Ethylenecarbodiimide-treated splenocytes carrying male CD4 epitopes confer histocompatibility Y chromosome antigen transplant protection by inhibiting CD154 upregulation.

In humans and certain strains of laboratory mice, male tissue is recognized as nonself and destroyed by the female immune system via recognition of histocompatibility Y chromosome Ag (Hya). Male tissue destruction is thought to be accomplished by CTLs in a helper-dependent manner. We show that graft protection induced with the immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SPs) with the chemical cross-linker ethylenecarbodiimide significantly, and often indefinitely, prolongs the survival of male skin graft transplants in an Ag-specific manner. In contrast, treatments with the Hya CD8 epitopes (Uty-/Smcy-SPs) failed to prolong graft survival. Dby-SP-tolerized CD4(+) T cells fail to proliferate, secrete IFN-gamma, or effectively prime a CD8 response in recipients of male grafts. Ag-coupled splenocyte treatment is associated with defective CD40-CD40L interactions as demonstrated by the observation that CD4 cells from treated animals exhibit a defect in CD40L upregulation following in vitro Ag challenge. Furthermore, treatment with an agonistic anti-CD40 Ab at the time of transplantation abrogates protection from graft rejection. Interestingly, anti-CD40 treatment completely restores the function of Dby-specific CD4 cells but not Uty- or Smcy-specific CD8 cells.

MHC restriction in contact hypersensitivity to dicyclohexylcarbodiimide.

Mouse ear swelling tests were performed using different strains of mice with dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), di-p-tolylcarbodiimide (DTC), and positive control chemicals, such as dinitrochlorobenzene (DNCB) and oxazolone (OXA). The chemicals were examined at different doses up to the minimal irritating concentration determined in a irritancy assay. While BALB/c mice exhibited strong responses for the carbodiimide compounds, C3H/HeN mice demonstrated no reactions. Other strains, C57BL/6 and DBA/1, also showed responses to DCC, but CBA/J mice with the same haplotype as C3H/HeN (H-2(k)) did not. Based on our present findings, there may be a specific unresponsiveness to DCC dependent on the H-2(k) haplotype.

Distribution of taurine in the rat cerebellum and insect brain: application of a new antiserum against carbodiimide-conjugated taurine.

The production, specificity and application of an antiserum against taurine conjugated to succinylated ovalbumin by means of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide is reported. The antiserum was produced in rabbits. The carbodiimide was used also as a tissue fixative. The development of the antibody titre was followed by dot-blot tests on nitrocellulose filters using different amino acid conjugates and with immunohistochemical reaction in the rat and insect brain. Blocking controls were also used. Taurine antiserum, sufficiently specific and sensitive, developed after the fourth booster injection, after which the antiserum was characterized. In the insect brain, intense taurine-like immunoreactivity was observed in the photoreceptors, in the Kenyon cells and the neuropile of the mushroom bodies, in the lower part of the central body and in the antennal lobes. In the rat carebellum, intense taurine-like immunoreactivity was seen in the Purkinje cells. Immunoreaction was seen also in small cells most probably corresponding to the basket cells. The use of the carbodiimide in the production of antisera against taurine provides a parallel method for comparison of the distribution of taurine-like immunoreactivity obtained with antisera made against conjugates prepared with aldehydes.

Analytical, toxicological and immunological consequences of the use of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide as coupling reagent for the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate as vaccine for human use.

For the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate the reactive reagent N-ethyl-N'-(dimethylaminopropyl) carbodiimide is used. The application of this reagent results in a number of stable linkages (viz. "peptide" linkages between the polysaccharide and tetanus toxoid, intrachain ester linkages in the polysaccharide component and binding of the N-acylurea derivative of the reagent) and less stable ones (viz. anhydride linkages). As a consequence of the reaction, the reagent is converted to a non-reactive urea derivative. The toxic properties of the reagent and of the converted reagent were studied. These properties do not contraindicate the use of the coupling reagent for the preparation of vaccines for human use. In addition analytical methods have been developed for the quantitative evaluation of the coupling reagent, the reaction products and for the N-acylurea derivative of the reagent and of the residual reactivity of conjugates for primary aminogroups. Although no test was performed for the assay of ester linkages in the polysaccharide component of the conjugate, evidence is presented that such linkages may be present. The results of the test for residual reactivity indicated a spontaneous rearrangement of linkages after the preparation of the conjugate. In addition the influence of the ratio of coupling reagent-to-polysaccharide and tetanus toxoid on antigenic and immunogenic activities of the conjugate was studied. An increase of the ratio resulted in a decrease of the antigenic activity of the polysaccharide component but in an increase of its immunogenic activity as to the induction of IgG antibodies to the polysaccharide. The immunogenic activity of the polysaccharide component correlated rather well with the antigenic activity measured in heterologous enzyme-linked immunosorbent assay using antibodies to both components.

Antisera specificities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide adducts of proteins.

Animals were immunized with either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-coupled peptide hapten-protein carrier conjugates or with N-acylurea EDCI derived protein carrier. Sera from all animals demonstrated antibody to N-acylurea EDCI when tested by double immunodiffusion using N-acylurea EDCI coupled to a heterologous carrier. Two rabbit antisera, one produced to EDCI-coupled peptide-24-thyroglobulin and another produced to N-acylurea EDCI derived thyroglobulin were further characterized using an enzyme-linked immunosorbent assay (ELISA). Competitive binding assays using either EDCI-coupled peptide-protein conjugates or N-acylurea EDCI derived proteins demonstrated the presence of N-acylurea EDCI reactive antibodies. Such antibodies were also shown to react with the EDCI isourea employed as inhibitor. The specificity of the anti-EDCI antibodies to portions of the EDCI molecule was demonstrated using structural analogues of EDCI. Both 3-dimethylaminopropylamine and 2-dimethylaminoethanol effectively inhibited anti-N-acylurea EDCI reactivity. These results show that when EDCI is used as a coupling agent, antibody is produced to N-acylurea EDCI-carrier, the antibody being primarily directed to the 3-dimethylaminopropyl end of the EDCI molecule.

Succinylation of hapten-protein conjugates facilitates coupling to erythrocytes by water soluble carbodiimide: preparation of stable and sensitive target cells for use in hemolytic assays.

A facile method is described for the preparation of haptenated sheep red blood cells (SRBC) for use as targets in hemolytic spot and plaque assays for the detection of anti-hapten antibody. The method involves the use of the water soluble 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDCI) as a reagent to couple hapten-succinyl-rabbit serum albumin conjugates to SRBC. The presence of the succinyl groups on such conjugates is shown to increase the efficacy of the resulting target cells, presumably by acting as a substrate for the EDCI and thus increasing the extent of coupling to SRBC.