Sequential double immunocytochemical staining for in situ identification of an auto-anti-allotype immune response in allotype-suppressed rabbits

Immunocytochemical staining has been used to detect putative autoimmune B-cells in rabbits undergoing chronic allotype suppression. This condition is seen in heterozygous rabbits exposed perinatally to antibody against the paternal immunoglobulin allotype. Such animals develop lifelong suppression for this allotype and have been used as models for study of antibody-induced disturbance of immune regulation. Normal rabbits deliberately immunized against a heterologous allotype were used to establish the feasibility of identifying cells forming anti-allotypic antibodies in cryostat sections of rabbit lymphoid tissues. Incubation and staining of tissue sections from suppressed rabbits then revealed the presence of autoimmune B-cells, with antibody specificity for the suppressed allotype, in all chronically suppressed adult rabbits tested. Sequential incubation and staining with allotype- and anti-allotype-enzyme conjugates established that such cells were of non-suppressed origin. Auto-anti-allotype antibody-forming cells were not found in normal heterozygotes or in chimeric rabbits. The immunocytochemical techniques described here permitted simultaneous detection of specificity (i.e., anti-allotype) and origin (allotype) of antibody-forming cells involved in an autoimmune response, as well as their anatomical correlation with other B-cells of suppressed or non-suppressed origin. Since the method described can be adapted to detection of alternate cell markers, we believe it to have potential application to the study of other autoimmune phenomena.     

Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity

Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.     

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping.

Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents. SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules. We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen. The relevance of these results for physiological follicular trapping of protein antigens is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)