Intestinal Bacteria and Rabbit Antibody Diversification

肠道细菌和兔抗体多样化

• 批准号: 7081299
• 负责人: DENNIS K LANNING
• 金额: $ 21.68万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081299
• 关键词: antibody formation; developmental immunology; enteric bacteria; gene rearrangement; gut associated lymphoid tissue; host organism interaction; immunoglobulin genes; laboratory rabbit

DESCRIPTION (provided by applicant): The rabbit primary antibody repertoire is generated through somatic diversification of a limited initial repertoire in which most VDJ gene rearrangements utilize the 3'-most VH gene segment, VH1. The initial repertoire is diversified by somatic hypermutation and a somatic gene conversion-like mechanism in gut-associated lymphoid tissue (GALT) 1 to 2 months after birth. Studies of germ-free rabbits and of rabbits in which the cecum was ligated at birth to prevent entry of intestinal contents into the appendix have shown that GALT development is dependent on an exogenous factor(s) derived from the gut. Our laboratory has extended these observations by showing, in rabbits in which the appendix was ligated at birth and all other organized GALT was removed (ligApx rabbits), that repertoire diversification is also dependent on an exogenous factor(s) derived from the gut. In order to identify this exogenous factor(s), we have hand-raised sterilely derived rabbits on a sterile diet away from contact with conventional rabbits and their microflora (remote colony rabbits) and found that most of these rabbits do not develop normal GALT or diversify their antibody repertoires. Since these remote colony rabbits are not germ-free, these results suggest that specific members of normal intestinal microflora are required for both normal GALT development and repertoire diversification. Using conventional culture methods to examine differences in the intestinal microflora of conventional and remote colony rabbits, we have not been able to identify bacterial species that drive rabbit GALT development and repertoire diversification. Approaches to the study of microbial diversity based on bacterial 16S rDNA have overcome many of the limitations of culture-based methods and made available sensitive techniques for studying intestinal microflora. We propose in this grant application to use one such approach, in combination with detailed temporal analysis of GALT development and VDJ gene diversification, to identify bacterial species that stimulate rabbit GALT development and repertoire diversification, and determine the mechanism by which they influence these developmental processes.

描述（由申请人提供）：兔一抗库是通过有限的初始库的体细胞多样化产生的，其中大多数VDJ基因重排利用了3'-most VH基因片段VH1。出生后1至2个月，肠道相关淋巴组织（GALT）的体细胞超突变和体细胞基因转换样机制使初始的储备多样化。对无菌家兔和出生时结扎盲肠以防止肠道内容物进入阑尾的家兔的研究表明，GALT的发展依赖于来自肠道的外源因素。我们的实验室扩展了这些观察结果，在出生时结扎阑尾并去除所有其他组织的GALT的兔子（ligApx兔）中，储备多样化也依赖于来自肠道的外源因素。为了确定这种外源性因素，我们用无菌饲料饲养无菌来源的家兔，远离与传统家兔及其微生物群（远程菌落家兔）的接触，发现这些家兔中的大多数不会产生正常的GALT或使其抗体库多样化。由于这些远程集落兔并非无菌，这些结果表明正常肠道菌群的特定成员对于正常的GALT发育和种群多样化都是必需的。使用传统的培养方法来检查传统和远程集落兔肠道菌群的差异，我们还不能确定驱动兔GALT发展和种群多样化的细菌种类。基于细菌16S rDNA的微生物多样性研究方法克服了基于培养方法的许多局限性，为研究肠道微生物群提供了灵敏的技术。我们在本次拨款申请中建议使用一种这样的方法，结合GALT发育和VDJ基因多样化的详细时间分析，确定刺激兔GALT发育和库多样化的细菌种类，并确定它们影响这些发育过程的机制。