Commensal intestinal bacterial strains trigger ankylosing enthesopathy of the ankle in inbred B10.BR (H-2(k)) male mice.

Joint disease ankylosing enthesopathy (ANKENT) naturally occurs in inbred mice with C57Bl/10 genetic background. ANKENT has many parallels to human ankylosing spondylitis (AS) and represents an animal model for AS. Environmental conditions (i.e., microbial load of the organism) are among the risk factors for ANKENT, similar to AS. The role of microflora in the development of ANKENT was investigated. ANKENT was tested in four experimental groups of germ-free mice associated with different numbers of various intestinal microbes and three control groups: germ-free, specific pathogen-free, and conventional (CV) mice. Mice were colonized either with anaerobic bacteria isolated from the intestine of a CV mouse or with bacterial strains obtained from the collection of microorganisms. Microbes were characterized and checked by microbiological cultivation methods and with the use of polymerase chain reaction amplification and rDNA sequence analysis. Joint disease developed in GF mice colonized with a mixture containing Bacteroides spp. and Enterococcus sp., and/or Veillonella sp. and Staphylococcus sp. No ANKENT appeared in males colonized with probiotic bacterium Lactobacillus sp. In control groups ANKENT occurred in SPF and CV animals; the GF animals remained healthy. The results confirmed that the germ-free conditions protect from joint inflammation, and thus microbes are necessary for ANKENT development. In colonized mice the ANKENT was triggered by luminal anaerobic bacteria, which are common components of intestinal microflora.

CD27(+) peripheral blood B-cells are a useful biodosimetric marker in vitro.

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.

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets.

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially gammadelta T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of gammadelta T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of gammadelta T cells, but the percentage of the gammadeltaTCR+CD8+ subpopulation significantly increased. The tissue distribution of gammadeltaTCR+CD8+ and gammadeltaTCR+CD8- cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8+CD3+CD4-gammadeltaTCR-, which was compensated by a decreasing proportion of CD4+ lymphocytes. Double positive CD4+CD8+ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.

Cross-reactive anti-human monoclonal antibodies as a tool for B-cell identification in dogs and pigs.

We have characterized a panel of commercially available anti-human monoclonal antibodies (mAbs) suitable for B-cell identification in pigs and dogs. The specificities of the mAbs were against CD20, CD21, CD22, and CD86. In addition to HM57, originally raised against human CD79alpha the broad cross-reactivity of which was documented more than 10 years ago, we recommend here a panel of several other mAbs as a useful tool for immunophenotyping and multicolor flow cytometry of canine and porcine B-lymphocytes. All six investigated antibodies did bind weakly to either canine or porcine lymphocytes (or both), but considerable weaker than for the human control cells. Four of them did bind to canine or porcine spleen section in immunohistochemistry. Monoclonal antibody against CD22 (clone RFB-4) was the only antibody in the tested panel the cross-reactivity of which was confirmed by Western blot. The advantages and limits of cross-reactive mAbs in studies on animal B-cells are discussed.

Commercially available rabbit anti-human polyclonal antisera as a useful tool for immune system studies in veterinary species.

We have used selected rabbit anti-human polyclonal antibodies as an example of useful and easily available tools for studies on immune system structure and development in important veterinary species, many of which also represent animal models in biomedicine. The cocktail of anti-human Igkappa-FITC/anti-Iglambda-RPE F(ab')(2) fragments was used for two-colour and, in combination with the cross-reactive anti-CD79alpha monoclonal antibody HM-57, for three-colour flow cytometry of canine, feline, bovine and porcine peripheral B-cells. A possible application of such immunoreagents in studies on primary B-cell differentiation has been suggested in pigs; the same approach can be used in other species of interest. Rabbit anti-human lactoferrin-FITC F(ab')(2) fragment was used for visualizing neutrophils in dogs, pigs and cattle and an application for two-colour immunophenotyping of canine granulocyte subsets has been designed. Affinity isolated rabbit anti-human CD3 and anti-human TdT have been shown to represent a ready-to-use tool for in situ studies on primary T-lymphopoiesis in pigs with possible extensions both to the B-lineage development in pigs and other animal models. Altogether, our study show that carefully selected polyclonal antibodies available on the market may possess broad cross-reactivity with important applications in veterinary research.

CD8+ natural killer cells have a potential of a sensitive and reliable biodosimetric marker in vitro.

The aim of our work was to evaluate peripheral blood lymphocyte subsets as in vitro indicators of the received dose of ionizing radiation (biodosimetric markers) in the range of 3-20 Gy and to determine the appropriate time interval, during which a dose-dependent induction of apoptosis occurs upon gamma irradiation. In lymphocyte subsets characterized by double color surface immunophenotyping, four-color flow cytometry was used for visualizing cell death-associated increase in superficial phosphatidylserine exposure and cytoplasmic membrane permeability by fluorinated Annexin V and propidium iodide, respectively. No differences between sham-treated and lethal dose (7 Gy)-irradiated samples were observed upon 6 h cultivation in vitro. Ten and 18 h later, about 50 % of lymphocytes were apoptotic, but only the minority of them was in the late apoptotic phase. The only difference in radioresistance of the CD4(+)CD8(-) and CD4(-)CD8(+) lymphocyte subsets was seen upon 2-day cultivation when huge depletion of intact cells and prevalence of the late apoptotic population became obvious. A dose-dependence study in 16 and 48 h cultures confirmed the effectiveness of major T cell subsets as biodosimetric indicators. On the other hand, the minor CD8(+) subset of natural killer (NK) cells has been identified as a radiosensitive lymphocyte population the disappearance of which correlated with the received dose. We demonstrated that the CD3(-)CD8(+)NK subset can be used as a lethal/sublethal dose discriminator to 16 h cultivation. In addition, our data indicate that two-day cultivation followed by CD3/CD8 expression analysis in an intact lymphocyte population may provide a clue for low dosage biodosimetry.

Lymphatic organ development in dogs: major lymphocyte subsets and activity.

In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of three adult dogs. In neonatal pups, lower numbers of CD3(+) T-cells were detected in both the spleen and peripheral blood than in lymph nodes. In contrast to the other compartments, CD21(+) B-cells prevailed in the spleen, which resulted in low values (<1) of the CD3(+)/CD21(+) ratio. Low numbers of CD8(+) lymphocytes were characteristic in all compartments immediately after birth; consequently a high CD4(+)/CD8(+) ratio has been calculated. Postnatal development was characterized by an increasing frequency of CD8(+) lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen. DNA synthesis in newborn pups' cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively competent immune system the structure of which, however, markedly develops during a few postnatal weeks.

Experimental Actinobacillus pleuropneumoniae infection in piglets with different types and levels of specific protection: immunophenotypic analysis of lymphocyte subsets in the circulation and respiratory mucosal lymphoid tissue.

Actinobacillus pleuropneumoniae (APP) infection in piglets results in severe and fatal fibrinous hemorrhagic necrotizing pneumoniae. The aim of our study was to analyze changes in lymphocyte subset distribution in peripheral blood, bronchoalveolar lavage fluid (BALF) and tracheobronchal lymph nodes (TLN) in non-immune piglets upon a challenge with a high dose of APP and to compare the quality of such changes in unprotected piglets with counterparts exhibiting specific immunity mediated by high titers of colostrum-derived APP-specific antibodies and/or a low dose APP infection in the early postnatal period. Challenge with APP resulted in a massive increase in CD8-negative gammadelta T-cells in parallel with a reduction in numbers of CD3-CD8low cells in BALF independent of the type and level of immunity and this seems to be a general phenomenon associated with experimental infection. An increase in B-lymphocyte numbers in TLN was another characteristic feature accompanying APP infection in all experimental groups. In piglets with colostrum-derived APP-specific antibodies, this was associated with higher relative numbers of IgM+CD2+ lymphocytes in TLN, while B-cells with the CD2- surface phenotype apparently expanded in the absence of passive humoral immunity.

Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease.

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.

Specific antibody and immunoglobulin responses after intestinal colonization of germ-free piglets with non-pathogenic Escherichia coli O86.

Colonization of the gut with components of commensal microflora profoundly affects the development of the immune system. The aim of the present study was to investigate mucosal and systemic B cell responses during the first few days after intestinal association of colostrum-deprived piglets reared in germ-free (GF) conditions with non-pathogenic Escherichia coli O86. Specific intestinal anti-E. coli antibodies (Ab), among which IgA Ab prevailed, were found 4 days after colonization (72% of standard) and their amount decreased 11 days later reaching 22% of standard. In contrast to mucosal Ab, specific serum Ab remained at the level of GF animals at day 4 (less than 10% of standard) and markedly increased 15 days after colonization (156% of standard). In addition to the occurrence of specific Ab, increased amounts of total immunoglobulins (Ig) of all isotypes were detected in sera and intestinal washings. Using the ELISPOT method an increased number of IgM, IgG and IgA-secreting lymphocytes were found in spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP) in colonized animals as compared to GF piglets. Contrary to cells from these lymphatic organs, B cells from thymus were not affected by E. coli stimulation. Our results show that at the onset of intestinal colonization, non-pathogenic E. coli specifically and polyclonally stimulate the mucosal and systemic humoral immunity, but relatively soon after stimulation, mucosal-specific responses in gut decreases, indicating the possible beginning of inhibition mechanisms (oral tolerance).

Early development of immune system in pigs.

Prenatal and early postnatal immune system development has been studied in minipigs. First leukocytes were observed in the yolk sac and fetal liver (FL) on the 17th day of gestation, the majority of them being SWC3(+). The colonization of the thymus (TH) with leukocytes was observed 21 days later. Two waves of fetal TH colonization with pro-T cells were deduced from the frequency of thymocyte subsets. Thymic B cells and immunoglobulin-secreting cells (Ig-SC) were studied by flow cytometry and ELISPOT, respectively. When the total numbers of fetal Ig-SC were compared, the TH was identified as the main source of natural antibodies and the only site of IgA and IgG synthesis. In germ-free animals, the TH also represented the major site of IgG and IgA production and the number of Ig-SC was not influenced by colonization with microflora. FL and bone marrow were identified as primary B lymphopoietic sites. The phenotype of B precursors was characterized and pre-B II cells were shown to be the dominant mononuclear fraction between DG50 and DG105. In the periphery, relative proportions of lymphocyte subsets were determined. Studies in gnotobiotic piglets have revealed that the appearance of CD4(+)CD8(+) T cells and CD2(-) B cells is absolutely dependent on the contact of immune system with live viruses and bacteria, respectively.

Postnatal development of leukocyte subset composition and activity in dogs.

The aim of the presentation is to summarise our data on the counts and activity of circulating canine leukocytes at birth and on their changes in the first 3 months of life. On day 1, neutrophil counts were almost three times higher than lymphocyte counts. During the first week of life, a decrease of neutrophil and an increase of lymphocyte counts, resulting in a predominance of lymphocytes, were observed. Neutrophil counts reached values comparable with those in adults in 1 month. Lymphocyte counts were higher than those in adults during the first 3 months. From birth to the age of 3 months, the phagocytic activity of neutrophils was nonsignificantly higher than in young adults. When compared with adults, the peripheral blood of new-born pups contained a lower proportion of T lymphocytes (detected by CD3 and CD5 markers), with a very low percentage of CD8(+) cells and a higher proportion of CD21(+) B lymphocytes. The counts of individual subsets levelled out during the first 3 months of life, although the proportion of CD21(+) B cells remained higher all the time. Lymphocytes of new-born pups were able to respond to nonspecific mitogen stimulation. Spontaneous proliferation in vitro was higher during the first week of life. Although in vitro stimulation of lymphocytes with Concanavalin A in some pups was comparable with that of adult dogs, mean activity was weaker. Pups with zero or very low levels of maternal antibodies were able to develop specific immune responses to a parvovirus antigen as early as at 2 weeks of age. On the basis of these data, we assume that pups are born with an immune system that can respond to external stimuli. Nevertheless its development continues in the postnatal period and some parameters differ from adult values for at least 3 months after birth.

Summary of workshop findings for porcine B-cell markers.

Based on cluster groups from the first-round analyses of the Third International Swine CD Workshop, 38 monoclonal antibodies (MAbs) including eight internal controls were analysed by flow cytometry (FCM) and immunohistochemistry (IH) in the second-round analysis of the B-cell section of this workshop. Targets in this section included peripheral blood lymphocytes and cells isolated from ileal Peyer's patches (PP), mesenteric lymph nodes (MLN) of adult animals, bone marrow cells from newborn piglets and thymus cells isolated from foetuses at day 105 of gestation. Immunohistochemistry of these 38 MAbs identified four sets, whose ligands were co-expressed with CD21, which showed a tissue distribution compatible with specificity for cells including those of the B-cell lineage. Another group of miscellaneous antibodies appeared to identify other cells, several antibodies were negative. Two-colour flow cytometry (2C-FCM) was carried out by pairing each antibody of interest with antibodies to SWC7, CD21, sIgM and a polyclonal rabbit anti-swine immunoglobulin antiserum (RaSwIg). The anti-CD21 MAb BB6-11C9 (no. 20) and IAH-CC51 (no. 19), established in previous workshops, as well as the cross-reactive anti-human CD21 B-1y4 (no. 146), clustered together in FCM analyses of the first round and showed similar cellular distribution in IH. A further cluster was formed by the standard CC55 (no. 55) and 2A10/8 (no. 102) submitted as SWC7 specific. The second SWC7 standard 2F6/8 (no. 100) clustered separately, but IH showed an identical pattern of reactivity to the other SWC7 MAb.Unfortunately, this work could not identify any other novel clusters with specificity for B-cells, as the statistical clustering of other MAbs could not be substantiated by IH or subsequent two-colour-FCM work. However, we could identify MAb with similar cellular distribution. The ligands for the cross-reactive anti-human CD40 G28.5 (no. 25) and STH224 (no. 153) were expressed on very similar targets, similarly the ligands for the MAb pair JM1H1 (no. 139) with BB6-10A10 (no. 142) and the MAb pair 3F7/11 (no. 115) with 1C2F10 (no. 187).

Characterization of monoclonal antibodies recognizing immunoglobulin kappa and lambda chains in pigs by flow cytometry.

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igkappa) and Ig lambda (Iglambda). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igkappa and Iglambda and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Iglambda antiserum and were negative when tested using anti-human Igkappa antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Iglambda-specific whereas mAb 27.2.1 recognizes the Igkappa chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Iglambda(+) B cells in most pigs. In summary, we identified two pan-specific anti-pig Iglambda mAbs, one anti-lambda mAb that recognizes a lambda-light chain subset and one anti-pig Igkappa mAb.

Monoclonal antibodies putatively recognising activation and differentiation antigens.

In the activation/maturation section, 46 monoclonal antibodies (mAbs) were analysed using freshly isolated as well as mitogen activated and recall antigen re-stimulated cells. A total of 10 internal standards as well as 6 antibodies with established reactivity for human cells, reported to cross-react with porcine leukocytes, were included in the panel. The standard antibodies were anti-CD25, CD44, CD45, SLA II, SWC1, SWC2, SWC7 and SWC8 reagents. The test panel contained antibodies with putative reactivity to CD25, SLA II and other mAbs directed against ill-defined targets. Single and double colour surface staining was performed in the attempt to group the mAbs tested into clusters of differentiation. Five new anti-class II reagents, two directed to SLA-DQ and three to SLA-DR, could be added to the previously established ones. One new anti-CD25 as well as two new antibodies with SWC7 and SWC8 specificities, respectively, could also be added to the previously established ones. The identity of the two latter antibodies was also confirmed in other sections of this workshop (B-cell section for SWC7 antibodies and myeloid section for the SWC8 antibodies). The antibody JM2F12, in our hands, has shown strong similarities to the cross-reactive anti human-CD49f reagent. No other clusters were identified, as all remaining antibodies behaved in a different way on different target leukocyte populations. The second purpose of the section was fulfilled: interesting staining profiles of several antibodies on differentiating lymphocytes were recorded and are discussed here.

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop.

As a result of the first-round cluster analysis, a panel of 16 novel monoclonal antibodies (mAbs) was assigned for detailed analysis to the CD45 subgroup of the Third International Swine CD Workshop. The specificity of the mAbs was initially determined by examining their reactivity with Chinese hamster ovary (CHO) cells engineered to express individual isoforms of porcine CD45. These analyses indicated that seven of the mAbs (PG77A, PG96A, PG167A, PGB78A, 3C/9, MIL13, NHT 101) recognized the portion of the CD45 molecule encoded by the A exon (CD45RA), while one (MIL15) was specific for that portion encoded by the C exon (CD45RC). In each case, the designation was supported by the demonstration that the molecular weight(s) of the recognized antigen(s) in porcine mononuclear cells, as determined by immunoprecipitation, corresponded to the predicted size(s) according to their specificity. As expected, a similar correlation was obtained for five standard mAbs whose specificity for either common or restricted epitopes of porcine CD45 had been established in previous workshops. Screening of the remaining 174 mAbs that comprised this workshop but were excluded from the CD45 subgroup by cluster analysis failed to detect any additional ones reactive with the porcine CD45-expressing cells.

Analysis of monoclonal antibodies reactive with the porcine CD2 antigen.

As result of the First International Swine CD Workshop, six monoclonal antibodies (mAbs) (numbers 014, 023, 024, 057, 128, and 130) clustered closely to the internal standard anti-porcine CD2 mAb, MSA4. Despite the close clustering, the cluster was split into two subgroups. To further characterize the relationship between these mAbs, they were used in flow cytometry to inhibit binding of MSA4 to porcine lymphocytes. mAbs 014 (1038-8-31), 023 (MAC83), 024 (MAC80), 057 (PG168), and 128 (MSA4) completely inhibited the binding of MSA4, mAb 130 (MSA2) failed to inhibit MSA4 binding. On dual parameter flow cytometry comparing MSA2 with MSA4, all MSA2+ cells were MSA4+, two thirds of the MSA4+ cells were MSA2-. We conclude that five of the mAbs bind to the same or a closely related epitope on porcine lymphocytes. mAb 130 appears to have aberrantly clustered with the CD2 group of mAb.

Summary of workshop findings for porcine myelomonocytic markers.

About 65 monoclonal antibodies (mAb) including 17 internal controls were analyzed for their ability to recognize and bind to various cells of the myelomonocytic lineage. Flow cytometry (FCM) utilizing both single and double staining, and immunoprecipitation (IP) assays were used in the analysis. About 38 of the mAb were reactive with myelomonocytic cells, resulting in nine clusters of interest. Although the exact identity of many of the molecules on the cells bound by the mAb remains undetermined, information obtained about the mAb analyzed in this workshop should be helpful in further identifying various populations of myelomonocytic cells and their stages of differentiation. Out of 12 mAbs with potential CD11 specificity, seven were assigned to three different swine specific alpha chains of the CD11/CD18 integrin heterodimer, the assignment of the remaining four was tentative. One antibody had a binding specificity consistent with SWC3 and one with SWC8. CD14 expression on pig cells was characterized with a panel of CD14-positive antibodies, two of these antibodies were assigned to swine CD14. Two antibodies were assigned to CD163. Further work is required to determine the antigens recognized by many of the other mAb.

Switch recombination in fetal porcine thymus is uncoupled from somatic mutation.

Since fetal serum Ig isotype profiles suggested that IgG and IgA could be of de novo origin, we studied their transcription and secretion. IgM transcripts were present at 50 days of gestation in major fetal lymphoid tissues, IgG and IgA transcription was pronounced at 60 days in fetal thymus and both transcription and secretion in this organ increased in late fetal life. The CDR3 spectratype of thymic IgG and IgA transcripts was as polyclonal as that of IgM already at 70 days in utero indicating a broad repertoire of switched B-cells. However, VDJs transcribed with the switched isotypes were not hypermutated as were those from immunized fetuses, indicating that switch recombination and somatic mutation are not coupled in utero in piglets. This finding and the fact that the oligoclonal IgA and IgM repertoires in a non-inductive site of the mucosal immune system (parotid gland) becomes polyclonal in piglets reared germ-free, suggest that initial expansion of switched B-cells in fetal and neonatal piglets is not driven by environmental antigen. Our findings collectively suggest that all IgA and IgM may result from de novo synthesis while some IgG probably results from selective transport. The latter is consistent with the gradual decline in serum IgG concentration in germ-free isolator piglets and the expression of FcRn in the porcine placenta.

Early ontogeny of immune cells and their functions in the fetal pig.

The origin of immune cells and their products have been studied in the prenatal period in miniature pigs. Macrophages were first detected on day 25, and myelocytes and lymphoid cells by day 28. Membrane antigens SLA-DR and CD45 were found by day 22, membrane molecules MG-7, 8/1, CD1, CD2 and 74-22 by day 28, Gamma/delta T cells were found initially in extrathymic sites (in the liver). The first gamma/delta T cells were detected as early as 40 days of gestation. The expression of fibronectin, Thy-1 and its message, Ig isotypes and the first induction of IFN alpha were described.