Onset of physiological immunocompetence, i.e. ability to respond to antigen by production of specific antibodies without disturbance of the physiological integrity of the organism was studied in pig fetuses. Closed surgery technique was used to expose the fetuses. Flagellin in non-complete adjuvant was used as antigen. Fetuses were immunized on day 54 of gestation, i.e. in the period when T-B dichotomy in secondary lymphatic organs as principal sign of differentiation is already present. Besides the specific immunological response the intrauterine immunization resulted in increased numbers of agranulocytes within secondary lymphatic organs and in circulating blood. No maturation to their plasma cells but increased activity of gamma-glutamyl transpeptidase (GGT) was observed. Increased concentration of total proteins with predominant increase of albumins and gamma globulins along with a decrease of actual amount of alpha-1-fetoprotein was found in blood serum of immunized fetuses. Shift of maturation curves of erythroid and myeloid line to younger developmental forms appeared in haemopoietic organs of these fetuses. In older fetuses immunized on day 74 of gestation, i.e. when a basis of follicular organisation of lymphatic tissue is present, a typical cytological immune reaction "of adult type" was observed. It was characterized by multiplication of lymphocytes of all size categories and their differentiation to plasma cells. Pig fetuses, development of immunocompetence, lymphatic and hemopoietic organsDevelopmental immunology has an ideal experimental model in porcine fetuses because of the character of placentation in this species (placenta epitheliochorialis, diffuse type). This type of placenta not only prevents transfer of immunoglobulins between mother and fetus but it also markedly constrains the non-controllable antigenic stimulation during intrauterine development (·terzl and Silverstein 1967; KováfiÛ et al. 1969). However, developmental immunology is focused not only on the study of spontaneous development of effector systems of immunity but it also takes advantage of possible induction of immune response at particular developmental stages under precisely defined conditions. For this purpose a unique ontogenetic model of development of physiological immunocompetence has been elaborated. It is based on the ability of developing fetuses to respond to specific antigenic stimulus after intrauterine immunization without affecting their basic physiological indices and integrity (KováfiÛ et al. 1971; KováfiÛ and StoÏick˘ 1986;¤eháková et al. 1996).The onset of physiological immunocompetence against sheep erythrocytes (corpuscular antigen) and φX phage 174 (model of viral antigen) on day 70 of gestation has been demonstrated (·terzl and KováfiÛ 1877). The aim of this experiment was to study the antigenic stimulation during an earlier prenatal period (from day 54 of gestation), i.e. during
Changes in individual subpopulations of lymphocytes (mainly CD2 + , CD4 + and CD8 + ) in primary and secondary lymphatic organs and circulating blood were observed in pig fetuses between days 51 and 112 of gestation, and in circulating blood in postnatal piglets. The technique of flow cytofluorimetry was used and binding of specific monoclonal antibodies was visualised using polyclonal antibodies against mouse or rat Ig marked with fluorochroms (PE and FITC). As soon as on day 51 of gestation, CD4 + and CD8 + T lymphocytes were demonstrated in porcine thymus. Their relative and actual numbers continued to increase markedly. When this increase was expressed in terms of age with subsequent intrapolation, the changes in CD4 + /CD8 + phenotype expression could be expected around day 40, i.e. in the period, when thymus cortex and medulla are not yet morphologically differentiated.In the spleen only CD2 + cells were found on day 51 of gestation. Expression of lymphocytes with CD4 + and CD8 + receptors was shown on day 60. Their relative and actual numbers increased with age. This increase when expressed per whole organ made a difference of three orders of magnitude. In lymph nodes, only changes from day 90 were followed. In this secondary lymphatic organ, the percentage of T lymphocytes with CD4 + and CD8 + markers was higher than that in the spleen. The CD4 + /CD8 + ratio in spleen and thymus gradually decreased with advancing age to 1 with a slightly dominant CD4 + lymphocyte subpopulation. On the other hand, in lymph nodes of pig fetuses CD8 + lymphocytes prevailed (index 0.85). In the postnatal period, a marked increase of cytotoxic CD8 + lymphocytes occurred in peripheral blood of 28-day-old piglets. Thus the CD4 + /CD8 + index decreased from 1 to 0.2. This characteristic of lymphocyte subpopulations in circulating blood is also typical of adult individuals. The numbers of B lymphocytes with IgM receptors in circulating blood increased gradually from day 90 of prenatal development until day 28 h of postnatal life both in relative and actual terms. Prenatal ontogenesis, pig fetuses, lymphatic organs, quantitative cytology
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