Intestinal Bacteria and Rabbit Antibody Diversification

肠道细菌和兔抗体多样化

• 批准号: 6749539
• 负责人: DENNIS K LANNING
• 金额: $ 22.2万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6749539
• 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3'端VH基因片段VH1。出生后 1 至 2 个月，肠道相关淋巴组织 (GALT) 中的体细胞超突变和体细胞基因转换样机制使最初的基因库变得多样化。对无菌兔子和出生时盲肠结扎以防止肠内容物进入阑尾的兔子的研究表明，GALT 发育依赖于来自肠道的外源因子。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有组织的 GALT 的兔子（ligApx 兔子）中，所有功能的多样化也依赖于源自肠道的外源因子。为了识别这种外源性因素，我们采用无菌饮食手工饲养无菌兔子，使其远离传统兔子及其微生物区系（远程兔群），并发现这些兔子中的大多数不会产生正常的 GALT 或使其抗体库多样化。由于这些偏远群体的兔子并非无菌，这些结果表明正常肠道微生物群的特定成员对于正常 GALT 发育和功能多样化是必需的。使用传统的培养方法来检查传统和远程群体兔子肠道微生物区系的差异，我们无法识别驱动兔子 GALT 发育和菌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的方法的许多局限性，并为研究肠道微生物区系提供了灵敏的技术。我们建议在这项拨款申请中使用这样一种方法，结合 GALT 发育和 VDJ 基因多样化的详细时间分析，以确定刺激兔 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。