Neuroprotective and antiamnestic effects of mutant molecules of erythropoietin on model of photochemical thrombosis of rat brain prefrontal cortex.

Mutant EPO molecules, deprived of erythropoietic activity, but possessing cytoprotective action, were created by the method of genetic engineering. The assessment of the therapeutic effectiveness of the received mutant proteins was carried out by the retention of the conditioned reflex of passive avoidance (PA), developed before the ischemic injury of rat brain prefrontal cortex, and by the MRI-analysis of ischemic damage volume. Antiamnestic and neuroprotective action of mutant molecules - MERO-Fc and MEPO-TR is investigated on model of photothrombosis of rat brain prefrontal cortex at single intranasal introduction in 1 h after cortex ischemic damage. The neuroprotective (MRI) and antiamnestic (PA) effects of mutant molecules of erythropoietin derivatives are shown.

Neuroprotective and antiamnesic effect of erythropoietin derivatives after experimental ischemic injury of cerebral cortex.

Using the model of bilateral photothrombosis of the blood vessels in the prefrontal cortex we have shown that new hybrid proteins derived from recombinant human erythropoietin, carbamylated EPO-Fc and EPO-TR fusion proteins, injected intraperitoneally 1 h after ischemic injury contribute to restoration of passive avoidance response formed before photothrombotic injury and reduction in the volume of the ischemic focus. These data attest to nootropic and neuroprotective activities of these hybrid proteins. Carbamylated glycopeptide derivative ЕPO-TR exhibited prolonged neuroprotective properties.

Immunoglobulin-specific T-B cell interaction. I. Presentation of self immunoglobulin determinants by B lymphocytes.

Immunoglobulin (Ig)-specific T-B cell interactions have been studied in the model of T cell recognition of the kappa chain Ig kappa-1b allotype in Ig kappa-1-congeneic rat strains. An efficient presentation of endogenous Ig allotypic determinants by irradiated spleen cells from (WAG.1b x August)F1 (RT-1u/c; Ig kappa-1b/1a) rats to Ig kappa-1b-specific lymph node T cells from Ig kappa-1-congeneic (WAG x August)F1 (RT-1u/c; Ig kappa-1a) rats was demonstrated. This presentation was found to be sensitive to high irradiation doses (greater than 1000 rad). By fractionation of Ig kappa-1b+ F1 spleen cells on Percoll density gradient we have shown that a radioresistant, low-density fraction, consisting mainly of macrophages (M phi) and dendritic cells, triggers only weak Ig kappa-1b-specific T cell response. The high level of response was observed against radiosensitive spleen cell fractions of intermediate and high density, suggesting that B cells were the main antigen-presenting cells (APC) of Ig kappa-1b determinants of endogenous Ig. This conclusion was confirmed in the experiments using purified B cells from Ig kappa-1b-bearing rats. Earlier we have shown that the responsiveness of August (RT-1c; Ig kappa-1a) and WAG (RT-1u; Ig kappa-1a) rats to Ig kappa-1b in vivo is controlled by the dominant allele of an RT-1-linked Ir gene. August and (August X WAG)F1 rats were found to be responders to Ig kappa-1b while WAG rats were nonresponders. The same pattern of Ir gene-controlled reactivity was demonstrated using an Ig kappa-1b-specific T cell proliferation assay. Ig kappa-1b-specific F1 T cell response was only observed when Ig kappa-1b+ B cells or IgG (Ig kappa-1b)-pulsed M phi-bearing responder major histocompatibility complex (MHC) haplotype were used as the APC. Anti-RT-1 monoclonal antibody inhibition studies suggested that the RT-1Bc molecule is the main restricting element of T cell recognition of Ig kappa-1b+ B cell as well as exogenous IgG (Ig kappa-1b). We have demonstrated allelic exclusion of Ig kappa-1b presenting function by negatively and positively selecting for Ig kappa-1b+ and Ig kappa-1a+ B cells from heterozygous F1(Ig kappa-1b/1a) rats. This clearly indicate that the B cells presented exclusively Ig kappa-1b allotypic determinants of their own Ig.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones.

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-1-congenic rats. Using Ig kappa-1b-recognizing major histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Ig kappa-1b+ B cells from congenic August.1b rats presented Ig kappa-1b epitope of the processed self-synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(Ig kappa-1b) synthesis. Small Ig kappa-1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Ig kappa-1b+ B cell population is involved in this interaction. The bystander activation of Ig kappa-1a+ B cells by T clones in the presence of irradiated Ig kappa-1b+ spleen cells, if observed, was less than the level of specific Ig kappa-1b+ B cell proliferation. In contrast to a 20-fold increase of Ig(Ig kappa-1b) levels upon stimulation of Ig kappa-1b+/1a+ B cell population from heterozygous (August x August.1b)F1 rats by T clones, a "nonspecific" increase of Ig(Ig kappa-1a) was not observed. This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.

Presentation of endogenous immunoglobulin determinant to immunoglobulin-recognizing T cell clones by the thymic cells.

Using immunoglobulin (Ig)-recognizing T helper clones the expression of Ig peptide/major histocompatibility complex class II complexes derived by the processing of endogeneous Ig molecules in the thymus was demonstrated. It was found that thymic B cells but not "classic" thymic antigen-presenting cells and macrophages represent the major antigen-presenting cell type of determinants of endogenously synthesized surface Ig (Ig kappa-1b) and anti-surface Ig antibodies (IdC3B9). The Ig kappa-1b-presenting activity in the thymus appears relatively late, only after 3 weeks of postnatal life, while in the spleen an efficient presentation of endogenous Ig kappa-1b epitope is observed very early after birth. This difference between thymic and peripheral presentation of endogeneous Ig determinant could be important for understanding the mechanisms of T cell tolerance to self Ig and the role of self Ig in negative and positive selection of T cell repertoire.

Separation of rosette-forming cells with the aid of an immunosorbent based on sephadex G-25 and G-75.

A method of separating rosette-forming cells on columns with an immunosorbent based on sephadex G-75 is described and has been tested. The protein antigen (bovine serum albumin-BSA) is attatched by covalent bonds to the surface of the Sephadex granules oxidized by sodium periodate. Cells with receptors on their surface were tested by the rosette method. Up to 88% of the rosette-forming cells were retained on a column packed with BSA-Sephadex granules. Nonspecific retention of the cells was relatively small, about 4%.

Immunoglobulin-specific T-B cell interaction. IV. B cell presentation of idiotypic determinant(s) of monoclonal anti-surface immunoglobulin antibody to idiotope-recognizing helper T clones.

Previously, we have demonstrated T-B cell interactions mediated by T cell recognition of immunoglobulin (Ig) peptide/major histocompatibility complex (MHC) class II complexes derived by the B cell processing of endogenously synthesized Ig molecules. In this report Ig-specific T-B cell interaction mediated by B cell presentation of idiotopes (Id) of anti-sIg antibodies to Id-specific T cell clones has been studied in Ig kappa-1-congenic rat strains. A panel of August (RT-1c; Ig kappa-1a) rat T helper clones specific for Id of syngeneic anti-Ig kappa-1b C3B9 monoclonal antibody (mAb) has been developed to study IdC3B9 presentation by Ig kappa-1b-bearing B cells from congenic August.1b (RT-1c; Ig kappa-1b) rats. Five of seven IdC3B9-specific T clones responded even at very low concentrations (100-200 pg/ml) of C3B9 mAb presented by Ig kappa-1b+ B cells. In contrast, the presentation of intact C3B9 mAb by nonspecific antigen-presenting cells (macrophages, Ig kappa-1a+ B cells, etc.) to IdC3B9-specific T cells was of low efficiency. The IdC3B9-specific T cell response to idiotopes of anti-Ig kappa-1b C3B9 mAb was found to be restricted by RT-1B molecule and required the processing of intact C3B9 molecule. IdC3B9 epitope recognized by C31 and C5 clones was mapped to the heavy chain of C3B9 mAb. Thus, B cells are able to present peptides related to the V region of anti-sIg Ab, i.e. Id peptide/MHC class II complexes, to Id-recognizing T cells. IdC3B9-presenting B cells are specifically activated both to proliferation and Ig production upon interaction with IdC3B9-specific T clones. Based on the results of our studies on B cell presentation of Ig epitopes to T cells a hypothetical model of Ig peptide-driven T-B cell interaction has been proposed.

Immunoglobulin-specific T-B cell interaction. II. T cell clones recognize the processed form of B cells' own surface immunoglobulin in the context of the major histocompatibility complex class II molecule.

In the preceding report (Eur. J. Immunol. 1989. 19: 1677) we have demonstrated that normal B cells, including small B cells, are capable of presenting Ig kappa-1b allotypic determinants of their endogeneously synthesized Ig+ to Ig kappa-1b-immune major histocompatibility complex (MHC) class II-restricted T cells. A panel of Ig kappa-1b allotype-specific T cell clones from August rats has been developed to study further the presentation of self surface Ig by B cells from Ig kappa-1-congeneic August.1b rats. All the clones studied were of the T helper/inducer phenotype (W3/25+,OX8-) and restricted by the RT-1Bc molecule. These clones responded both to the serum IgG(Ig kappa-1b) in the presence of irradiated spleen cells (SC) from August rats and to the Ig kappa-1b-bearing irradiated B cells from August.1b rats. SC presentation of secreted IgG was much less effective than B cell presentation of membrane Ig. Using CNBr cleavage of isolated C kappa (Ig kappa-1b) domain followed by high-performance liquid chromatography fractionation of the derived antigenic peptides, the kappa chain sequence between amino acids 176 and 214 has been identified as the T cell epitope recognized by all T cell clones in association with RT-1Bc. The fragment 176-214 of the Ig kappa-1b allotype differs from that of Ig kappa-1a allotype by three amino acid substitutions at positions 184, 185, 188. T cell recognition of pL kappa-1b(176-214) required no additional processing by the antigen-presenting cell: the efficient presentation of the peptide but not of intact IgG(Ig kappa-1b) by the paraformaldehyde-fixed SC was observed. These data provide clear-cut evidence for an absolute requirement of the processing of Ig molecules for T cell recognition to occur in our experimental system. Although the fixation of B cells from August.1b rats diminished their Ig kappa-1b-presenting ability, fixed Ig kappa-1b+ B cells were still able to induce Ig kappa-1b-specific T cell clone responses. Our results suggest that B cells can express the processed form of self-synthesized surface Ig in addition to intact surface Ig molecules. The former can be recognized by MHC-restricted T cell.