To date, thirty-nine asthmatics with chronic, stable asthma and 11 controls underwent evaluation of the upper and lower airways and serologic analysis to determine the presence of M. pneumoniae, C. pneumoniae and seven respiratory viruses through culture, enzyme-linked immunoassay (EIA) and polymerase chain reaction (PCR). Asthmatics were then randomized to clarithromycin at 500 mg twice daily or placebo for six weeks in a double-blind fashion with repeat airway and serologic analysis performed. M. pneumoniae was detected by PCR in 20/39 asthmatics and 1/11 controls (p=0.007). In 13 of the 20 patients, the organism was detected in bronchoalveolar lavage (BAL) and/or bronchial biopsies. In addition, 7/39 asthmatics and 0/11 controls were positive for C. pneumoniae by PCR (p<0.05). All patients' cultures, EIAs, and serology were negative for M. pneumoniae. All cultures were negative for C. pneumoniae and all EIAs for respiratory viruses were negative in all subjects. Eighteen asthmatics and one control exhibited positive serology for C. pneumoniae (p= 0.02). Analysis of the treatment arm of the protocol revealed an improvement in the percentage predicted forced expiratory volume in one second (FEV1) from 2.65 1 0.12 L to 2.73 1 0.11 L (p=0.015) in the clarithromycin treated group. The forced vital capacity (FVC) also increased from 4.05 1 0.13L to 4.19 1 0.13L, p=0.02. No significant change in FEV1 or FVC was seen in the placebo group (FEV1 : 2.81 1 0.19 L to 2.73 1 0.14 L, p=0.58; FVC: 4.35 1 0.21 to 4.44 1 0.34 L, p=0.56). When the M. pneumoniae PCR positive and negative subjects who received clarithromycin were compared, the FEV1 of the M. pneumoniae PCR positive subjects increased from 2.79 1 0.19 L to 2.97 1 0.23 L, p=0.04. The M. pneumoniae PCR negative subjects who received clarithromycin did not experience such an improvement (2.23 1 0.19 L to 2.34 1 0.21 L, p=0.51). In addition, the M. pneumoniae positive subjects exhibited a reduction in BAL lymphocytes, and this trended toward significance (1.42 1 0.38 x103 to 0.77 1 0.13 x 103, p=0.06). When subjects who were positive for either M. pneumoniae or C. pneumoniae who received clarithromycin were evaluated, they also experienced an increase in their FEV1 from 2.52 1 0.19 L to 2.78 1 0.22 L (p=0.03). This significant increase was not appreciated in the subjects who were negative for either M. pneumoniae or C. pneumoniae and received clarithromycin (FEV1: 2.49 1 0.32 L to 2.62 1 0.24, p=0.24). Thus, these observations continue to support the hypothesis that M. pneumoniae was present in the lower airways of chronic, stable asthmatics with greater frequency than controls. In addition, several asthmatic subjects also exhibited PCR positivity for C. pneumoniae, which may also be contributing to the pathogenesis of asthma. Finally, those subjects who exhibited presence of M. pneumoniae or C. pneumoniae in the upper and lower airways appeared to respond better to clarithromycin than those who were negative for either organism. Plans for year 3 include continued evaluation of airway tissue and bronchoalveolar lavage cells via immunohistochemistry and in situ hybridization from subjects partipating in specific aims 1 and 2. Evaluation of mycoplasma and chlamydia postive and negative subjects will also be performed to determine the local immune response in those subjects as compared to non-colonized/infected asthmatics. Specifically, the presence of T cells (CD3, CD4, CD8), activated T cells (CD25) and cytokines felt to be involved in the infectious process (tumor necrosis factor-alpha [TNF-alpha], interleukin 2 [IL-2], IL-6 and interferon-gamma will be determined(specific aim 3). In addition, the presence of cytokines known to be involved in asthma pathogenesis will also be evaluated (IL-3, IL-4, IL-5 and granulocyte-macrophage colony stimulating factor).
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