Immunochemical studies on lens protein-protein complexes I. The heterogeneity and structure of complexed alpha-crystallin.
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Biomedical subjects
Publications and source records attributed to W Manski.
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In stimultaneous experiments, the ability of inbred Wistar-Furth rat corneal cells to stimulate inbred Fisher rat lymphocytes in mixed culture was compared with the stimulatory capacity of the same number of Wistar-Furth skin, kidney, heart and lymphocyte cells. The tissue cells were dissociated and after inhibition by mitomycin C cultured with an equal number of allogeneic spleen lymphocytes for 5 days. In all of these mixed cell cultures, the allogeneic lymphocytic response was mainly to the major Ag-B2 histocompatibility antigen. The stimulatory effect of corneal cells was found to be the same as that of heart, kidney and skin cells. The data indicate a lack of differences between the density of histocompatibility antigens on the surfaces of these cells.
A dependence of second-set corneal graft reactions on the size of the sensitizing skin allografts was established in different rat donor-recipient pairs, each of which involved a response to the B2 major histocompatibility antigen. Sensitizing skin sizes larger or smaller than the optimal size were found to result in less intense reaction in subsequently placed second-set corneal allografts. The data obtained support the view that a persisting skin allograft not only sensitizes, but constitutes a competitive inflammatory site. The latter role of a first-set skin allograft may be facilitated by being both more accessible to inflammatory cells and larger than corneal grafts. The relative strength of the sensitizing and competitive effects of a first-set skin allograft apparently influences the fate of a second-set corneal allograft.
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A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 micrometer pore size in a specially designed multisample apparatus, and (2) spectrophotometric determination at 210 nm of the amount of proteins in the antigen-antibody precipitates dissolved in 0.01 N HCl. At this wavelength, the = C = O group of the polypeptide chains constitutes the main chromophoric group. In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 microgram of antigen. The concentration range of protein solutions in 0.01 N HCl which is measured at low UV can be regulated by a factor of 8--10 by changing absorption between 200 and 230 nm. Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5--6 h. The standard precipitin technique, by contrast, requires a 5--7-day reaction period for completion.
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Histamine or prostaglandin (PG) E1 or E2 administered to rabbits topically alone in high doses produced conjunctival vasodilation associated with little or no edema while their mixture at lower concentrations produced conjunctival vasodilation associated with profound edema. Sections of tissues treated with the mixture of histamine and PGE1 or PGE2 showed widespread epithelial and subepithelial inflammatory cellular infiltration. Conjunctival smears from eyes treated with the histamine/PG mixture contained small lymphocytes and polymorphonuclear leukocytes, including eosinophilic and occasionally basophilic cells. Differential staining of the polymorphs demonstrated both eosinophils and pseudoeosinophils. Histological examination of the conjunctival smears and sections of the lids obtained from eyes treated with either histamine or PGE1 or PGE2 alone did not show any detectable increase of inflammatory cells when compared to normal controls. The clinical and histological results indicate that the synergistic effect of histamine with PGs of the E-type in the conjunctiva produces an inflammatory response similar to that seen in various clinical forms of human allergic conjunctivitis. Such a response could not be produced by histamine or PGE1 or PGE2 alone even at much higher doses than in the mixture. The data indicate that an interplay of several different mediators may be crucial in the conjunctival response in allergy.
In the native alpha-crystallin molecule, 45.9% of all reactive antigenic determinants were found to be located on SH-containing subunits. Of these, the majority (35.3%) were reaggregation dependent, and 10.6% were reactive on monomeric subunits. By contrast, only 10.9% of all antigenic determinants were located on SH-free subunits, and the ratio of aggregation-dependent determinants (4.4%) to those of monomeric subunits (6.5%) was reversed compared to SH-containing subunits. Among all antigenic determinants reactive in native alpha-crystallin, 44.1% were dependent on the presence of both types of subunits. These data indicate that the antigenic determinants requiring subunit interaction were formed from SH-containing and SH-free subunits in a ratio of 1:1. Direct analysis showed that in the alpha-crystallin molecule, the ratio of these subunits is 2:1. The experiments indicate that some conformations of subunits in the native molecule persist in separated subunits. The relative immunogenicity of each type of antigenic determinant expressed as the ratio of the percentage of the determinant reactive in the native calf lens alpha-crystallin to the percentage of corresponding antibodies induced by native alpha-crystallin was found to be close to 1.
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In contrast to metabolically dependent tissue-specific cell surface antigens (MDA) which are available for reactions with antibodies only on surfaces of metabolically active cells, the availability of the universally distributed blood group A or Forssman-type antigens on cell surfaces was found to be independent of the metabolic activity of cultured cells. In case of the MDA, cytotoxic reactions were induced by antibodies alone, resulting in a disorganization of the cellular sheet without a significant release of radioactive label. Radioactive release in this cytotoxic reaction was increased by the addition of complement without additionally affecting the degree of cell sheet disorganization. In case of Forssman and blood group A antigens, such morphologically demonstrable cytotoxicity required complement, and the resulting pathology was always accompanied by extensive release of cellular contents. The ability of anti-MDA antibodies to induce cytotoxic reactions in the absence of complement may be related to a vital role of MDA in cellular function.
Two types of corneal cell membrane receptors are described, one occurs on the surface of only regenerating or cultured cells, that is, actively metabolizing and/or proliferating ones. The other type of corneal membrane receptors occurs on the cell surface independent of the metabolic state, e.g. on excised resting cells. In contrast to the restricted ontogenic specificity of the metabolically dependent corneal cell membrane antigens, the metabolically independent cell surface antigens were found to be universally distributed across ontogenic or species barriers. Such metabolically independent corneal cell surface antigens include Forssman or blood group antigens. The immuno-cytotoxic reaction of corneal cells involving the metabolically independent cell surface antigens was enhanced by complement, whereas a similar immunocytotoxic reaction involving the metabolically dependent antigens was not.