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

这个为期2年的项目旨在建立一个7天的外周血单个核细胞（PBMNC）培养系统，这些细胞对膳食蛋白抗原如卵清蛋白（OVA）、牛血清白蛋白（BSA）、卡帕酪蛋白（k-C）和牛γ -球蛋白（BGG）有增殖反应。如果在多发性硬化症（MS）和重症肌无力（MG）患者中，一种简单的检查肠道黏膜免疫的筛选试验可行，这种试验在正常情况下会诱导对通过肠道摄取的抗原的全身免疫反应性降低，这种方法将有助于选择新的治疗策略（即口服耐受）的最佳候选者。得到了以下结果：培养PBMNC的最佳饲料抗原浓度为50和100mg/ml。在与膳食抗原一起培养7天的PBMNC的最后16小时内，氚标记胸苷的掺入被用来衡量对每种抗原的增殖反应。用这种方法不仅在MS患者中发现了OVA和BGG的反应，在MG患者和健康对照者中也发现了。后两组对BSA没有反应，而对k-C的高反应可能是ms特异性肠道黏膜免疫的代表。通过胸腺嘧啶掺入测量的增殖反应与细胞培养7天后CD4^+CD26^+记忆辅助T细胞的扩增相关。为了研究细胞因子水平与上述结果的关系，每种抗原的培养上清液在-70 C.3下保存。用锁孔帽贝血青素（KLH）法测定口服耐受的前体细胞频率是监测口服耐受诱导最灵敏的方法，结果可重复性好。

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.