Faculty Bibliography

The interaction of Mycobacterium tuberculosis (MTB) with the immune system is mediated by cytokine and chemokine responses of macrophages and/or dendritic cells. Chemokine (C-C motif) ligand 18 (CCL18) and interleukin (IL)-10 are major factors secreted by phagocytes, postulated to recruit naive T lymphocytes and inhibit pro-inflammatory cells. Our study investigated the role of CCL18 and IL-10 in an in vitro model of infection by MTB in human macrophages. CD14(+) monocytes, obtained from the peripheral blood of eight healthy donors, differentiated in monocyte-derived macrophages (MDM) with monocyte-colony stimulating factor (100 ng/ml) for 6 days, were stimulated in vitro with lipopolysaccharide (LPS) (1 microg/ml) and with heat killed MTB Hv37Ra (multiplicity of infection 1:5) for 24 h. Alveolar macrophages from five healthy donors were infected with MTB Hv37RA. CCL18 protein and mRNA were detected by enzyme-linked immunosorbent assay (ELISA) and real-time PCR, IL-10 levels by ELISA. Stimulation of MDM with LPS or MTB led to a significant increase in CCL18 protein (control 2.67 +/- 0.46 ng/ml, LPS 4.05 +/- 0.56 ng/ml, with MTB 6.70 +/- 1.59 ng/ml, n = 5, P < 0.05) and specific mRNA levels (control 0.09 +/- 0.01, LPS 0.24 +/- 0.11, with MTB 0.34 +/- 0.08 CCL18/Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), n = 3, P < 0.05). A significant increase of the production of CCL18 was observed in infected alveolar macrophages. IL-10 levels increased from 38.52 +/- 26.38 pg/ml in control cells to 1129.32 +/- 235.00 and 974.25 +/- 164.46 pg/ml in LPS and MTB treated cells, respectively (P < 0.05). Up-regulation of CCL18 and IL-10 in macrophages by MTB may be involved in the recruitment of naive T cells in association with local suppressive immunity against intracellular pathogens. This could represent a mechanism of tolerance during the early phases of infection

Estimating the burden of exposure-related diffuse lung disease in terms of health effects and economic burden remains challenging. Labor statistics are inadequate to define the scope of the problem, and few studies have analyzed the prevalence of exposure-related illnesses and the subsequent health care cost. Well-defined exposures, such as those associated with coal mines, asbestos mines, and stonecutting, have led to more accurate assessment of prevalence and cost. As governmental regulation of workplace exposure has increased, the prevalence of diseases such as silicosis and coal workers' pneumoconiosis has diminished. However, the health and economic effects of diseases with long latency periods, such as asbestosis and mesothelioma, continue to increase in the short term. Newer exposures, such as those related to air pollution, nylon flock, and the World Trade Center collapse, have added to these costs. As a result, estimates of cost for occupational diseases, including respiratory illnesses, exceed $26 billion annually, and the true economic burden is likely much higher

Pulmonary tuberculosis may alter volatile organic compounds (VOCs) in breath because Mycobacteria and oxidative stress resulting from Mycobacterial infection both generate distinctive VOCs. The objective of this study was to determine if breath VOCs contain biomarkers of active pulmonary tuberculosis. Head space VOCs from cultured Mycobacterium tuberculosis were captured on sorbent traps and assayed by gas chromatography/mass spectroscopy (GC/MS). One hundred and thirty different VOCs were consistently detected. The most abundant were naphthalene, 1-methyl-, 3-heptanone, methylcyclododecane, heptane, 2,2,4,6,6-pentamethyl-, benzene, 1-methyl-4-(1-methylethyl)-, and cyclohexane, 1,4-dimethyl-. Breath VOCs were assayed by GC/MS in 42 patients hospitalized for suspicion of pulmonary tuberculosis and in 59 healthy controls. Sputum cultures were positive for Mycobacteria in 23/42 and negative in19/42 patients. Breath markers of oxidative stress were increased in all hospitalized patients (p<0.04). Pattern recognition analysis and fuzzy logic analysis of breath VOCs independently distinguished healthy controls from hospitalized patients with 100% sensitivity and 100% specificity. Fuzzy logic analysis identified patients with positive sputum cultures with 100% sensitivity and 100% specificity (95.7% sensitivity and 78.9% specificity on leave-one-out cross-validation); breath VOC markers were similar to those observed in vitro, including naphthalene, 1-methyl- and cyclohexane, 1,4-dimethyl-. Pattern recognition analysis identified patients with positive sputum cultures with 82.6% sensitivity (19/23) and 100% specificity (18/18), employing 12 principal components from 134 breath VOCs. We conclude that volatile biomarkers in breath were sensitive and specific for pulmonary tuberculosis: the breath test distinguished between 'sick versus well' i.e. between normal controls and patients hospitalized for suspicion of pulmonary tuberculosis, and between infected versus non-infected patients i.e. between those whose sputum cultures were positive or negative for Mycobacteria

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from approximately 80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis

Quality assurance (QA) is essential for data accuracy and proper evaluation of study objectives in clinical trials. The Tuberculosis Trials Consortium (TBTC)-a collaboration of 28 clinical sites and the Centers for Disease Control and Prevention-has developed a comprehensive QA program that provides quantitative assessments of performance based on clearly defined standards that are communicated to data collectors through a feedback process. The Implementation and Quality Committee of the TBTC developed a Site Evaluation Report (SER) that assesses performance measures (PMs) critical to the accomplishment of study objectives. PMs are defined, quantified, and evaluated, and goals and minimum acceptable scores are specified. Sites not meeting a PM minimum must provide an explanation and develop a plan to meet the goal. Site-specific and system-wide problems can be readily identified through this process. The SER is used prospectively for all TBTC treatment trials, and a Web site has been developed to maximize the availability and usefulness of performance data. The TBTC's comprehensive QA program is an example of a successful method for ensuring high quality, evaluable data

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung

Tuberculosis leads to immune activation and increased human immunodeficiency virus type 1 (HIV-1) replication in the lung. However, in vitro models of mycobacterial infection of human macrophages do not fully reproduce these in vivo observations, suggesting that there are additional host factors. Surfactant protein A (SP-A) is an important mediator of innate immunity in the lung. SP-A levels were assayed in the human lung by using bronchoalveolar lavage (BAL). There was a threefold reduction in SP-A levels during tuberculosis only in the radiographically involved lung segments, and the levels returned to normal after 1 month of treatment. The SP-A levels were inversely correlated with the percentage of neutrophils in BAL fluid, suggesting that low SP-A levels were associated with increased inflammation in the lung. Differentiated THP-1 macrophages were used to test the effect of decreasing SP-A levels on immune function. In the absence of infection with Mycobacterium tuberculosis, SP-A at doses ranging from 5 to 0.01 micro g/ml inhibited both interleukin-6 (IL-6) production and HIV-1 long terminal repeat (LTR) activity. In macrophages infected with M. tuberculosis, SP-A augmented both IL-6 production and HIV-1 LTR activity. To better understand the effect of SP-A, we measured expression of CAAT/enhancer binding protein beta (C/EBPbeta), a transcription factor central to the regulation of IL-6 and the HIV-1 LTR. In macrophages infected with M. tuberculosis, SP-A reduced expression of a dominant negative isoform of C/EBPbeta. These data suggest that SP-A has pleiotropic effects even at the low concentrations found in tuberculosis patients. This protein augments inflammation in the presence of infection and inhibits inflammation in uninfected macrophages, protecting uninvolved lung segments from the deleterious effects of inflammation

STUDY OBJECTIVES: Aerosol interferon-gamma (IFN-gamma) is a potential immunomodulator in the treatment of pulmonary tuberculosis (TB). Previous investigations demonstrated conversion of sputum smears in five patients with multidrug-resistant TB after 12 treatments over 1 month, and induction of signaling molecules in 10 of 11 drug-sensitive TB patients using BAL. The objective of the current study was to evaluate particle size and deposition pattern in patients with TB receiving aerosol IFN-gamma treatment. DESIGN: Particle size was determined with a cascade impactor, and deposition of IFN-gamma mixed with (99m)Tc-labeled human serum albumin was assessed using a gamma camera. Local levels of IFN-gamma were measured in BAL using enzyme-linked immunosorbent assays. Study patients/intervention: Fourteen patients with pulmonary TB received IFN-gamma aerosol (500 micro g) for 12 treatments in addition to antimycobacterial therapy with BAL before and after IFN-gamma aerosol treatment. Eight patients with minimal-to-moderate parenchymal involvement underwent deposition studies. Deposited (99m)Tc-labeled IFN-gamma aerosol was partitioned between upper airways and lungs using attenuation correction measurements. (133)Xe equilibrium scanning, (133)Xe washout, and (99m)Tc- macroaggregate injection defined regional lung volume, ventilation, and perfusion. RESULTS: Upper airway deposition was significant often exceeding lung deposition (53.9 +/- 7.09 micro g vs 35.8 +/- 2.73 micro g, respectively [mean +/- SE]). IFN-gamma levels measured in BAL fluid were significantly increased with aerosol treatment (0.83 +/- 0.43 micro g before vs 24.76 +/- 8.71 micro g after, p </= 0.017), and IFN-gamma levels correlated with regional deposition of IFN-gamma aerosol (r = 0.823). Four-quadrant analysis of regional lung deposition best correlated with regional perfusion (r = 0.422, p = 0.013) with penetration of aerosol into areas of obvious radiographic infiltration on chest radiograph. CONCLUSIONS: Aerosol therapy with IFN-gamma in patients with pulmonary TB is widely distributed and results in significant enhancement of IFN-gamma levels in the lower respiratory tract. In patients without lung destruction, IFN-gamma aerosol may be an adjuvant to enhance the local immune response