Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella Typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine.

The colonization, translocation and protective effect of two intestinal bacteria - PR4 (pig commensal strain of Bifidobacterium choerinum) or EcN (probiotic Escherichia coli strain Nissle 1917) - against subsequent infection with a virulent LT2 strain of Salmonella enterica serovar Typhimurium were studied in gnotobiotic pigs after oral association. The clinical state of experimental animals correlated with bacterial translocation and levels of inflammatory cytokines [a chemokine, interleukin (IL)-8, a proinflammatory cytokine, tumour necrosis factor (TNF)-α and an anti-inflammatory cytokine, IL-10] in plasma and intestinal lavages. Gnotobiotic pigs orally mono-associated with either PR4 or EcN thrived, and bacteria were not found in their blood. No significant inflammatory cytokine response was observed. Mono-association with Salmonella caused devastating septicaemia characterized by high levels of IL-10 and TNF-α in plasma and TNF-α in the intestine. Di-associated gnotobiotic pigs were given PR4 or EcN for 24 h. Subsequently, they were infected orally with Salmonella and euthanized 24 h later. Pigs associated with bifidobacteria before Salmonella infection suffered from severe systemic infection and mounted similar cytokine responses as pigs infected with Salmonella alone. In contrast, EcN interfered with translocation of Salmonella into mesenteric lymph nodes and systemic circulation. Pigs pre-associated with EcN thrived and their clinical condition correlated with the absence of IL-10 in their plasma and a decrease of TNF-α in plasma and ileum.

Cytokine response to Escherichia coli in gnotobiotic pigs.

One-week-old-germ-free pigs were inoculated with 10(8) CFU of E.coli bacteria-either commensal 086 strain or virulent 055 strain for 1 d. Bacteria were counted in the small intestine, mesenteric lymph nodes, blood and lungs. The O55 strain reached higher levels in circulation and lungs. IL-8, IL-10 and TNF-alpha concentrations were determined by ELISA in plasma and intestinal washes . No difference in cytokine levels was found between control germ-free pigs and their counterparts associated with commensal O86 strain in spite of its high concentration in the gut and circulation.

Probiotics manipulate host cytokine response and induce antimicrobial peptides.

Probiotics modulate production of both cytokine and antimicrobial peptides. This effect can be regarded as a part of complex interplay between them and the host.

Ontogeny of J chain expression in pig lymphocytes.

The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.

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

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.

Cells adherent to IUDs produce tumor necrosis factor alpha.

The nature of adherent cells obtained from the surface of intrauterine contraceptive devices (IUD) was investigated by immunofluorescence using antibodies directed against tumor necrosis factor alpha (TNF), leukocyte differentiation antigens, immunoglobulin isotypes and J chain. Macrophages/monocytes, lymphocytes and neutrophils were found in all fifty samples. Macrophages were characterized by CD14, CD15 and CD71 positivity, DAKO macrophage antibody and HLA-DR, CD11c and CD45 expression. B cells contained secretory IgA with J chain or IgG and some cells expressed surface IgM. Neither IgD nor IgE were found. T-antigens CD3, CD4 and CD5 were not detected, the CD8 was faintly expressed. The CD56 molecule identifying NK cells was found on small lymphocytes. Cytoplasmic C3 protein was detected in neutrophils. Tumor necrosis factor alpha (TNF) was visualized in 14.3 +/- 8.8% of nucleated IUD cells. This cytokine was localized in the cytoplasm of the macrophages and the lymphocytes.

Effect of peroral anti-bacterial antiserum treatment on intestinal immune parameters of germ-free piglets intragastrically infected with virulent Salmonella typhimurium or enteropathogenic E. coli.

Germ-free piglets were orally infected with either enteropathogenic E. coli 055 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-alpha 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.

Development of immune responses in early pig ontogeny.

Low amounts of immunoglobulins, produced without any known cause of stimulation, can be detected in sera and cells of fetal and colostrum deprived newborn pigs. These immunoglobulins are believed to represent the preimmune antibody repertoire on the basis of their polyspecificity and reactivity against self antigens. In vitro activation of liver and spleen cells with various polyclonal B cell activators (PBA) results in pronounced immunoglobulins synthesis as measured in the culture media by enzyme-linked immunosorbent assay. Intrauterine injection of fetal and germfree pigs with PBA led to increased IgM, IgG and IgA levels in sera. Specific responses during fetal development were studied after intrauterine immunization. Antibodies to the heapten and its carrier flagellin, could be detected 7 days after the immunization of 55-day-old fetuses. Fetal and colostrum germfree pigs may be useful experimental models in which developmental immunity can be studied in the absence of maternal antibodies and environmental antigens.

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.

Analysis of monoclonal antibodies reactive with the porcine CD4 antigen.

As result of the First International Swine CD Workshop, four monoclonal antibodies (mAb) (#002, 054, 118, and 127) clustered closely to the internal standard anti-porcine CD4 mAb, 74-12-4. To further characterize the relationship between these mAb, they were used in flow cytometry to inhibit binding of 74-12-4 to porcine lymphocytes. mAb #002 (74-12-4) and #054 (PT90) completely inhibited, while mAb #127 (10.2H2) and #118 (b38c6) partially inhibited (57% and 77% respectively), the binding of 74-12-4. Furthermore, none of the mAbs bound to a 74-12-4 negative strain of pigs. We conclude that the four mAb bind to the same, or a closely related, epitope on porcine lymphocytes.

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3 epsilon-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 epsilon designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop.

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8+ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8+ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8low and CD8high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8low and CD8high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4-/CD8high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8+ lymphocyte subsets in health and disease.

Analyses of monoclonal antibodies reacting with porcine CD5: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group, three mAbs fell within cluster T13 including the CD5a standard b53b7 (No. 174). The two new mAbs 1H6/8 (No. 058) and BB6-9G12 (No. 166) both precipitated 55 and 60 kDa proteins that were of similar molecular weights as the standard. Staining patterns on the various cell types were similar. Both new antibodies inhibited the binding of the CD5a reference mAbs b53b7 to peripheral lymphocytes. These mAbs, therefore both react with the CD5a epitope bringing the number of anti-porcine CD5 mAbs to eight, all of which appear to recognize the same epitope.

Analyses of monoclonal antibodies reacting with porcine wCD6: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.

Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop.

After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer's patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.

Calprotectin - a pleiotropic molecule in acute and chronic inflammation.

Calprotectin (MRP8/14, S100A8/S100A9, 27E10 antigen) is a heterodimer of two calcium-binding proteins present in the cytoplasm of neutrophils and expressed on the membrane of monocytes. Upon neutrophil activation or endothelial adhesion of monocytes, calprotectin is released and may be detected in serum or body fluids as potentially useful clinical inflammatory marker. The soluble form of calprotectin provides both bacteriostatic and cytokine-like effects in the local environment. When calprotectin metabolism is affected on a systemic level, the zinc-binding properties of protein may induce severe dysregulation of zinc homeostasis with severe clinical symptoms. The distribution of membrane form of calprotectin is restricted to monocytes and immature macrophages and the presence of calprotectin-positive infiltrating cells reflects the influx of mononuclear phagocytes to the site of inflammation. Calprotectin expression and release seems to be of particular importance in immune and immunopathological reactions.

Escherichia coli administered into pig amniotic cavity appear in fetal airways and attract macrophages into fetal lungs.

Escherichia coli (2 x 10(4) bacteria) of the non-pathogenic O86 strain or enteropathogenic O55 strain were administered into the pig amniotic cavity at 79 to 86 days of gestation for six or ten hours. Translocation of bacteria into fetal lungs was confirmed by cultivation as well as by light and electron microscopy. Infection caused an influx of macrophages that were immunostained in cryostat sections by monoclonal antibody recognizing calprotectin.

Early ontogeny of monocytes and macrophages in the pig.

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.

Successive immunofluorescent, cytological, and ultrastructural examination of the same cell.

A method is described for processing of cells with accurate orientation of their position. Marked surface of the polyester film has been utilized as an orientation plane for examined biological structures.

Expression of HLA-DR molecules and some other differentiation antigens within early human fetus.

Leukocyte membrane markers have been examined within cryostat sections and cell suspensions of human fetuses aged between 4 and 18 weeks of gestation using a panel of monoclonal antibodies. The HLA-DR molecule was present on a population of erythroid cells and macrophages in liver sinuses at 34 days and periarteriolar splenic macrophages at 60 days of gestation. CD5, CD15 and CD45 antigens were detected at 6 weeks of gestation, whereas CD4, CD8, CD10, CD11c, CD14 and IgM were not expressed at this stage. These findings and ultrastructural examination of embryonic liver confirm early differentiation of macrophages in the pre-lymphatic developmental period.