Difference in gut mucosal immunity in multiple sclerosis and myasthenia gravis and the safety in oral tolerance induction.

本研究为期2年，旨在建立一个7天的外周血单个核细胞（PBMNC）培养系统，PBMNC可对卵清蛋白（OVA）、牛血清白蛋白（BSA）、κ-酪蛋白（k-C）和牛γ-球蛋白（BGG）等膳食蛋白抗原产生增殖反应。如果用于检查肠道粘膜免疫的简单筛选试验在多发性硬化症（MS）和重症肌无力（MG）患者中变得可行，则该试验在正常条件下引起针对通过肠道摄取的抗原的全身免疫中的低反应性，该方法将有助于选择新治疗策略的最佳候选者的患者，即，口服耐受性结果表明：1.培养PBMNC的最适抗原浓度为50和100 mg/ml。氚标记的胸苷掺入在最后16个小时的PBMNC与饮食抗原的7天培养被用来作为一个措施的增殖反应，每种抗原。用这种方法不仅在MS患者中发现了对OVA和BGG的反应，而且在MG患者和健康对照者中也发现了对OVA和BGG的反应。后两组对BSA无反应，而对k-C的高反应性可能代表了对MS特异性的肠粘膜免疫。2.细胞培养7天后，通过胸苷掺入测定的增殖反应与CD 4 ^+ CD 26 ^+记忆辅助性T细胞的扩增相关。为了研究细胞因子水平与上述结果之间的关系，将每种抗原的培养上清液储存在-70 ℃下。3.通过使用匙孔血蓝蛋白（KLH）证明，测量人类受试者口服耐受的KLH特异性前体细胞频率是监测口服耐受诱导的最灵敏的方法，并且给出了可重复的结果。

This 2-year project aimed at establishing a 7-day culture system for peripheral blood mononuclear cells (PBMNC) which proliferate in response to dietary protein antigens such as ovalbumin (OVA), bovine serum albumin (BSA), kappa-casein (k-C), and bovine gamma-globulin (BGG). If a simple screening test for checking gut mucosal immunity, which under the normal condition renders induction of hyporesponsiveness in systemic immunity against an antigen taken up through the gut, becomes feasible among patients with multiple sclerosis (MS) and myasthenia gravis (MG), this approach would be of help to select patients who are the best candidates of a new treatment strategy, i.e., oral tolerance. The following results were obtained.1.The optimum concentrations of dietary antigens in culturing PBMNC were 50 and 100mg/ml. Tritium-labelled thymidine incorporation during the final 16 hours of a 7-day culture of PBMNC with dietary antigens was used as a measure of proliferative response to each antigen. With this method responses to OVA and BGG were found not only in patients with MS but also in those with MG and healthy controls. There was no response to BSA in the latter two groups, whereas high responsiveness to k-C may be representative of the gut mucosal immunity specific to MS.2.A proliferative response as measured by thymidine incorporation correlated with an expansion of CD4^+CD26^+ memory helper T cells after seven days of cell culture. In order to investigate the relationship between cytokine levels and aforementioned results, the culture supernatants for each antigen were stored at -70ﾟC.3.It was proved by using keyhole limpet hemocyanin (KLH) that measurement of precursor cell frequency specific with KLH against which human subjects were orally tolerized was the most sensitive method for monitoring induction of oral tolerance and gave reproducible results.