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Gastrointestinal form is the second stage of the Acute Radiation Syndrome (ARS) with a threshold dose of 8 Gy. It represents an absolutely lethal clinical-pathological unit, enteritis necrohemorrhagica (duodenitis, jejunitis, ileitis, respectively) with unknown causal therapy. The purpose of our study has been to evaluate the morphological changes in a model of radiationinduced enteritis in rats and estimate the significance of changes in biodosimetry. Wistar rats were randomly divided into 21 groups, 10 animals per group. Samples of the jejunum were taken 24, 48, 72, and 96 h after the whole-body γ-irradiation with the doses of 1, 5, 10, 15, and 20 Gy, and routinely stained with hematoxylin and eosin. Five morphometric markers – intercryptal distance, enterocytal height on the top and base of villus, length of basal lamina of 10 enterocytes and enterocytal width – in irradiated rat jejunum were examined. The results were compared with sham-irradiated control group. After lethal doses of irradiation, all morphometric parameters of jejunum significantly changed. With the exception of intercryptal distance, they might be considered as suitable biodosimetric markers under these experimental conditions. Our morphometry results in radiation-induced jejunitis are in accordance with those in other studies. We were the first who quantified morphological postirradiation changes in animal jejunum. Some of them might be used under experimental conditions. This experimental study is a predecessor of the clinical assessment of a specific marker. Under clinical practice, the sensitive biodosimetric parameter could serve as one of the guidance for evaluation of the absorbed dose in irradiated troops as well as rescue workers. This is in accordance with tasks and Standardization Agreement of the North Atlantic Treaty Organization.

Gastrointestinal form is the second stage of acute radiation syndrome (ARS) with a threshold dose of 8 Gy in man. It represents an absolutely lethal clinical-pathological unit, necrohemorrhagic enteritis and proctocolitis, with unknown causal therapy. Elk-1 is a protein acting as a transcription factor activating specified genes. The purpose of our study was to examine the expression of phospho-Elk-1 in irradiated jejunum and transversal colon of rats with radiation-induced enterocolitis and to assess the importance of this transcriptional factor as a biodosimetric marker of radiation-induced enteropathy. The laboratory rats were randomly divided into 21 groups, 10 animals per group, and irradiated with whole body γ-irradiation of 1, 5, 10, 15, and 20 Gy. Samples of jejunum and transversal colon were taken 24, 48, 72, and 96 hours later, immunohistochemically stained, and the phospho-Elk-1 expression was examined using computer image analysis. A group of 10 shamirradiated animals was used as control. Significantly increased expression of phospho-Elk-1 in rat jejunum has been found in all time intervals after irradiation by sublethal doses of 1 and 5 Gy, whereas after the irradiation by lethal doses, the expression of phospho-Elk-1 in rat jejunum varied considerably. Significantly increased expression of phospho-Elk-1 in transversal colon has also been found in the first days after irradiation by sublethal doses of 1 and 5 Gy. After irradiation by lethal doses, tere was no uniform pattern of the changes in the expression of phospho-Elk1 in rat transversal colon. The detection of phospho-Elk-1 might be considered as a suitable and very sensitive biodosimetric marker of radiation-induced injury of small and large intestine. According to our knowledge, this is the first study on the phospho-Elk-1 expression in irradiated jejunum and transversal colon in the rat.

A possible relationship between intestinal inflammation and joint disease development was investigated. Clinical symptoms of colitis--diarrhea and rectal bleeding--were confirmed by findings of inflammatory processes in the colon in dextran sodium sulfate-treated mice and joint ankylosing enthesopathy (ANKENT) developed in 12.8 % mice with chronic colitis and 13.6 % mice in the control group. Consequently no significant difference in ANKENT frequency was found between mice with and without chronic colitis and the occurrence of ANKENT in both groups was typical for conventional conditions. ANKENT cannot be triggered solely a generalized inflammatory process in the gut.

The prenatal development of the porcine gamma/delta TCR repertoire was studied by complementarity-determining region 3 (CDR3) spectratyping and sequencing of TRDV1-DV5 transcripts. Specimens from the small and large intestine, spleen, thymus, liver, bone marrow and PBMC from fetal piglets between 38 and 114 days of gestation (DG) were examined. The TCR delta repertoire was highly restricted early in gestation (DG38-DG57) and an invariant TRDV3 transcript, lacking the N/D region, was found in different fetuses throughout gestation and dominated the TRDV3 repertoires of all organs at mid gestation ( approximately DG55). Near the end of gestation, this invariant TRDV3 transcript was absent from the thymus but was still present, in a less dominant manner, in the intestine and spleen. The average CDR3 length of all Vdelta subgroups increased with ontogeny, suggesting an increase in activity of TdT. Thus, the persistence of fetal gamma/delta T cells expressing an invariant TRDV3 chain throughout development is especially surprising since TdT is active early in gestation in swine. We speculate that these gamma/delta T cells might have been selectively expanded by (self)-ligands and may have an important function throughout fetal development.

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.

Surface binding of the Galanthus nivalis agglutinin (GNA) to thymocyte subsets has been studied in pigs and rodents by multicolour flow cytometry. In all the species examined, analogous staining profiles have been recorded. Counter-staining with anti-CD3epsilon, anti-CD4 and anti-CD8 monoclonal antibodies (MoAb) revealed that a significant increase of the GNA targets on the cell surface occurred during early thymocyte differentiation and reached its maximum at the level of the CD3loCD4+CD8+ small cortical thymocyte. This was followed by a decrease in the GNA binding capacity upon terminal maturation to the single positive thymocytes. PAGE analysis has revealed a dominant GNA-binding glycoprotein (molar mass approx. 90 kDa) present on thymocyte plasma membranes and absent on the surface of splenic lymphocytes, although both the whole cell lysates from both organs contained GNA ligands of the same size. Our findings are in agreement with previous data showing that immature thymocytes differ from their mature counterparts and peripheral T lymphocytes in the surface glycosylation pattern, and support the hypothesis that lectin-glycoprotein interaction plays a significant role in the cell-to-cell crosstalk in the thymic cortex.

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.

The CD8(+) natural killer (NK) subpopulation has recently been identified as a fast and reliable biodosimetric indicator within human peripheral blood mononuclear cells (PBMC) in vitro. In irradiated and subsequently cultivated PBMC, a decrease of the relative number of intact CD3(-)CD8(+) lymphocytes 16 and 48 h after treatment has allowed for estimating the received dose in the range of 0 - 10 Gy and lethal/sublethal dose discrimination, respectively. Here we show that suitable biodosimeters can also be found in the peripheral blood B-cell compartment. Multiparameter flow cytometric analysis of irradiated and subsequently cultivated human PBMC revealed that both the CD27(+) and CD21(-) B-cell subpopulations can be used as biodosimeters and the CD19(+)CD27(+) lymphocytes have proved useful for retrospective determination of the received dose in the range of 0 - 6 Gy. In addition, several CD19(+) lymphocyte subsets characterized by co expression of CD21, CD27 and CD38 have been shown to bear biodosimetric potential, too. However, when important parameters like the original size within the CD19(+) compartment, its radiation-induced changes and data variation had been taken into account, the CD27(+) subpopulation proved superior to the other B-cell subpopulations and subsets. It appears that, in the dose range of 0 - 6 Gy, the relative decrease of CD27(+) B lymphocytes provides more sensitive and reliable data than that of CD8(+) NK-cells due mainly to lower data variation. In contrast to CD27(+) B cells, the proportions of CD27(+) subpopulations of T-cells were not affected by irradiation. We have also proposed a simple experimental protocol based on full blood cultivation and three-color CD27/CD3/CD19 immuno-phenotyping as a time-saving and inexpensive approach for practical biodosimetric evaluations on simple, three-to-four color flow cytometers.

Ankylosing enthesopathy (ANKENT) is a naturally occurring joint disease in mice with numerous parallels to human ankylosing spondylitis (AS). Similarities between AS and ANKENT include not only affected tissue (joint entheses) but also association of the disease with genetic background, including MHC genes, gender, and age. Young males with the C57Bl/10 background have been described to suffer from ANKENT and, among H-2 congenic strains, high frequency of afflicted joints has been recorded in B10.BR (H-2(k)) males. Interestingly, the incidence of ANKENT is higher in conventional (CV) males that in their specific-pathogen-free (SPF) counterparts. The latter finding suggests that microbes could play a role as an ANKENT-triggering agent. To further examine this hypothesis we have established a germ-free (GF) colony of B10.BR mice and observed ANKENT incidence in both GF males and their conventionalized (ex-GF) male littermates; 20% of ex-GF males developed ANKENT before 1 year of age. In contrast, no joint disease was observed under GF conditions (p < 0.0001). Our results show that live microflora is required in ANKENT pathogenesis.

Germ-free piglets were orally infected with either enteropathogenic E. coli O55 or a virulent strain of Salmonella typhimurium. Orally applied antiserum against E. coli protected infected animals in spite of the fact that the bacteria were consistently found in mesenteric lymph nodes and other organs. By contrast, the application of an antiserum against S. typhimurium was without any effect on the outcome of infection. The treatment with anti-bacterial antiserum prevented inflammation of ileal mucosa (TNF-α and heat shock protein 65 expression) only in piglets infected with E. coli. A decrease in the frequency of ileal MAC320+ cells was observed in all infected piglets treated with antiserum.

The effect of controlled antigenic stimulation in immunologically virgin organisms, i.e. pig fetuses treated with NDCM (Nocardia delipidated cell mitogen) and germ-free (GF) piglets associated with a non-pathogenic E. coli 086, on peripheral blood lymphocyte subsets defined by the expression of CD5 and CD8 was studied by double color flow cytometry. Stimulation of both fetuses and GF piglets increased the frequency of CD8low+ lymphocytes. A prominent subset of CD5-CD8low+NK cells was present in GF and E. coli associated piglets and their frequency was slightly higher in E. coli associated animals. The most pronounced difference between stimulated and non-stimulated animals was in a relative proportion of an ill-defined lymphocyte subset with an unusual CD5low+CD8low+ expression. Both NDCM injection into fetal blood circulation and association of GF piglets with E. coli resulted in a marked increase of frequency of CD5low+CD8low+ lymphocytes in peripheral blood.