Mirian Galmarini

Delayed hypersensitivity and lesions following isoimmunization with modified rat male accessory glands: kinetics of induction

The kinetics of the cellular immune response to rat male accessory glands were studied in Wistar rats isoimmunized with modified rat male accessory glands extract and complete Freunds adjuvant at 0, 30 and 45 days. The animals were divided into seven groups, and each group was sacrificed weekly. One immunization was sufficient for the induction of 2-, 6- and 24-h footpad reactivity. The reaction increased until 21 days post-immunization. After the second injection the reaction decreased and was negative 12 days later. Migration inhibitory factor (MIF) activity monitored by a mixed-direct assay was demonstrated in rats from all groups except in the animals studied at day 42 in which macrophage migration was markedly stimulated. The absence of MIF activity correlated with a lack of delayed type hypersensitivity (DTH) response. The humoral response was studied and detected by passive hemagglutination in a few sera after the first immunization. A second injection was necessary to obtain a more frequent occurrence and higher titres of antibodies. Histological modifications in the target organs started to appear in the group of animals studied at 35 days and were characterized by a mononuclear infiltrate in the prostate, coagulating glands and seminal vesicles. In several cases there was also infiltration of polymorphonuclear cells. Specimens obtained at 35 days showed the most severe lesions.

Humoral autoimmune response to rat male accessory glands. Effect of castration on the humoral response.

The ability to induce antibodies to rat male accessory glands in male and female rats was demonstrated, but a higher response with wider specificity was revealed in female animals. In order to investigate whether this different response may be influenced by sexual hormones we castrated male and female rats at 4 or 30 days after birth. After that we studied the course and specificity of their humoral response to male accessory glands comparing them with that of sham-operated, sex-matched, littermate controls. Orchidectomy in male or oophorectomy in female rats changed neither the course nor specificity of humoral immune response. We conclude, therefore, that hormonal factors do not play any important role in the experimental model under study.

Specific Suppression of Humoral and Delayed Hypersensitivity Responses by Cyclophosphamide in an Experimental Model of Autoimmunity

ABSTRACT: The aim of this report is to investigate the effect of cyclophosphamide (CY) in an experimental model of autoimmunity to rat male accessory glands. The results indicated that 100 mg/kg of this drug suppressed humoral immune response that persisted for at least 45 days when administered 3 days after the first immunization of rats with modified rat male accessory glands (MRAG) in complete Freunds adjuvant (CFA). Administration of the drug 3 days before ID injection of antigen caused a shorter suppression of antibody formation. Delayed type hypersensitivity (DTH) studied 13 days after the first immunization was suppressed only in the animals that were administered CY after the antigen.

The effect of cyclophosphamide on autoimmune response to rats immunized with modified accessory glands.

It was found that the organ specific hemagglutinating autoantibodies to rat male accessory glands can be suppressed by the injection of a single dose of cyclophosphamide applied 3 days after the first immunization. On the other hand, injection of the drug 3 days before immunization did not modify the response, that is, there were comparable incidence and titers in both treated and the control animals. Cyclophosphamide did not appear to act on the homocytotropic antibodies. These results indicate that cyclophosphamide acts only when it is administered after antigenic stimulation, to suppress hemagglutinating antibodies production.

Evaluation of the immunosuppression in an experimental model of autoimmunity: suppressor activity of spleen cells from cyclophosphamide-treated rats

Rats immunized with chemically modified rat male accessory glands (MRAG) and injected 3 days later with cyclophosphamide (CY) were unable to develop humoral and cellular immune response to the autoantigen of MRAG. The present report demonstrates that the spleen mononuclear (SpM) cells transference from rats injected with CY 3 days after the antigen to normal male or female syngeneic animals before immunization with MRAG did not suppress the immune response to this antigen, whereas the transference of SpM cells from suppressed animals to animals previously immunized, depressed the delayed type hypersensitivity (DTH) response against MRAG (suppression of the expression) only in male rats. Similar results were obtained by transference of purified T cells. SpM cells did not suppress an established humoral immune response induced in male or female rats. The results suggest that non-adherent cells present in the spleen of male suppressed rats might be one of the responsible mechanisms for suppression of the efferent phase of the cellular autoimmune response to MRAG.

Participation of different cellular types in the enhancement of autoimmune response of old animals to sex accessory glands in male rats: importance of macrophages

In a previous work, we showed that the immunization of male rats, 3 and 12 months old, with saline extract of rat male accessory glands chemically modified (MRAG) and human serum albumin (HSA) induced a higher humoral and cellular autoimmune response in old animals than in young ones. We have also demonstrated that the facilitation of the autoimmune response is transferred by spleen total cells of 12-month-old animals. The immune response to HSA was not modified. In this work, the cellular type involved in such facilitation was analyzed. For this transference experiment, cells enriched in T and B lymphocytes and macrophages were used. The results showed that the macrophage is the main cellular type involved. However, the transference was only total with the three cellular types together. The study, performed with macrophages pulsed in vivo with MRAG-HSA and then transferred to normal recipients, indicated that although the macrophages from young and old animals were capable of presenting the antigens, the latter did this with significantly greater efficiency for the autoantigen.

Effect of aging on the autoimmune response to sex accessory glands in male rats

The effect of aging on the immune response to autoantigen of rat male accessory glands (RAG) was studied in Wistar rats. Male and female rats, 3 and 12 months old, were immunized with chemically modified RAG and heterologous antigen (human serum albumin, HSA). The study of delayed type hypersensitivity (DTH) and antibodies against RAG revealed a higher response in 12-month-old animals than in 3-month-old animals (P less than 0.005), regardless of their sexes. No differences in DTH and humoral responses to HSA were observed. Experiments on the transfer of spleen cells showed an increase in response elicited by RAG immunization in young recipients of cells from normal or immunized old syngeneic donors. On the contrary, old recipients of spleen cells from normal or immunized young donors maintained their high response the same as non-transferred old rats. Therefore, both the lymphoid cells and the environment in which the response was elicited seem to be involved in the increase of the autoimmune response.

Characterization of an Antigen from Rat Male Accessory Glands

An isolated soluble antigen involved in the autoimmune response against rat male accessory glands migrated as a double band when it was submitted to analytical disc polyacrylamide gel electrophoresis and as a homogeneous molecular species in SDS electrophoresis. The purified antigen had a molecular weight of approximately 78 K by SDS electrophoresis and 80 K by gel filtration chromatography. The antigen was identified as a protein and displays androgen dependency. The purified fraction was pathogenically active in microgram doses and induced humoral and delayed-type hypersensitivity responses. Besides, lesions were observed in the target organ. The cellular infiltration was located in prostate glands according to the localization of antigen by immunofluorescence findings.

Localization of Immunoglobulin A in Electrophoretic Patterns of Proteins from Human Parotid Saliva

Proteins from human parotid saliva were separated by use of a standardized technique of electrophoresis on polyacrylamide gel. Several fractions were eluted from the gel and studied by immunochemical methods (immunodiffusion, hemagglutination, and immunoelectrophoresis). The presence of IgA was demonstrated in the three slowest bands of the pattern.

Detection of Autoimmune Response to Rabbit Epididymal and Seminal Spermatozoa

Conventional and anaphylactic autoantibodies to rabbit epididymal and seminal spermatozoa were detected in sera of rabbits autoimmunized by several procedures. Antibodies against epididymal spermatozoa were found in 85% of rabbits showing lesions of varying degrees in testes. When the comparison was made with antibodies to antigens of spermatozoa from semen, the correlation was markedly lower. The data prove the importance of a proper selection of the antigenic material when looking for antibodies to spermatozoa in studies of autoimmunity and fertility.