INVESTIGATION OF A POSSIBLE ASSOCIATION BETWEEN MYCOPLASMA SPECIES AND ASTHMA

迄今为止，39例慢性、稳定哮喘患者和11例对照患者接受了上、下气道评估和血清学分析，通过培养、酶联免疫测定（EIA）和聚合酶链反应（PCR）确定肺炎支原体、肺炎支原体和7种呼吸道病毒的存在。然后，哮喘患者随机接受克拉霉素500毫克，每日两次或安慰剂治疗，为期6周，双盲方式，重复气道和血清学分析。肺炎支原体PCR检出率为20/39，对照组为1/11 （p=0.007）。20例患者中有13例在支气管肺泡灌洗（BAL）和/或支气管活检中检测到该菌。另外，7/39例哮喘患者和0/11例对照组的PCR检测结果均为肺炎原体阳性（p<0.05）。所有患者的培养、eia和血清学均为肺炎支原体阴性。所有受试者的培养均为肺炎原体阴性，所有呼吸道病毒的eia均为阴性。18例哮喘患者和1例对照组肺炎原体血清学阳性（p= 0.02）。该方案治疗组的分析显示，克拉霉素治疗组的预测用力呼气量（FEV1）百分比从2.65 1.0.12 L提高到2.73 1.0.11 L （p=0.015）。用力肺活量（FVC）由4.05 1 0.13L增加至4.19 1 0.13L, p=0.02。安慰剂组FEV1和FVC无显著变化（FEV1: 2.81 1 0.19 L ~ 2.73 1 0.14 L, p=0.58; FVC: 4.35 1 0.21 ~ 4.44 1 0.34 L, p=0.56）。与使用克拉霉素的肺炎支原体PCR阳性组和阴性组比较，PCR阳性组的FEV1由2.79 1 0.19 L上升至2.97 1 0.23 L, p=0.04。肺炎支原体PCR阴性受试者接受克拉霉素治疗后，无明显改善（2.23 1 0.19 L ~ 2.34 1 0.21 L, p=0.51）。此外，肺炎支原体阳性受试者BAL淋巴细胞减少，且有显著性趋势（1.42 1 0.38 × 103 ~ 0.77 1 0.13 × 103, p=0.06）。当接受克拉霉素治疗的肺炎支原体或肺炎原体呈阳性的受试者进行评估时，他们的FEV1也从2.52 1 0.19 L增加到2.78 1 0.22 L （p=0.03）。肺炎支原体或肺炎支原体检测均为阴性并接受克拉霉素治疗的受试者（FEV1: 2.49 1 0.32 L至2.62 1 0.24,p=0.24）没有发现这种显著增加。因此，这些观察结果继续支持肺炎支原体存在于慢性、稳定哮喘患者的下气道的假设，其频率高于对照组。此外，一些哮喘患者也表现出肺炎原体PCR阳性，这也可能与哮喘的发病机制有关。最后，那些在上呼吸道和下呼吸道表现出肺炎支原体或肺炎支原体存在的受试者对克拉霉素的反应似乎比那些两者均阴性的受试者更好。第三年的计划包括通过免疫组织化学和原位杂交对参与特定目的1和2的受试者的气道组织和支气管肺泡灌洗液细胞进行持续评估。还将对支原体和衣原体阳性和阴性受试者进行评估，以确定与未定植/感染哮喘患者相比，这些受试者的局部免疫反应。具体来说，将确定T细胞（CD3、CD4、CD8）、活化T细胞（CD25）和参与感染过程的细胞因子（肿瘤坏死因子- α [tnf - α]、白细胞介素2 [IL-2]、IL-6和干扰素- γ）的存在（特异性目的3）。此外，还将评估已知参与哮喘发病的细胞因子（IL-3、IL-4、IL-5和粒细胞-巨噬细胞集落刺激因子）的存在。

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).