Intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated Salmonella enterica serovar enteritidis antigen.

Induction of intestinal mucosal immune responses against Salmonella enterica serovar enteritidis was studied by immunizing chickens with liposome-associated antigen. An ultrasonicated whole cell extract of the bacteria was used for immunizing antigen. Intraocular immunization induced serum IgA, IgG and IgM responses. Also, significant IgA and IgG antibodies were detected in the intestinal tract. Immunization with antigen alone induced only IgG response in the intestine. Salmonella enteritidis-specific antibody-secreting lymphocytes were detected in the spleen and lamina propria of the intestinal tract of immunized chickens. Immunoglobulin (Ig) fractions extracted from intestines of immunized chickens inhibited the adherence of S. enteritidis to cultured HeLa cells. These results indicate that intraocular immunization with liposome-associated S. enteritidis elicits specific antibody-producing lymphocytes in the intestinal tract, and that Ig secreted in the intestine inhibits adherence of the bacteria to intestinal epithelial cells, suppressing the spread of bacterial infection in the host.

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio).

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.

Perinatal development of the rat kidney: proliferative activity and epidermal growth factor.

The immunolocalization of proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF) and its receptor (EGFR) was examined in the perinatal rat kidney. As the index of proliferative activity, PCNA-positive cell ratios in glomeruli and proximal tubules were determined. The PCNA-positive ratios in both glomeruli and proximal tubules decreased significantly during the perinatal period and tended to decrease as the glomerular developmental stage proceeded as well. PCNA-positive cells were seen predominantly in the nephrogenic zone of the kidney throughout the period examined. They were noted in the collecting ducts of the nephrogenic zone and were rarely seen in those of the central zone of the kidney. On the other hand, PCNA-positive cells were noted in the straight portion of the proximal tubules and were rarely seen in the convoluted one. EGF-positive cells were seen in the proximal tubules, distal tubules and collecting ducts, though EGF-positive cells in the proximal tubules decreased after birth. EGFR-positive cells were seen along the entire length of the proximal tubules and collecting ducts as well as in immature glomeruli, not in mature ones. These results indicate that marked cell proliferation occurs in the collecting ducts in the peripheral area and in the proximal tubules in the central area of the kidney, that the proliferative activity decreases with age during the perinatal days and that EGF plays an important role in the proliferation of glomerular cells, and in both proliferation and maturation of the cells in the proximal tubules and collecting ducts.

Regulation of early chicken thymocyte proliferation by transforming growth factor-beta from thymic stromal cells and thymocytes.

We examined expression of TGF-betas in chicken thymic stromal cells and thymocytes and roles of TGF-betas in thymocyte development within the thymus. Thymic stromal cells expressed TGF-beta 2 and 3 genes but not TGF-beta 4 gene. Thymocytes showed expressions of TGF-beta 2, 3 and 4 genes and each TGF-beta gene was expressed more strongly in CD3- than CD3+ thymocytes. When anti-TGF-beta antibody was added with supernatants of stromal cells into thymocyte culture, only proliferative activity of CD3- thymocytes was enhanced and the cells in S and G2/M compartments of cell cycle increased. These results suggest that TGF-beta which is expressed in the thymus may regulate the ability of immature thymocytes to progress through the cell cycle and to differentiate to CD3+ thymocytes.

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).

Uterine NK cells produce epidermal growth factor in the murine pregnant uterus.

Uterine natural killer (uNK) cells belong to the large granular lymphocytes in the murine pregnant uterus and play essential roles in pregnancy success. We defined whether uNK cells can produce epidermal growth factor (EGF) important for implantation and embryo growth. The uNK cells were immunohistochemically positive for anti-EGF antibody especially during days 6 to 9 and at day 15 of pregnancy. Immunoreaction for EGF receptor was observed on the stromal cells in the metrial gland and trophoblasts in the placental labyrinth. EGF secretion (72.1 +/- 2.25 ng/10(40 cells) was noted in cultured uNK cells isolated from the metrial gland at day 15 of pregnancy. Treatment of anti-asialo-GM I antibody raised the level of EGF (129 +/- 21.5 ng/10(4) cells). These results suggest uNK cell can produce and release EGF for placental development.

Effects of chicken thymic stromal cells on the growth and differentiation of thymocytes in vitro.

We examined contact-mediated effects of chicken thymic stromal cells (TSC) on thymocyte differentiation by co-cultivation of these cell populations. The primary cultures of TSC isolated from thymus mainly have consisted of epithelial cells which were polygonal in shape, possessed long processes and expressed MHC class II antigen. When thymocytes were co-cultured with TSC, 60% to 70% of thymocytes attached to TSC and some of them engulfed underneath TSC. These attached thymocytes were CD4-CD8- and CD4+CD8+ subsets and expressed alpha/beta TCRhigh or gamma/delta TCRlow. Some of the thymocytes attaching to TSC showed an increase of intracellular and nuclear density, fragmentation of cytoplasm and nuclei, and DNA fragmentation. And also, thymocytes attaching to TSC contained a higher percentage of cycling (S and G2 + M phase) cells than nonattaching cells. These results indicate that specific subsets in thymocytes selectively bind to TSC and undergo apoptotic death or proliferation because of interaction with TSC. Chicken TSC may play an important role in thymic differentiation by direct contact within the thymus as in mammals.

Effects of cytokines from thymocytes and thymic stromal cells on chicken intrathymic T cell development.

We have studied the ability of thymic stromal cells (TSC) and thymocytes to produce cytokines and the involvement of cytokines in intrathymic T cell development. When thymocytes were co-cultured with thymic stromal cells in absence of direct contact and mitogenic stimulation, induction of thymocyte proliferation was observed. Supernatants of cultured stromal cells (TSC-CS) promoted a high proliferative response on CD3- thymocytes but had little effect on CD3+ thymocytes. These results indicate that stromal cells have produced a cytokine which can induce immature thymocyte proliferation. Moreover, stromal cells express the MRNA for stem cell factor (SCF) and c-kit (the receptor for SCF) was detected on CD3- thymocytes but not on CD3+ thymocytes. Since SCF can enhance the proliferation of immature thymocytes in synergy with IL-7 in mammals, there is a possibility that chicken stromal cells may produce a IL-7-like factor. Thymocytes have clearly expressed interferon (IFN)-gamma. In contrast, thymic stromal cells showed no detectable expression of IFN-gamma. CD3+ thymocytes express IFN-gamma MRNA more strongly than CD3 thymocytes, suggesting that IFN-gamma from thymocytes may operate on stromal cells and then may indirectly induce clonal elimination of CD3+ cells on stromal cells. The expression of these cytokines and receptors by thymic stromal cells and thymocyte subpopulations suggests that these cytokines participate in paracrine interactions between these cell populations during thymocyte differentiation.

Enhancement of phagocytic and chemokinetic activities of rainbow trout head kidney cells by C-reactive protein.

OBJECTIVE: To investigate immunostimulating activity of purified rainbow trout (Oncorhynchus mykiss) C-reactive protein (CRP) on trout phagocytic cells. ANIMALS: 20 rainbow trout and 2 rabbits. PROCEDURE: The effect of CRP on phagocytic activity of head kidney (HK) cells was examined by use of a phagocytosis assay with plastic particles. The enhancing effect of CRP on migration activity of HK cells was examined by use of the blind well assay. RESULTS: Glass-adherent cells from clinically normal trout had increased dose-dependent phagocytic activity against plastic particles when cells were incubated in the presence of CRP. Pretreatment of particles with CRP also enhanced phagocytic activity of the cells, indicating an opsonic effect of CRP. Rabbit anti-trout CRP serum suppressed the enhancing activity of CRP. The HK cells had significant dose-dependent chemokinetic activity against CRP that was not inhibited by anti-CRP serum, indicating that a CRP-antibody complex also could be chemokinetic. CONCLUSIONS: Rainbow trout CRP has immunostimulating activity for HK cells, resulting in enhanced phagocytic and chemokinetic activities.

Cytotoxicity induced by recombinant human tumor necrosis factor-alpha dependent on the types of its receptors on canine cells.

Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-alpha has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-alpha on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-alpha receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-alpha induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-alpha. Combined with ACT-D, rh-TNF-alpha showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-alpha depends on the presence of TNF RI receptors on canine tumor cells.

Effect of tylosin tartrate (Tylan Soluble) on cellular immune responses in chickens.

Although many antimicrobial agents have been reported to cause immunosuppression in animals, macrolide antibiotics enhance immune function. Tylosin is a macrolide antibiotic approved for the control of mycoplasmosis in poultry. The purpose of this investigation was to determine the effect of tylosin on cellular immune functions in chickens. There was no significant difference in adherent splenocyte chemotaxis between tylosin-treated and untreated (control) chickens. Tylosin increased splenocyte proliferation and splenocyte conditioned medium (CM) proliferative activity above control levels. Removal of adherent splenocytes before preparation of CM caused a reduction in CM proliferative activity. Tylosin also increased antitumor activity of splenocytes. These data are the first to suggest that the macrolide antibiotic, tylosin tartrate, has a modulatory effect in chickens on the immune parameters studied.

Effect of tylosin tartrate on humoral immune responses in chickens.

While many antimicrobial agents have been reported to cause immunosuppression in animals, macrolide antibiotics enhance the immune function. Tylosin tartrate is a macrolide antibiotic approved for the control of mycoplasmosis in poultry. The purpose of this investigation was to determine the effect of tylosin on the humoral immune functions in chickens. Three days after oral tylosin tartrate administration, 4- or 8-week-old chickens were immunized intravenously with carbolic acid-killed Brucella abortus bacterin or sheep red blood cells. Seven days (plasma antibodies titre) or 4 days (antibody forming cells) post-immunization, there was a significant increase in antibody production as well as in the numbers of antibody-producing cells in tylosin tartrate-administered chickens compared with the untreated controls. However, 3 days after tylosin tartrate administration, there was no difference in the distribution of T-lymphocyte subpopulations (CD4 or CD8 positive cells) or B lymphocytes (surface immunoglobulin positive cells) in either the peripheral blood or spleens or untreated control chickens.

Thymic stromal cells produce soluble factors which increase polarization of chicken thymocytes.

The effects were examined of soluble factors present in culture supernatant (CS) of thymic stromal cells (TSC) on the differentiation and locomotor activity of chicken thymocytes. The locomotor activity of thymocytes was assessed by cell polarization assay. When thymocytes were incubated in the presence of TSC-CS, the proportion of polarized cells increased. This indicates that thymocytes acquired a potent locomotor activity. A high proportion of peanut agglutinin-positive (PNA + ) thymocytes, as well as CD4(-)CD8(-) and CD4(+)CD8(+) thymocytes showed polarization in TSC-CS. Bone marrow cells exhibited higher level of polarizing activity compared to CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes and spleen T cells. These results suggest that thymic stromal cells secrete a soluble factor(s) which enhances mobilizing activity of immature T cells. The factor may take part in the intrathymic migration of progenitor T cells into the site of differentiation.

Immunopotentiating activities of polysaccharide fraction from pine seed shell extract.

The function of polysaccharide (PSE) extracted from pine seed shells as a immunopotentiator was investigated. The phagocytosis and chemotaxis of mouse peritoneal macrophages (MP) were augmented by oral administration of PSE. The incorporation of 3H-thymidine by Con A-stimulated mouse splenic cells was significantly intensified by administration of PSE but the adherent-cells (MP)-eliminated splenic lymphocytes was not increased. It is shown that the existence of activated MP is needed for the activation (proliferation) of T lymphocytes. The responsiveness of mouse T cells to alloantigen was also augmented by administration of PSE. The number of anti-SRBC plaque-forming cells in the spleen of mouse and chicken was also significantly increased. The results indicate that MP are first activated by oral administration of PSE and that the activation of T cells is then initiated indirectly by humoral factors (cytokines) produced by activated MP.

Possible migration of harderian gland immunoglobulin A bearing lymphocytes into the caecal tonsil in chickens.

This study was designed to evaluate the significance of the Harderian gland (HG) in the chicken IgA-production system by investigating the influence of HG on the increase of surface IgA (sIgA)-positive cells in other lymphoid organs and also the possibility of migration of HG lymphocytes into other organs. Non-contact culture of splenic or caecal tonsil (CT) lymphocytes with HG whole cells did not enhance the expression of sIgA. The experiment of HG lymphocyte transfer showed that the transferred HG lymphocytes migrated mainly into the CT in the 3-week-old recipient, and mainly into the CT and bursa of Fabricius (BF) in the 6-week-old recipient. It was also found that the transferred sIgA-positive HG lymphocytes migrated selectively into CT in both 3-week-old and 6-week-old recipients. These results indicated that chicken HG plays a central role not only in governing the local immunity in the eyes and respiratory tract but also as an organ which supplies precursory IgA-producing cells involved in local immunity in the intestine.

Detection of Salmonella DNA in chicken embryos and environmental samples by polymerase chain reaction.

By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.

Chicken thymocyte antigen which participates in cell proliferations of thymocytes and tumour-derived lymphoid cell lines.

A novel antigen on chicken thymocytes was defined by CETHB1 and CETH46 monoclonal antibodies (mAbs) that were prepared against chick embryonic thymocytes. CETHB1 and CETH46 mAbs recognized different epitopes on the same membrane antigen (molecular weight: 76.2 kDa). These mAbs reacted with >80% of thymocytes of 14-day-old embryos to 8-week-old chickens. Almost all splenocytes, peripheral blood lymphocytes and bursa cells were negative, and only 7.7% of bone marrow cells were positive for both antibodies. In two-colour analysis with mAbs reacting to T cell markers (CD4 or CD8), most CETHB1 positive cells were CD4- CD8- or CD4+ CD8+. However, a proportion of CD4+ CD8- and CD4- CD8+ cells were negative for CETHB1 mAb. The proportion of thymocytes reacting with CETHB1 in chickens immunosuppressed by cyclophosphamide treatment increased gradually in parallel with the restoration of the thymus. An increase of CETHB1-positive cells was observed in thymocytes stimulated with Con A. Hence, it seems that the CETHB1 antigen expression on thymocytes is influenced by the thymic micro-environment and that the antigen may take part in thymic differentiation. Interestingly, CETHB1 antigen was expressed not only on T cell tumour-derived lymphoid cell lines, but also B-lymphoma-derived cell lines. The antigen expressions on these cell lines were observed only in the proliferative phase of the cells. Hence, the molecule which reacted with CETHB1 may be an antigen commonly expressed on lymphoma cells and may be involved in cell proliferation.

Participation of a chicken thymocyte antigen in intrathymic differentiation of T cells in vivo.

The participation of CETHB1 antigen in the intrathymic development of T cells was analysed in sublethally X-ray irradiated chickens and normal embryos which were inoculated with anti-thymocyte monoclonal antibody CETHB1. When chickens were given a sublethal X-ray irradiation on the day of hatchings immature thymocytes existed prominently in the thymus up to 3 weeks after irradiation. The inoculation of CETHB1 monoclonal antibody on days 14 and 18 after irradiation caused an increase of CD4- CD8- cells and a decrease of CD4+ CD8+ cells as compared to changes in control chickens. However, expression of T cell receptors CD3, alphabetaTCR, and gammadeltaTCR was not influenced. Inoculation of the monoclonal antibody on embryonic days 13 to 16 resulted in a decrease of CD4+ CD8- cells and an increase of CD4-CD8- and CD4+ CD8+ cells after hatching. Thus, the binding of CETHB1 monoclonal antibody to thymocytes can inhibit the differentiation of both CD4-CD8- and CD4+ CD8+ cells to CD4+ CD8+ and CD4+ CD8- or CD4-CD8+ cells, respectively. These results suggest that CETHB1 antigen participated in the growth and differentiation of thymocytes in the thymic microenvironment.

Suppression of T lymphocyte proliferation by peritoneal macrophages in chickens.

The mechanism of the suppression of lymphocyte proliferation by peritoneal macrophages (MPs) in chickens and the presence of a suppressive substance in the culture supernatant of MPs were studied. The results showed that gelatin- and peptone-induced peritoneal MPs had an inhibitory accessory function against blastogenesis of mitogen-stimulated splenic lymphocytes and that resident peritoneal MPs had an enhanced accessory function. The suppressive factor produced by gelatin-induced peritoneal MPs is a protein having a pI of 3.6 and a molecular weight of 64,000 and it is physically and chemically a relatively stable substance. Moreover, it has no cytotoxic effect against lymphocytes and it was verified that the suppressor in the culture supernatant of MPs was not prostaglandin E2 or alpha-acid glycoprotein.

Inhibition of cytotoxic activity of carp lymphocytes (Cyprinus carpio) by anti-thymocyte monoclonal antibodies.

A series of monoclonal antibodies (mAbs) were developed against carp thymocytes and carp immunoglobulin (IgM). Anti-thymocyte mAbs reacted with thymocytes, head kidney lymphocytes (HKL) as well as peripheral blood lymphocytes (PBL) at various proportions. Anti-IgM antibody reacted with HKL and PBL but not with thymocytes. PBL from carp immunized with xenogeneic mouse myeloma cells exhibited cytotoxic activity against target cells but PBL from unimmunized fish showed weak cytotoxicity. Treatment of PBL with anti-thymocyte mAbs and guinea pig complement suppressed the cytotoxic activity dose dependently; however, anti-IgM mAb did not. These results indicate the cytotoxic cells in PBL possess cell surface antigens common to thymocytes.