廣東住血線蟲造成嗜伊紅性腦膜炎之臨床及致病機轉研究

Clinical and pathogenetic study of eosinophilic meningitis caused by Angiostrongylus cantonensis

蔡宏津(Hung-Chin Tsai)

指導教授 ： 陳瑩霖 ； 顏全敏

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

摘要

本研究想進一步了解人體感染廣東住血線蟲引起嗜伊紅性腦膜炎之臨床病變及致病機轉，在1998，1999 及2001年我們診療三個群突發，因感染廣東住血線蟲引起嗜伊紅性腦膜炎共22個病人，我們採取了三種不同的治療方法，包括8例合併用類固醇及mebendazole，9例用保守止痛藥療法，及5例用類固醇(prednisolone 60mg) 兩週治療，三組均有不錯的療效。其中在第二次及第三次群突發中，我們使用的世代研究(cohort study)定期追蹤病人CSF/serum及腦部磁振造影檢查，發現了腦部磁振造影T1-WEIGHTED 影像在 basal ganglion及大腦角(cerebral peduncle) 上有 increased intensity 的現象，且與病人的臨床症狀及腦髓液異常有正相關，這是ㄧ個全新的發現。另外在第三次群突發中我們首次證實生機飲食精力湯中隱藏感染廣東住血線蟲症之風險，雖沒有找到蟲體證實但是統計學有明顯相關。
在致病機轉我們首先想了解血管內皮生長因子(vascular endothelial growth factor，VEGF) 在廣東住血線蟲嗜伊紅性腦膜炎所扮演角色。在我們9個泰勞的世代研究中所有病人僅接受止痛劑及或glycerol治療。每一個病人接受完整之理學、神經學、眼科檢查，在入院時及住院中每個均接受腰椎穿刺腦脊髓液檢查直到出院， 90％病人在初入院時腦脊髓液VEGF的量有增加現象，至於血清中VEGF的量在6個月追蹤期則呈上下波動(fluctuation)，腦脊髓液VEGF濃度與腦脊髓液蛋白質濃度、白血球數及嗜伊紅性球數均呈正相關(p＜0.05，Pearson correlation test)，這些結果顯示VEGF在嗜伊紅性腦膜炎扮演重要的角色。另一方面我们為了要評估九號基質金屬蛋白分解酵素(MMP-9)及基質金屬蛋白抑制成份(TIMPs)，如何在廣東住血線蟲引起之嗜伊紅性腦膜炎病人上影響血腦屏障，我們分析了40個病人及28個對照組的腦脊髓液發現病人組的MMP-2，MMP-9，TIMP-1及CSF/serum白蛋白比值(QAlb)顯著比對照組增加，但是TIMP-4在病人組卻顯著降低。我們以Gelatin zymography來偵測基質金屬蛋白分解酵素的蛋白分解能力(proteolytic activity)，發現只有9號基質金屬蛋白分解酵素(相對於2號基質金屬蛋白分解酵素)，在病人的腦脊髓液中具有強烈的蛋白分解能力。我們亦觀察到在病人的腦脊髓液中對廣東住血線蟲的抗體值愈高，同時QAlb及MMP-9的值也愈高，同時QAlb的增加與病人腦脊髓液中MMP-9的增加呈現正相關。當腦脊髓液中MMP-9增加時，我們臨床上亦觀察到腦脊髓液中，白血球數有增加現象。我們追蹤了6個病人一系列的腦脊髓液變化，發現在恢復期時QAlb、MMP-9及TIMP-1會漸漸減少，相反的TIMP-4卻是漸漸增加。這些結果顯示嗜伊紅性腦膜炎腦脊髓液中之MMP-9的來源可能自週邊血液遷移(migration)至中樞神經系統，至於TIMP-4在恢復期增高的現象，需要進一步研究證實。

關鍵字

廣東住血線蟲 ； 嗜伊紅性腦膜炎 ； 血腦屏障 ； 血管內皮生長因子 ； 基質金屬蛋白分解酵素 ； 腦部磁振造影 ； 基質金屬蛋白分解酵素抑制劑

並列摘要

Angiostrongylus cantonensis is the most common cause of eosinophilic meningitis worldwide. Human infection occurs following ingestion of the worms in raw snails or fish that serve as intermediate hosts. Three outbreaks of central nervous system infection with A. cantonensis occurred in Kaoshiung, Taiwan during 1998, 1999 and 2001 among 17 Thai laborers who ate raw snails and 5 native Taiwanese who drank raw vegetable juice. We performed a retrospective cohort study to enhance case findings with eosinophilic meningitis, as well as environmental surveillance of larvae in snails and testing for antibodies of A. cantonensis in the blood and cerebrospinal fluids. We enrolled 22 patients with severe headache and eosinophilia within 4 to 90 days after eating raw snails and drinking raw vegetable juice. Sixteen (73 %) developed eosinophilic meningitis. Third stage larvae were found in the cerebrospinal fluids of 2 patients and 12/12 snails. Specific antibodies were detected in serum and cerebrospinal fluids. The severity of illness and eosinophilia were associated with amount of snails ingested. Eighteen of twenty-two of these cases underwent magnetic resonance imaging (MRI) of the brain. MRI imaging revealed high signal intensities over the globus pallidus on TI-weighted imaging, leptomeningeal enhancement, ventriculomegaly and punctate areas of abnormal enhancement within the cerebral and cerebellar hemisphere on gadolinium enhancing T1 imaging, and a hyperintense signal on T2-weighted images. There was a significant correlation between severity of headache, CSF pleocystosis and CSF and blood eosinophilia with MRI signal intensity in T1-weighted imaging (p <0.05). Among the five native Taiwanese who drank raw vegetable juice, risk factors identification showed that consumption of raw vegetable juice was associated with illness (Pearson correlation test r = 0.867, P= 0.01). There was association between the presence of raw vegetable juice and CSF eosinophilia (Spearman’s correlation test r=0.816, P= 0.004).
Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor and a mediator of brain edema. To assess the role of VEGF in eosinophilic meningitis, VEGF was measured in the cerebrospinal fluid (CSF) and blood of 9 patients with eosinophilic meningitis in a cohort study. VEGF CSF was detected in 8 (90%) of 9 eosinophilic meningitis patients (range, 45–2190 pg/mL) at presentation. The mean VEGF CSF at presentation, one week and two weeks after admission was 568 pg/mL, 751 pg/mL and 1031 pg/mL, respectively. There was an association between VEGFCSF, CSF protein, white cell count, and eosinophil counts. The VEGFSERUM fluctuated during the 6-month follow up period. These results indicate that VEGF may be associated with blood-brain barrier disruption in patients with eosinophilic meningitis.
Our another study is designed to evaluate the possibility of blood-brain barrier (BBB) dysfunction caused by matrix metalloproteinase-9 (MMP-9) and its regulation by tissue inhibitors of metalloproteinase (TIMPs) in patients with eosinophilic meningitis caused by the infection with A. cantonensis, 40 patients and 28 controls were included in this study. Concentrations of MMP-2, MMP-9, TIMP-1 and CSF/serum albumin ratio (QAlb values) are significantly increased in patients than those in controls, however, TIMP-4 is significantly lower in patients. Contrarily to MMP-2, proteolytic activity of MMP-9 detected by gelatin zymography was only observed in patients with eosinophilic meningitis. It was observed that the higher the antibody levels specific to A. cantonensis in CSF of patients with eosinophilic meningitis, the higher the QAlb values and MMP-9 concentrations in CSF of patients. Furthermore, the increase in QAlb value was significantly correlated with the increase of MMP-9 in patients. In parallel with CSF MMP-9, patients also showed an increase of CSF white blood cell counts. Gradual decrease levels of QAlb, MMP-9 and TIMP-1 and increase levels of TIMP-4 were observed in 6 patients during recovery from eosinophilic meningitis. These results propose that the source of MMP-9 in CSF of patients with eosinophilic meningitis was probably associated with leukocytes migrating from peripheral blood to CSF. The activity of MMP-9 in CSF of patients could not completely inhibited because of the decrease of TIMP-4 which had the chance to cause BBB dysfunction as proven by the higher QAlb values found in patients.
In conclusion, BBB dysfunction may occur in patients with eosinophilic meningitis due to the infection of A. cantonensis because patients show significantly higher QAlb which significantly correlates with the concentrations of MMP-9 in CSF of patients. And also VEGF may be associated with blood-brain barrier disruption in patients with eosinophilic meningitis. The increase of MMP-9 in CSF of patients probably derives from leukocytes because they appeared a large number in CSF. Although TIMP-1 increased in the CSF of patients to neutralize the MMP-9, the proteolytic activity of MMP-9 still showed high levels. The reason why TIMP-4 in CSF of patients suddenly steeply dropped in the acute phase of eosinophilic meningitis and its role in BBB dysfunction of this parasitic disease should be clarified in further studies.

並列關鍵字

Angiostrongylus cantonensis ； blood brain barrier ； eosinophilic meningitis ； inhibitors of matrix metalloproteinase ； matrix metalloproteinase ； MRI ； vascular endothelial growth factor ； zymography

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