Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.     

Inhibition of yeast adenylate cyclase by antibodies to ras p21.

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.     

Heterogeneity of EBV-transformable human B lymphocyte populations

Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil-derived B cells respond to EBV, we re-examined the properties of EBV-transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM-committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti-immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of soluble antigens of blood group ABH using immunoenzyme analysis

The use of the indirect ELISA techniques did not ensure the sharp differentiation of the antigens of the blood groups A and B on the polystyrene sorbent by means of heteroimmune sera, though such differentiation could be achieved by means of monoclonal antibodies. The test system known as "the lectin-antibody sandwich" was found to have the optimum sensitivity and specificity permitting the detection of soluble ABH antigens. This variant of ELISA permitted the detection of blood group A antigen both in native biological materials and in traces of blood and saliva, thus making it possible to carry out its quantitative determination.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.