Our studies in the rabbit have shown that the young appendix is an important site of development of the cells destined to produce protective antibodies. The rabbit appendix has functions similar to those of the avian bursa of Fabricius. In the young rabbit, there is rapid growth and expansion of B lymphocytes in the appendix, accompanied by positive and negative selection. We believe that cells surviving selection in the appendix travel to the peripheral lymphoid system and renew themselves to maintain the primary repertoire. The observations on rabbit appendix led us to study the role of human appendix and gut associated lymphoid tissue (GALT) in development of the human immune repertoire and mucosal immunity. Our previous studies compared development of normal human appendix germinal centers (GC) with rabbit appendix GC at different ages. In both human and rabbit appendix, the lymphoid follicles were just beginning to form at birth. Germinal centers became evident at about 1 month in human and at 1 week in rabbit appendix. Most lymphoid development occurred within the first year in the human and within the first 12 weeks in the rabbit appendix. Lymphoid tissue began to atrophy by adulthood in both human and rabbit appendix. The follicles became smaller and fewer in number whereas the connective tissue and smooth muscle increased. The appendix did not entirely involute; germinal centers were still present in 5 human samples from individuals 75 years or older and in 6- and 7-year-old rabbit specimens. The human appendix shares some characteristics of a """"""""mammalian bursa equivalent""""""""; it is a B- cell rich organ in the young and the morphology of the follicles and GC changes with age. In order to further investigate whether the appendix of man plays a role as a primary lymphoid organ we microdissected single cells from normal appendix tissues collected from children of 3 months, 4 years and 9 years of age for PCR amplification of rearranged VHDHJH genes, and direct DNA sequencing. Collections and analyses of single cell sequences are still in progress (see also Z01 AI00226-21 LI). For collection of appendix B cells, we conducted comparative studies of single cell microdissection by hydraulic micromanipulation, laser capture microdissection (LCM) (2) and the Leica UV laser microdissection system (LMD) (3). The LMD system seems well suited for collection of specific cell clusters; but more work is needed to achieve better single cell collection efficiency (3) (see also Z01 AI00226-21 LI). An automated microsphere-based flow cytometric assay (FlowMetrixTM system) was compared with a conventional ELISA for quantifying human Ig classes in serum and stool samples. The purpose of the assays was to determine whether appendectomy reduced intestinal immunoglobulin levels in children. Age-matched children treated surgically for abdominal hernias were used as controls. The results of a pilot study of the effect of appendectomy indicate that significant differences between appendectomized and control patients' total fecal IgA, IgA1 and IgA2 levels could not be demonstrated between 1 and 6 months after surgery (1). Measurement of specific responses to oral immunization may be a more sensitive means of detecting significant effects. Tests of the reproducibility of coupling capture antibodies to FlowMetrix microspheres showed that using independently coupled microspheres did not increase the variation of assay results relative to using the same bead set in repeated assays. However, slight variations in the coupling procedures can profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect assay precision. Although the ELISA was more sensitive and did not have problems with instrument performance encountered with the FlowMetrixTM assay, the latter was more reproducible, had a greater dynamic range of measurement, and took considerably less preparation time than the ELISA. Replicate ELISA and FlowMetrixTM assays of IgA, IgA1 and IgA2 in stool samples demonstrated considerable intra- and inter-subject variation of IgA levels in both control and appendectomized patients. Replicate assays were less variable in the Flow MetrixTM assays than in ELISAs for five of six assays which is especially important for measuring highly variable fecal Ig levels. Thus, in addition to its multi-analyte capability, the FlowMetrixTM assay system has definite advantages over a conventional ELISA. Mechanical problems such as microspheres settling to the bottom of wells during analysis by an automated plate reader, will likely be overcome, and sensitivity improved as this technology develops.
| Obiakor, Harold; Sehgal, Devinder; Dasso, Joseph F et al. (2002) A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Anal Biochem 306:55-62 |
| Dasso, Joseph; Lee, Juliet; Bach, Hanh et al. (2002) A comparison of ELISA and flow microsphere-based assays for quantification of immunoglobulins. J Immunol Methods 263:23-33 |
| Dasso, J F; Obiakor, H; Bach, H et al. (2000) A morphological and immunohistological study of the human and rabbit appendix for comparison with the avian bursa. Dev Comp Immunol 24:797-814 |
