Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens

The impact of live and killed Salmonella vaccines on cell-mediated immunity (CMI) was investigated in 18- and 32-week-old White Leghorn chickens, by assessing splenic lymphocyte proliferation, expression of IL-2 mRNA in concanavalin A (Con A) stimulated cells and flow cytometric analysis of cell subpopulations. Con A and Salmonella enteritidis (SE) flagella induced proliferation of splenocytes were enhanced in the 18- and 32-week-old chickens treated with live vaccine, compared to the corresponding control chickens. Among the killed vaccine treated birds, Con A-mediated response was higher in the 18-week-old chickens compared to the corresponding control birds. Increased proliferation was accompanied by increased CD4 and reduced CD8 and gammadelta T-lymphocytes in the 18-week-old live vaccine treated chickens. Relative expression of IL-2 mRNA in Con A-stimulated splenocytes from 18-week-old birds was not affected by vaccine treatment. Overall, live vaccine was more effective in increasing the lymphocyte proliferation to Con A as well as SE antigen. This enhanced CMI may prove beneficial in protecting chickens against SE infection.     

The effect of Pasteurella haemolytica A1 leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes

The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the leukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemolytica leukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures.     

Differential cytokine gene expression in CD4+ and CD8+ T cell subsets of calves

The distinct patterns of cytokine expression in CD4+ and CD8+ T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4+ and CD8+ T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4+ and CD8+ T cells of calves with or without Concanavalin A (Con A) stimulation. CD4+ and CD8+ T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4+ T cells spontaneously expressed the mRNAs of interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta, and augmented the mRNA expression of IL-10, IFN-gamma and TNF-beta after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4+ T cells. CD8+ T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-alpha, TNF-beta and TGF-beta, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-gamma) and GM-CSF after Con A stimulation. It was found that CD4+ T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8+ T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4+ and CD8+ T cells of calves, indicating that CD4+ T cells can be distinguished from CD8+ T cells by the cytokine gene expression of IL-1alpha, IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4+ and CD8+ T cells serve to interpret an individual function of T cell subsets in the immune system of calves.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Parameters of production and partial characterization of feline interleukin 2

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 micrograms/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promoter, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL-2 production. Fetal calf serum (FCS) was not required for IL-2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con A-activated feline PBL at a final concentration of 0.3 to 0.9 units/ml.     

Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus

A system for a reproducible in vitro restimulation of bovine viral diarrhea virus (BVDV)-specific cytotoxic T-cells (CTL) was developed. Lymphocyte cultures of BVDV-immunised cattle were stimulated with infectious BVDV isolate PT810 and recombinant bovine interleukin-2 for 12 to 25 days. A specific lysis of Concanavalin A-stimulated BVDV-infected autologous target cells was observed, whereas allogeneic BVDV-infected target cells were only marginally lysed as detected by flow cytometry. BVDV-specific lymphocyte transformation was further characterised by the expression of bovine lymphocyte activation antigens and bovine MHC class-II molecules. Secondary stimulation of CTL was influenced by in vitro production of BVDV-specific neutralising antibodies, which were secreted exclusively in BVDV-inoculated lymphocyte cultures of immunised cattle. These results demonstrate the presence of CTL in peripheral blood mononuclear cells (PBMC) of immunised cattle which can kill autologous BVDV-infected antigen-presenting cells after in vitro restimulation.     

Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.     

重组牛白细胞介素－2对不同种属动物淋巴细胞的作用

以重组牛白细胞介素－２作用于ＣｏｎＡ活化的不同种属动物的淋巴细胞，结果发现重组牛白细胞介素－２对人、犬、猪、绵羊以及鸡淋巴细胞没有作用；对山羊、豚鼠及小鼠淋巴细胞具有显著作用；对牛、驴和兔淋巴细胞具有极显著的作用。     

Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.     

Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro

Dendritic cells (DCs) are professional antigen presenting cells cooperating with other immune cells for the activation of innate and adaptive immune responses. The objective of the present study was to investigate the replication activity of porcine circovirus type 2 (PCV2) in DCs and/or lymphocytes during their cross talk and its possible mechanism. Two models were set, herein. Swine blood monocyte (Mo)-derived DCs (MoDCs) or peripheral blood lymphocytes (PBLs) were inoculated with PCV2 prior to their co-cultivation. Bacterial lipopolysaccharide (LPS) and concanavalin A (Con A) were used to stimulate MoDCs and PBLs, respectively. During 6 days of cultivation, a high PCV2 antigen-containing rate without detectable intranuclear signals and a slight but significant increase in the copy number of PCV2 genome were detected in PCV2-inoculated MoDCs. The presence of LPS alone or PCV2-free PBLs, however, had no effect on the location of PCV2 antigens or copy number of PCV2 genome in PCV2-inoculated MoDCs. On the contrary, active PCV2 replication occurred in Con A-stimulated PCV2-inoculated PBLs. When compared with blood Mos, MoDCs induced significantly higher cell proliferation and intensified PCV2 replication in Con A-stimulated PCV2-inoculated PBLs, for which direct contact between MoDCs and lymphocytes was required. Among the cytokines secreted by Con A-activated PBLs, interleukin (IL)-2, but not IL-4 or interferon-γ, could induce cell proliferation and PCV2 replication in PCV2-inoculated PBLs. The findings suggest that although MoDCs support only limited PCV2 replication in themselves, their accessory cell function is required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes.     

Differential cytokine expression in T cell subsets from bovine peripheral blood

Although distinct cytokine expression in T cell subsets is well understood in mice and humans, limited information is available on bovine T cell subsets. In the present study, we analyzed the mRNA expression of 10 kinds of cytokines and CD25 expression in CD4⁺, CD8⁺, WC1⁺ and WC1^-γδ T cell subsets in bovine peripheral blood by Concanavalin A (Con A) stimulation. CD25 expression was significantly increased in CD4⁺, CD8⁺ and WC1⁺γδ T cells, but not in WC1^-γδ T cells by Con A stimulation. In CD4⁺ T cells, the mRNAs of Interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β and transforming growth factor (TGF)-β were expressed in control cultures, and IL-3, IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were newly expressed when the cells were stimulated with Con A. CD8⁺ T cells expressed the mRNAs of IL-6, TNF-α, TNF-β and TGF-β in control cultures, and newly expressed those of IL-2, IFN-γ and GM-CSF, but did not express those of IL-3, IL-4 or IL-10 after Con A stimulation. The cytokine expression profile of WC1⁺γδ T cells was similar to that of CD8⁺ T cells. However, WC1^-γδ T cells did not express any cytokine mRNA except TGF-â mRNA. These results will contribute to elucidate the participation of T cell subsets in immune responses against infectious disease in cattle.     

Immunomodulation in cattle immunized with Brucella abortus strain 19

Regulation of the bovine immune response to immunization with Brucella abortus Strain 19 (S19) was investigated through application of a modification of an assay to measure suppressor T lymphocyte activities in humans and through development and characterization of antigen-stimulated T lymphocyte lines in vitro. A total of nine of steers were alloted into two groups: control (n = 4) and S19-immunized (n = 5). Peripheral blood mononuclear cells (PBMC) from each animal were cultured in vitro with mitogens (concanavalin A (Con A) and pokeweed mitogen (PWM], B. abortus antigens (B. abortus soluble antigen (BASA) and whole heat-killed B. abortus cells (HKC)) and media alone periodically from days 4 through 49 of the experiment. Supernates from these cultures were assayed for immunomodulatory activity(s) by addition to indicator cultures stimulated with suboptimal concentrations of Con A. Supernates from PBMC of S19-immunized steers generated with B. abortus antigens significantly (P less than 0.05) suppressed indicator cell responses as compared to those from control steers on days 35 and 49 post-immunization. This suppressive activity from PBMC of immunized cattle with respect to that of control cattle could also be induced through mitogenic stimulation with Con A or PWM. On day 49 of the study, suppressive activity was spontaneously released from the PBMC of immunized cattle. T lymphocyte lines were initiated from two S19-immunized steers at 2 and 9 weeks post-immunization. These T cell lines were characterized with respect to proliferative responses to B. abortus antigens through in vitro assay and surface marker expression through indirect immunofluorescence with a limited panel of monoclonal antibodies. Results from the present study indicated that S19 immunization induces a subpopulation(s) of cells in the PBMC of cattle capable of regulating the in vitro response to B. abortus. This regulatory activity is detectable by in vitro assay as early as 7 weeks post-immunization. Furthermore, the regulatory cell(s) appear to involve BoCD8+ T, lymphocytes which are specific for B. abortus antigens.     

Immunosuppression by canine distemper virus: modulation of in vitro immunoglobulin synthesis, interleukin release and prostaglandin E2 production

In vitro or in vivo infection of canine mononuclear cells by canine distemper virus (CDV) in short-term microcultures resulted in suppression of lectin-induced 3H-thymidine incorporation. This suppressive effect was also evident in pokeweed mitogen-driven in vitro immunoglobulin synthesis and release. Lectin-induced interleukin-2 production by monocyte-depleted lymphocyte cultures was marginally affected by CDV, whereas interleukin-1 production by adherent mononuclear cells was significantly depressed. Monocyte cultures established from viremic dogs released prostaglandin (PG)E2. The results suggest that, in addition to a direct viral effect upon lectin responsive cellular population(s), CDV modulates monocyte functions by inhibition of interleukin-1 production and by enhancing PGE2 release.     

Pathogenic and immunosuppressive effects of avian pneumovirus in turkeys

Avian pneumovirus (APV) causes a respiratory disease in turkeys. The virus has been associated with morbidity and mortality due to secondary infections. Our objective was to determine if APV caused immunosuppression in the T-cell or B-cell compartments and to study the pathogenesis of the disease in APV maternal antibody-lacking 2-wk-old commercial turkeys. APV was administered by the eyedrop/intranasal route. Observations were made for gross lesions, viral genome, and T-cell mitogenesis and cytokine secretion at 3, 5, 7, 14, and 21 days postinoculation (DPI). During the acute phase of the disease that lasted for about 1 wk, the turkeys exposed to APV showed clinical signs characterized by nasal discharge and sinus swelling. Virus genome was detected by in situ hybridization in cells of turbinates and trachea at 3 and 5 DPI. At 3 and 5 DPI, spleen cells of the birds infected with APV markedly decreased proliferative response to concanavalin A (Con A). Con A and lipopolysaccharide stimulation of spleen cells from virus-exposed turkeys resulted in accumulation of nitric oxide-inducing factors (NOIF) in the culture fluid. NOIF were not detected in culture fluids of Con A-stimulated spleen cells of virus-free turkeys. APV did not compromise the antibody-producing ability of turkeys against several extraneous antigens such as Brucella abortus and tetanus toxoid.     

Cytokine modulation of the interaction between bluetongue virus and endothelial cells in vitro.

An in vitro model was developed to examine the interaction between endothelial cells and the host inflammatory response in bluetongue virus (BTV) infections. Whole cell enzyme-linked immunosorbent assays, a tritiated thymidine uptake assay, and a colorimetric assay of mitochondrial function were used to assess how four cytokines (interleukin-1, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) affect endothelial cell metabolism and susceptibility to BTV infection. Concurrent alterations in major histocompatibility complex (MHC) antigen expression were also examined. BTV infection suppressed target cell mitochondrial function and DNA synthesis and enhanced MHC class I expression. Interferon-gamma and tumor necrosis factor alpha suppressed viral antigen expression and were synergistic early in the infection. Interferon gamma enhanced MHC class I and induced MHC class II antigen expression in both BTV infected and uninfected endothelial cells. The other cytokines had minimal effect on endothelial cell surface antigen expression, although interleukin-1 (IL-1) did inhibit cell growth. Infected endothelial cell cultures produced interferon at 20 hours and 40 hours after infection. Electron microscopic analysis confirmed previous findings in other cell lines regarding BTV morphogenesis in endothelial cells, the putative target cell population in vivo.     

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.     

Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin.

OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.     

Evaluation of cell replication by bovine T cells in polyclonally activated cultures using carboxyfluorescein succinimidyl ester (CFSE) loading and flow cytometric analysis

Studies reported here demonstrated that carboxyfluorescein succinimidyl ester (CFSE) loading of lymphocytes and flow cytometric analysis is a powerful assay to assess the kinetics and extent of cellular replication by bovine T-cell subpopulations in heterogeneous cultures of peripheral blood mononuclear cells (PBMC) where subpopulation interactions can occur. As CFSE analysis allows determination of the proportion of lymphocytes that divided, as well as the number of cell divisions each cell underwent, distinctions in responses among mitogen-stimulated cultures could be made even when(3)H-thymidine incorporation was equivalent. When combined with surface staining for detection of differentiation antigens, differences among T-cell subpopulations with regard to the number of divisions their members had undergone, were found. Anti-CD3 mAb stimulated both CD8(+)and CD4(+)T cells to undergo several cell divisions in 72 hours, while there was essentially no division by gamma delta T cells. In contrast, in concanavalin A-stimulated cultures, all T-cell subpopulations had divided.