Chakravarti B, Lass J H, Diaconu E, Roy C E, Herring T A, Chakravarti D N, Greene B M
Department of Medicine, University of Alabama at Birmingham, USA.
Exp Eye Res. 1995 Apr;60(4):347-58. doi: 10.1016/s0014-4835(05)80092-2.
Sclerosing keratitis is the predominant cause of blindness due to onchocerciasis which is a major human parasitic disease caused by the filarial parasite Onchocerca volvulus. In the present investigation, native pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea pig model. Following demonstration of the protein nature of these antigens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each stage of purification, pooled antigen fractions were injected into one cornea of presensitized guinea pigs followed by clinical evaluation of stromal inflammation and vascularization at different intervals of time after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chromatography on Bio-gel A-5m. anion exchange chromatography on Mono Q followed by DEAE-Sepharose CL-6B and cation exchange chromatography on Mono S. Two out of six different pools from the Mono S column (pool a eluted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM-NaCl) were found to be most pathogenic. Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate molecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely responsible for induction of experimental interstitial keratitis. We have demonstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and hence the relevance of the purified antigens to ocular onchocerciasis in man since sclerosing keratitis is associated with invasion of the cornea by O. volvulus microfilariae. Isolation of these two pathogenic antigen pools represents the practical limits of purification and subsequent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the absence of a suitable non-human host or of an in vitro culture system for O. volvulus.
硬化性角膜炎是盘尾丝虫病导致失明的主要原因，盘尾丝虫病是由丝虫寄生虫旋盘尾丝虫引起的一种主要的人类寄生虫病。在本研究中，利用豚鼠模型对旋盘尾丝虫的天然致病抗原进行了表征，这些抗原在引起间质性角膜炎方面特别有效。在用链霉蛋白酶消化证明这些抗原的蛋白质性质后，对旋盘尾丝虫粗抗原提取物进行了逐步的蛋白质纯化程序。在纯化的每个阶段，将合并的抗原级分注射到预先致敏的豚鼠的一只角膜中，然后在基质内激发后的不同时间间隔对基质炎症和血管化进行临床评估。致病抗原的初步纯化按以下顺序进行：在Bio-gel A-5m上进行分子筛色谱，在Mono Q上进行阴离子交换色谱，随后在DEAE-Sepharose CL-6B上进行，以及在Mono S上进行阳离子交换色谱。来自Mono S柱的六个不同级分中有两个（级分a在10 mM氯化钠时未结合洗脱，级分e在130 mM至475 mM氯化钠之间洗脱）被发现致病性最强。使用Superose 12通过凝胶过滤色谱分别对Mono S级分a和级分e进行进一步纯化，结果表明，在诱导间质性角膜炎方面最有效的级分含有分子量约为100至200 kDa的蛋白质。这些结果表明，旋盘尾丝虫总粗抗原的微小亚级分在很大程度上负责诱导实验性间质性角膜炎。我们通过它们与抗微丝蚴抗体的交叉反应证明了这些抗原在旋盘尾丝虫微丝蚴中的存在，因此这些纯化抗原与人类眼部盘尾丝虫病相关，因为硬化性角膜炎与旋盘尾丝虫微丝蚴侵入角膜有关。分离这两个致病抗原级分代表了在没有合适的非人类宿主或旋盘尾丝虫体外培养系统的情况下，利用从受感染人类个体获得的可用量天然寄生虫材料进行纯化及后续动物实验的实际限度。
