Zuzana Rehakova

Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells

Background: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RBhigh subset of CD4+T cells isolated from the spleen of normal BALB/c mice. Methods: A CD4+CD45RBhigh subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ‐free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen‐free (SPF) bacteria. Mice were evaluated 8–12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). Results: After the transfer of the CD4+CD45RBhigh T‐cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO‐1 (zona occludens). Conclusions: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB. (Inflamm Bowel Dis 2007)

Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38–110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

Early Ontogeny of Thymocytes in Pigs: Sequential Colonization of the Thymus by T Cell Progenitors

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3ε-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3εhigh and belonged to the γδ T cell lineage. Mature CD3εhigh αβ thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3εlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Rα (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRγδ+ thymocytes that were cycling late during their development in the thymus.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with w. As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

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.

Monoclonal antibodies putatively identifying porcine B cells

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Characterization of monoclonal antibodies recognizing immunoglobulin κ and λ chains in pigs by flow cytometry

Abstract 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 (Igκ) and Ig lambda (Igλ). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igκ and Igλ 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 Igλ antiserum and were negative when tested using anti-human Igκ 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 Igλ-specific whereas mAb 27.2.1 recognizes the Igκ 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 Igλ+ B cells in most pigs. In summary, we identified two pan-specific anti-pig Igλ mAbs, one anti-λ mAb that recognizes a λ-light chain subset and one anti-pig Igκ mAb.

Bifidobacterium bifidum monoassociation of gnotobiotic mice: Effect on enterocyte brush-border enzymes

The effect of intestinal colonization withBifidobacterium bifidum (Gram-positive anaerobic bacterium colonizing the intestine of healthy new-born mammals, exhibiting a probiotic effect, protecting the intestinal mucosa against colonization by pathogenic microflora) on enterocyte brush-border enzymes was examined in weaned 23-d- and in 2-month-old gnotobiotic inbred mice and compared with that in corresponding germ-free (GF) and conventional (CV) controls. The two groups of GF mice were associated with humanB. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes—lactase, alkaline phosphatase and γ-glutamyltranspeptidase was significantly higher in both age groups of GF mice in comparison with CV ones; on the other hand, sucrase and glucoamylase activities were higher in CV mice. Monoassociation withB. bifidum accelerates biochemical maturation of enterocytes resulting in a shift of specific activities of brush-border enzymes between the values found for GF and CV mice. This effect ofB. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase IV in immature gut of weaned mice than of 2-month-old ones.

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs.

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.

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 Peyers 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).