INVESTIGATION OF A POSSIBLE ASSOCIATION BETWEEN MYCOPLASMA SPECIES AND ASTHMA

迄今为止，39例慢性稳定型哮喘患者和11例对照者接受了上下气道评估和血清学分析，以确定是否存在M。pneumoniae、C.通过培养、酶联免疫测定（EIA）和聚合酶链反应（PCR）检测肺炎克雷伯氏菌和7种呼吸道病毒。哮喘患者随后以双盲方式随机接受克拉霉素500 mg每日两次或安慰剂治疗6周，并重复进行气道和血清学分析。M.在20/39例哮喘患者和1/11例对照中通过PCR检测到肺炎链球菌（p=0.007）。在20例患者中的13例中，在支气管肺泡灌洗（BAL）和/或支气管活检中检测到微生物。此外，7/39例哮喘患者和0/11例对照者C. pneumoniae的PCR产物（p<0.05）。所有患者的培养、EIA和血清学均为M阴性。肺炎。所有培养物均为C阴性。所有受试者的呼吸道病毒EIA均为阴性。18例哮喘患者和1例对照组血清C.肺炎（p= 0.02）。方案治疗组的分析显示，克拉霉素治疗组的1秒用力呼气量预测值百分比（FEV 1）从2.65 ± 0.12 L改善至2.73 ± 0.11 L（p=0.015）。用力肺活量（FVC）也从4.05 1 0.13 L增加至4.19 1 0.13 L，p=0.02。在安慰剂组中未观察到FEV 1或FVC的显著变化（FEV 1：2.81 1 0.19 L至2.73 1 0.14 L，p=0.58; FVC：4.35 1 0.21至4.44 1 0.34 L，p=0.56）。当M.肺炎支原体PCR阳性与阴性受试者接受克拉霉素治疗后，比较两组受试者的FEV 1。肺炎PCR阳性受试者从2.79 ± 0.19 L增加至2.97 ± 0.23 L，p=0.04。分枝接受克拉霉素治疗的肺炎克雷伯氏菌PCR阴性受试者未出现此类改善（2.23 ± 0.19 L至2.34 ± 0.21 L，p=0.51）。此外，M.肺炎球菌阳性受试者表现出BAL淋巴细胞减少，并且这趋向于显著性（1.42 ± 0.38 × 103至0.77 ± 0.13 × 103，p=0.06）。当受试者M. pneumoniae或C.对接受克拉霉素的肺炎患者进行了评价，他们的FEV 1也从2.52 ± 0.19 L增加到2.78 ± 0.22 L（p=0.03）。这种显著的增加在M阴性的受试者中并不明显。pneumoniae或C.肺炎并接受克拉霉素（FEV 1：2.49 ± 0.32 L至2.62 ± 0.24，p=0.24）。因此，这些观察结果继续支持M.慢性稳定型哮喘患者的下呼吸道中存在肺炎克雷伯氏菌，其频率高于对照组。此外，一些哮喘受试者也表现出C.肺炎，这也可能有助于哮喘的发病机制。最后，那些表现出M。pneumoniae或C.上呼吸道和下呼吸道中的肺炎克雷伯氏菌对克拉霉素的反应似乎比对任一种微生物阴性的人更好。第3年的计划包括通过免疫组织化学和原位杂交对特定目标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价。还将对支原体和衣原体阳性和阴性受试者进行评价，以确定这些受试者与非定植/感染哮喘患者相比的局部免疫应答。具体而言，将确定T细胞（CD 3、CD 4、CD 8）、活化T细胞（CD 25）和感觉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（具体目的3）。此外，还将评价已知参与哮喘发病机制的细胞因子（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).