INVESTIGATION OF A POSSIBLE ASSOCIATION BETWEEN MYCOPLASMA SPECIES AND ASTHMA

到目前为止，39例慢性哮喘缓解期哮喘患者和11例对照组接受了上、下呼吸道评估和血清学分析，以确定是否存在肺炎支原体、肺炎衣原体和7种呼吸道病毒，方法是培养、酶联免疫分析(EIA)和聚合酶链式反应(PCR)。然后，哮喘患者随机服用克拉霉素500 mg，每天两次，或以双盲方式服用安慰剂6周，并进行重复呼吸道和血清学分析。39例哮喘患者中有20例检出肺炎支原体，11例正常对照中检出1例肺炎支原体(p=0.007)。在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细菌。此外，7/39名哮喘患者和0/11名对照的肺炎衣原体聚合酶链式反应均为阳性(p&lt；0.05)。所有患者的培养、EIA和血清学均为肺炎支原体阴性。所有受试者的肺炎衣原体培养均为阴性，呼吸道病毒的所有EIA均为阴性。18名哮喘患者和1名对照组肺炎衣原体血清学检测呈阳性(p=0.02)。对方案治疗臂的分析显示，克拉霉素组1秒用力呼气容积(FEV1)预测百分比从2.651±0.12 L提高到2.73±0.11 L(p=0.015)。用力肺活量(FVC)由4.05±0.13升至4.19±0.13升，P=0.02。安慰剂组FEV1和FVC无明显变化(FEV1：2.81±0.19 L比2.73±0.14 L，p=0.58；FVC：4.35±0.21比4.44±0.34 L，p=0.56)。比较接受克拉霉素治疗的肺炎支原体聚合酶链式反应阳性和阴性患者，肺炎支原体聚合酶链式反应阳性患者的FEV1由2.79±0.19 L增加到2.97±0.23 L，P=0.04。接受克拉霉素治疗的肺炎支原体聚合酶链式反应阴性的受试者没有这样的改善(2.23±0.19 L比2.34±0.21 L，p=0.51)。此外，肺炎支原体阳性者的BAL淋巴细胞减少，有统计学意义(1.42±0.38×103比0.77±0.13×103，p=0.06)。当接受克拉霉素治疗的肺炎支原体或肺炎支原体阳性的受试者接受评估时，他们的FEV1也从2.521±0.19 L增加到2.78±0.22 L(p=0.03)。在肺炎支原体或肺炎衣原体阴性并接受克拉霉素治疗的受试者中，这种显著的增加并不明显(FEV1：2.49±0.32 L对2.62±0.24，p=0.24)。因此，这些观察结果继续支持肺炎支原体存在于慢性稳定期哮喘患者下呼吸道的假设，其频率比对照组更高。此外，几名哮喘受试者也表现出肺炎衣原体的聚合酶链式反应阳性，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出现肺炎支原体或肺炎支原体的受试者似乎比那些两者都阴性的受试者对克拉霉素的反应更好。第三年的计划包括继续通过免疫组织化学和原位杂交对参与特定目标1和2的受试者的呼吸道组织和支气管肺泡灌洗细胞进行评估。还将对支原体和衣原体阳性和阴性受试者进行评估，以确定这些受试者与未定居/感染的哮喘患者的局部免疫反应。具体地说，将确定T细胞(CD3、CD4、CD8)、激活的T细胞(CD25)和感觉参与感染过程的细胞因子(肿瘤坏死因子-α[TNF-α]、白介素2[IL-2]、白介素6和干扰素-γ)的存在(具体目标3)。此外，还将评估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).