F. Kovářů

Lymphatic tissue of the intestinal tract of germfree and conventional rabbits.

The lymphatic tissue of ileum, sacculus rotundus and appendix was poorly developed with-out germinal centres in germfree rabbits; on the other hand, in conventional rabbits, the lymphatic tissue was abundant with numerous germinal centers. In germfree rabbits, the stroma of villi of the jejunum and ileum possessed low cellularity, in conventional rabbits, the villi were wide with rich cellularity. The basal position of lymphocytes was predominant in the villi of ileum of germfree rabbits, in conventional rabbits a high percentage of lymphocytes was in the apical position.

Alpha-fetoprotein and phagocytosis in athymic nude mice

Alpha-fetoprotein (AFP) was found to suppress the phagocytic activity of the blood monocytes and neutrophils in vitro. The amounts of AFP detectable by immunofluorescence in the livers of nu/nu, nu/+ and +/+ mice were quite comparable, and thus could not have been responsible for the alterations in phagocytosis found in leukocytes of athymic nude mice during their ontogenetic development.

Immunoglobulin-producing cells in lymphatic tissues of germfree and conventional rabbits as detected by an immunofluorescence method

Lymphatic tissues of GF and CV rabbits were observed. No cells producing IgA and IgM antibodies were detected in appendix, sacculus rotundus, ileum terminale and thymu s of GF rabbits. IgA cells were found in lymph nodes of GF rabbits.

Identification of T and B lymphocytes in pigs by combined E-rosette test and surfage Ig labelling

A procedure for combining labelling of B lymphocytes with [125I]anti-Ig-antibodies and E-rosetting is described which permits simultaneous detection of B lymphocytes and T lymphocytes. Pig lymphocytes were incubated with iodinated anti-Ig antibodies and the E-rosette test with SRBC was performed. E-rosettes appeared stable after methanol fixation of cell smears on agarose-coated slides and treatment for autoradiography on stripping films. The percentage of cells classified as B or T lymphocytes in pig peripheral blood closely resembles that in man. No doubly marked cells were demonstrated provided cells were classified as E-rosettes only when 5 or more red cells were attached.

Morphology of germ-free piglets.

The postnatal ontogeny of primary (thymus) and secondary (spleen, lymph node, lingual tonsil) lymphoid tissues was studied in germ-free colostrum-deprived piglets up to age of 68 days. The thymus, which is morphologically fully developed by the end of gestation, showed no significant differences in the germ-free and conventional state. In germ-free piglets, slow development of periarteriolarly organized lymph follicles occurred in the spleen up to the end of the observation period. As distinct from the conditions in the spleen of conventional animals, the presence of a large number of pyroninophilic cells was not observed in germ-free piglets and no germinal centres were found. A similar situation was seen in the mesenteric lymph nodes, in which, in conventional piglets, cells belonging to the plasmacyte series, as well as the germinal centres, proliferate by the 13th day. Differences were also found in the organization of the follicular lymphoid tissue in the wall of the terminal ileum. In germ-free piglets, the lymph follicles increased only very slowly in size during the observation period and germinal centres were absent, while in conventional piglots germinal centres were present from the 12th day. The view is expressed that the intestinal lymphoid tissue ought rather to be classified as peripheral lymphoid tissue.

Development of lymphatic and haemopoietic organs in germfree models.

In the past, the problems of the morphological and functional development of primary and secondary lymphatic as well as haemopoietic organs were studied in a series of species reared under germfree conditions (guinea-pigs, chickens, mice, rats, piglets). The attention of research workers in this direction was concentrated primarily on those organ systems that are under conventional (CV) conditions in an intimate contact with the bacterial flora and where the absence of this stimulus could lead to morphological and functional changes (Luckey 1963). From a series of available data on this problem (Glimstedt 1936; Tajima 1955; Miyakawa et al. 1957; Gordon and Wostman 1958; Phillips 1959; Gordon 1959; ~terzl and Silverstein 1967; Pollard 1967; Plcasants 1968; Kruml et al. 1969; Cachava 1972; Rolls et al. 1975) we may summarize that the development of lymphatic system of germfree (GF) animals is retarded, at least with regard to secondary lymphatic organs; the most remarked differences between GF and CV animals were found in lymphatic organs draining the gastrointestinal tract. However, somewhat contradictory data are obtained already in a more detailed morphological description of these changes (GyllensSen 1950; Miyakawa 1957; Thorbecke 1959; Vaxler et al. 1978). I t is primarily the presence or absence of germinal centres in lymphatic follicles of GF animals and further the question of a possible detection of cells of the plasmocyte series in GF models. The differences in these data may be attributed mainly to the variability of experimental animal species but also to conditions of nutrition and adaptation of these animals to the environment of isolators for GF rearing (Gordon and Wostman 1958). The aim of our contribution to this problem is to compare the morphological and cytological development of lymphatic and haemopoietic organs in two of GF models that are most commonly used in this laboratory -GF precolostral piglets and GF rabbits, which markedly differ not only with regard

Lysosomal enzyme activities in polymorphonuclear leukocytes, macrophages, serum, and spleen of conventional, germ-free, and antigen-free minnesota miniature swine

o classification in the zoological system but also due to the ~ype of nutrition and the conditions of rearing. In morphological estimation of the development of lymphatic and haemopoietie organs the technique of quantitative cytology was used (Kov~fi 1971) which was supplemented with classical histological techniques. As far as the definition of experimental models, rearing conditions and nutrition of GF piglets and rabbits is concerned, they are given elsewhere in this Symposium (St~ps163 1979; Trs 5ek and Mandel et al. 1979).

Skin response to tuberculin in pig foetuses and germ-free piglets

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of guotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.

Ontogeny of T lymphocytes in the pig

Cellular response to intradermally administered PPD (2 TU) was demonstrated in pig foetuses of various ages and in germ-free piglets. CD2+, CD4+, CD8-, 86D-, SLA-D- T lymphocytes were the predominant cells in the skin tuberculin reaction in both foetuses and germ-free animals. Reactive T cells were observed as early as in mid-gestation, whereas SLA-D+ (porcine MHC class II) cells appeared only in older foetuses. Polymorphonuclear leukocytes were never observed in the PPD reaction.

The effect of an antigenic stimulation on proliferative response in athymic mice

Mononuclear cells isolated from pig fetal thymus (and thymus region), spleen and cord blood were examined for their reactivity with polyclonal sheep anti-pig T cell antiserum. First immunofluorescence-positive cells were detected after 28 d of gestation in the thymus region, cord blood and the liver.