosis

Pulmonary tuberculosis in guinea pigs is similar to the disease in humans and is accordingly widely used as a model to test tuberculosis vaccines. The primary site of expression of acquired immunity and the hallmark of tuberculosis is the granuloma. Granuloma morphology is well described, but there is limited information regarding T-cell subset influx. We monitored the course of pulmonary tuberculosis in guinea pigs and observed four distinct immunohistopathological stages. In all stages there were similar numbers and arrangement of CD4 and CD8 T cells. There were only small numbers of apoptotic lymphocytes, scattered around and within the necrotic core, and acid-fast bacilli were visible both within macrophages and free within airway debris. A key finding of the study was the observation that the development of the necrotic core was an early event and almost certainly preceded the emergence of the acquired immune response. This in turn suggests that innate mechanisms are the basis of the early lesions and that subsequent acquired responses are unable to moderate them. This hypothesis differs from the current dogma that excessive activity of T cells mediates delayed-type hypersensitivity and that cellular cytolysis is the root cause of the necrosis.     

肺结核病与非结核分枝杆菌肺病互相继发4例报告并文献复习

目的分析非结核分枝杆菌(Nontuberculous Mycobacteria,NTM)肺病及肺结核病与NTM肺病互相继发(或并发)的临床表现特点,提高认识,减少误诊。方法收集4例肺结核病与NTM肺病互相继发病例的有关资料,并文献资料一起分析。结果①NTM肺病的性别分布男多于女,发病年龄多见于40岁以上;②主要症状:咳嗽、咳痰、咯血(或血痰)、发热,③病程长,最长达50年,④多数患者有慢性病史,⑤X线胸片表现两侧达68%、单侧30%、空洞54%、胸膜累及40%,⑦对异烟肼(H)、利福平(R)、链霉素(S)、乙胺丁醇(E)的耐药性分别为:95%、73%、79%和74%。结论NTM肺病多见于有慢性病史,病程长,以咳嗽、咳痰、咯血(或血痰)、发热为主要症状,X线胸片,病灶分布两侧为多、多有空洞并累及胸膜,对常用的抗痨药物有高度耐药性;而患者病情较重与患肺结核或NTM肺病后容易互相继发(或并发)有一定关系。     

Magnetic resonance imaging of pulmonary lesions in guinea pigs infected with Mycobacterium tuberculosis

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines.     

Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension

The hypothesis for this study was that increased local expression of vascular angiotensin-converting enzyme (ACE) may contribute to the arterial remodelling which accompanies pulmonary hypertension, since angiotensin II (ANG II) is an important mediator of pulmonary vascular cell growth. The expression of ACE was studied by immunohistochemistry in paraffin-embedded lung sections from adults undergoing heart-lung transplantation for severe primary (n=6) and secondary (n=7) pulmonary arterial hypertension (PH), compared with age-matched controls (n=11). An antigen retrieval technique was used prior to incubating sections with the anti-ACE monoclonal antibody, CG2, or the endothelial marker, monoclonal anti-CD31. In control lungs, the highest level of ACE immunostaining was seen in the alveolar capillary endothelium, with less intense staining in small intra-acinar pulmonary arteries and relatively little staining in larger preacinar arteries. ACE immunostaining was virtually absent in lymphatics and veins. In both primary and secondary PH, there was an increase in ACE immunostaining in the endothelium of intra-acinar peripheral pulmonary arteries compared with control lungs, extending to the level of alveolar ducts, as confirmed by semi-quantitative analysis. The increase in endothelial ACE expression in the intra-acinar arteries of patients with primary and secondary PH is consistent with the hypothesis that locally increased production of ANG II may contribute to the process of pulmonary vascular remodelling.     

Cytokine profile of t lymphocytes from peripheral blood and bronchoalveolar lavage fluid in patients with active pulmonary tuberculosis

The possible immunological relationship between the pattern of Th1/Th2 cytokine production and tuberculin reactivity was assessed in patients with active Mycobacterium tuberculosis infection. The production of the intracellular cytokines interferon (IFN)-gamma and interleukin-4 (IL-4) was measured in CD4(+) and CD8(+) T cells obtained from peripheral blood and bronchoalveolar lavage fluid (BALF) of 20 tuberculin skin-positive patients and compared with the findings recorded in nine tuberculin skin-negative patients with active pulmonary tuberculosis. Upon stimulation with phorbol 12-myristate acetate/ionomycin for 6 h, tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in BALF than in peripheral blood, while both CD4(+) and CD8(+) T-lymphocyte subsets in BALF of tuberculin-positive patients secreted more IFN-gamma than their peripheral blood counterparts. Tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in peripheral blood than tuberculin-positive patients. There was no significant difference in the production of IFN-gamma by BALF CD4(+) T lymphocytes, or by either peripheral blood or BALF CD8(+) T lymphocytes. In two tuberculin-negative patients, peripheral blood CD4(+) T lymphocytes produced IL-4. Study results suggested a higher immune activity in the blood of tuberculin-negative patients, with an increased lymphocyte activity in BALF versus peripheral blood in both patient groups.     

Experimental production of pulmonary granulomas: III. Plasma-cell granulomas in mice

Endobronchial instillation of Freund's complete adjuvant (FCA) induced plasma-cell granulomas in the lungs of DD strain mice. One week after administration, plasma cells appeared in monolayered cell clusters around pulmonary arterioles running along the bronchial tree. The plasma cells increased in number with time to form granulomas consisting mainly of well developed plasma cells with an admixture of lymphocytes and macrophages. At the peak of production of granulomas (4-10 weeks), the plasma cells piled up around the arterioles and infiltrated the connective tissues surrounding the bronchioles. Germinal-centre-like foci could be demonstrated in the granulomas. The lymph nodes draining the treated lobe showed marked proliferation of plasma cells in the medullary cord, suggesting that the plasma cells passed through alveolar walls into the lung and migrated to periarteriolar connective tissue.     

Cytokine polarization in miliary and pleural tuberculosis

Cytokines were measured in patients with pleural effusion and miliary tuberculosis (TB). Patients with pleural effusion had significantly higher interferon-gamma (IFN-) levels (P < 0.001) in their pleural fluid as compared to that of peripheral blood of the same patients, thus exhibiting localization of predominantly Th1-type immunity in the pleural fluid. On the contrary, patients with miliary TB had higher IFN- levels in the peripheral blood as compared to their bronchoalveolar lavage fluid. Moreover, the median IFN-:IL-4 ratio in the peripheral blood of miliary TB patients was two-fold higher as compared to bronchoalveolar lavage fluid, suggesting that the cytokine profile at the disease site is skewed toward a Th2-like bias. Further, flow cytometry data revealed a significantly higher (P < 0.001) percentage of CD4⁺ pleural fluid lymphocytes expressing IFN-, whereas in the miliary TB, a nine-fold higher percentage of lymphocytes in bronchoalveolar lavage fluid expressed IL-4 in comparison with their peripheral CD4 T cells. Our data indicate, respectively, a Th1-like and Th2-like response in tuberculous pleural effusion and miliary TB, suggesting that these clinical forms of extrapulmonary tuberculosis probably reflect the extreme ends of a Th1–Th2 spectrum of the disease.     

Cytokine and lipid mediator networks in tuberculosis

A major approach for immunologic intervention in tuberculosis involves redirecting the outcome of the host immune response from the induction of disease to pathogen control. Cytokines and lipid mediators known as eicosanoids play key roles in regulating this balance and as such represent important targets for immunologic intervention. While the evidence for cytokine/eicosanoid function derives largely from the investigation of murine and zebrafish experimental infection models, clinical studies have confirmed the existence of many of the same pathways in tuberculosis patients. Here, we summarize new data that reveal important intersections between the cytokine and eicosanoid networks in the host response to mycobacteria and discuss how targeting this crosstalk can promote resistance to lethal Mycobacterium tuberculosis infection. This approach could lead to new host-directed therapies to be used either as an adjunct for improving the efficacy of standard antibiotic treatment or for the management of drug-resistant infections.     

Evolution of granulomas in lungs of mice infected aerogenically with Mycobacterium tuberculosis

Aerogenous infection of C57Bl/6 mice with a virulent strain of Mycobacterium tuberculosis (CL 511) leads to the formation of primary granulomas in the lung where neutrophils, macrophages and subsequently, lymphocytes accumulate progressively around an initial cluster of infected macrophages. The spread of infection through the lung parenchyma gives rise to secondary granulomas featuring numerous lymphocytes that surround a small number of infected macrophages. Afterwards, foamy macrophages add an outer layer to the granulomas, which characteristically respect the pulmonary interstitium and remain confined within the alveolar spaces. This feature, in conjunction with the constant presence of M. tuberculosis in the products of broncho-alveolar lavage, suggests that the upward bronchial migration of infected macrophages may contribute significantly to pulmonary dissemination of mycobacterial infection. The latter would be in agreement with the persistence of chronic pulmonary infection in spite of a concomitant strong T helper 1 cell response.     

Experimental production of pulmonary granulomas: III. Plasma-cell granulomas in mice.

Endobronchial instillation of Freund''s complete adjuvant (FCA) induced plasma-cell granulomas in the lungs of DD strain mice. One week after administration, plasma cells appeared in monolayered cell clusters around pulmonary arterioles running along the bronchial tree. The plasma cells increased in number with time to form granulomas consisting mainly of well developed plasma cells with an admixture of lymphocytes and macrophages. At the peak of production of granulomas (4-10 weeks), the plasma cells piled up around the arterioles and infiltrated the connective tissues surrounding the bronchioles. Germinal-centre-like foci could be demonstrated in the granulomas. The lymph nodes draining the treated lobe showed marked proliferation of plasma cells in the medullary cord, suggesting that the plasma cells passed through alveolar walls into the lung and migrated to periarteriolar connective tissue.     

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-alpha, IL6 and TGF-beta, IL10) or (interferon [IFN]-gamma and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1-2, whereas for PHA (predominantly T-cell-derived), they were on days 3-5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student's t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 (p=0.023) and IL10 (p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-gamma (p=0.028) and IL5 (p=0.012) secretion but increased IL10 (p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-gamma (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.     

Bone tuberculosis in a patient with pulmonary tuberculosis

A case of bone tuberculosis in 83 years old farmer was described. He was hospitalized because of pain, oedema and purulent fistula in carpal regio. He hadn't neither systemic symptoms neither from respiratory tract. Chest X-ray and X-ray of carpal bones revealed tuberculous lesions. In the pus from cutaneous fistula AFB were found in culture. Antituberculous treatment was successful.     

In situ study of abundant expression of proinflammatory chemokines and cytokines in pulmonary granulomas that develop in cynomolgus macaques experimentally infected with Mycobacterium tuberculosis

Tuberculosis remains a major public health problem worldwide. Chemokines and cytokines organize and direct infiltrating cells to sites of infection, and these molecules likely play crucial roles in granuloma formation and maintenance. To address this issue, we used in situ hybridization (ISH) to measure chemokine and cytokine mRNA expression levels and patterns directly in lung tissues from cynomolgus macaques (Macaca fascicularis) experimentally infected with a low dose of virulent Mycobacterium tuberculosis. We examined more than 300 granulomas and observed abundant expression of gamma interferon (IFN-gamma)-inducible chemokine mRNAs (CXCL9/monokine induced by IFN-gamma, CXCL10/IFN-gamma-inducible protein, and CXCL11/IFN-gamma-inducible T-cell alpha-chemoattractant) within solid and caseous granulomas, and there was only minimal expression in nongranulomatous regions of tissue. The mRNA expression patterns of IFN-gamma and tumor necrosis factor alpha were examined in parallel, and the results revealed that cytokine mRNA(+) cells were abundant and generally localized to the granulomas. Mycobacterial 16S rRNA expression was also measured by ISH, and the results revealed that there was localization predominantly to the granulomas and that the highest signal intensity was in caseous granulomas. We observed several granulomatous lesions with exceptionally high levels of RNA for mycobacterial 16S rRNA, IFN-gamma, and IFN-gamma-inducible chemokines, suggesting that the local presence of mycobacteria is partially responsible for the upregulation of IFN-gamma-inducible chemokines and recruitment of CXCR3(+) cells, which were also abundant in granulomatous lesions. These results suggest that expression of CXCR3 ligands and the subsequent recruitment of CXCR3(+) cells are involved in granuloma formation and maintenance.     

Altered Cytokine Production and Impaired Antimycobacterial Immunity in Protein-Malnourished Guinea Pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β), in response to PPD, despite the demonstration of higher serum levels of TNF-α and TGF-β after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-β 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Activated B cells in the granulomas of nonhuman primates infected with Mycobacterium tuberculosis

In an attempt to contain Mycobacterium tuberculosis, host immune cells form a granuloma as a physical and immunological barrier. To date, the contribution of humoral immunity, including antibodies and specific functions of B cells, to M. tuberculosis infection in humans remains largely unknown. Recent studies in mice show that humoral immunity can alter M. tuberculosis infection outcomes. M. tuberculosis infection in cynomolgus macaques recapitulates essentially all aspects of human tuberculosis. As a first step toward understanding the importance of humoral immunity to control of M. tuberculosis infection in primates, we characterized the B-cell and plasma-cell populations in infected animals and found that B cells are present primarily in clusters within the granuloma. The B-cell clusters are in close proximity to peripheral node addressin-positive cells and contain cells positive for Ki-67, a proliferation marker. Granuloma B cells also express CXCR5 and have elevated HLA-DR expression. Tissues containing M. tuberculosis bacilli had higher levels of M. tuberculosis-specific IgG, compared with uninvolved tissue from the same monkeys. Plasma cells detected within the granuloma produced mycobacteria-specific antibodies. Together, these data demonstrate that B cells are present and actively secreting antibodies specific for M. tuberculosis antigens at the site of infection, including lung granulomas and thoracic lymph nodes. These antibodies likely have the capacity to modulate local control of infection in tissues.