Progress 10/01/01 to 09/30/06
Outputs
This project terminated on September 30, 2006. The work conducted under this project evolved into new aspects and approaches to studying immunity in chickens and to determine appropriate organ/tissue sites for examination of inflammatory indicators. Since most environmental insults usually compromise the physiological reactivity of chickens, an additional response that exacerbates these conditions in inflammatory responses. In poultry operations, many of the environmental insults are either via oral or respiratory entry. The Haderian gland of the chicken was chosen as a immunological site of investigation due to its involvement in elaboration of immune mediators for protection of both the oral and respiratory passages. Generally, a compromise immune response in the Harderian gland also affects the detection of systemic protection, mostly in the form of blood antibodies. All together, work included examination of gene expression of inflammatory mediators in the
Harderian gland, a model cell line (C1-Vick), and eventually thrombocytes. It was found that the Harderian gland, when challenged with a Newcastle/Infectious bronchitis vacccine, expressed increased amounts of mRNA for prostaglandin D2 synthase (PGDS) and interleukin-6 (IL-6). The C1-Vick cells were challenged in culture with phorbol acetate (PMA). This induced the expression of mRNA for PGDS. However, treatment of the cells with inhibitors (calphostin and PD98059) did not effectively reduce induced expression of PDGS. Preliminary studies done with thrombocytes during the last nine months of the project showed good promise for using these cells as indicators of inflammatory reactivity. Lipopolysaccharide (LPS) stimulation of thrombocytes from broiler chickens showed that IL-1, -6 and -12 are all significantly increased. Therefore, this cell is being further investigated in a new project to evaluate its overall contribution to inflammation in chickens.
Impacts
Ongoing evaluation of inflammatory responses in chickens with specific immune indicators is required in order to have a reliable evaluative process. Based upon the expression of inflammtory cytokines and PDGS, these indicators will serve as excellent targets for future evaluations. Furthermore, the use of thrombocytes as target cells for evaluation of inflammatory capacity is proving to be ideal. This cell allows for a numerous, quickly recoverable immune cells that, at this point, are highly reactive to stimulation followed by dramatic inflammatory responses.
Publications
- Ferdous, F, D Maurice, and T Scott, 2007. Pro-Inflammatory response of broiler chicken thrombocytes. Poultry Sci. Suppl. 1. Submitted.
- Ferdous, F, 2007. Thrombocyte response to lipopolysaccharide in broiler chickens fed control, corticosterone-supplemented and vitamin C-supplemented diets. M.S. Thesis. Clemson University.
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Progress 01/01/05 to 12/31/05
Outputs
During the year, work continued with using tissues and cells from chickens to evaluate the responsiveness of certain genes under treatment conditions. The emphasis area of the project has shifted to examining indicators of inflammation in chickens. There has been recent development of sequence data for various pro-inflammatory cytokines of the chicken (e.g., IL-1beta, IL-12, IL-6). Additionally, our laboratory has sequenced and verified the presence of hematopoietic prostaglandin D2 synthase (H-PGDS) in tissues and T cells. This enzyme is an isomerase that converts PGH to PGD. The latter is a potent inducer of inflammatory responses through specific DP receptors found on other immune cells and epithelial cells. The increased or decreased expression of H-PGDS as determined by real time-PCR is a direct indicator of induced inflammation in tissues. Our model tissue is the Harderian gland (HG) of chickens and an avian T cell line. Manipulation of the enzymes expression of
the T cell line was difficult to affect with either inhibitors calphostin C (protein kinase C inhibitor) or PD-098059 (MAPKK inhibitor) or with stimulants phorbol myristate acetate plus calcium ionophore. However, vaccination of chickens with live Newcastle Disease/Infectious Bronchitis virus effectively induced the increased expression of H-PGDS three days post-vaccination. Immunohistochemical evaluation of frozen tissue sections of the HG indicated that Mast cells in the subepithelial areas of the ducts were the principal cells involved in the response. However, other immune cells are presumed to be involved in the overall immune responsiveness of the HG to vaccination. Continued work in this area will be expanded to include both Th1 (i.e., IFN gamma, IL-2) and Th2 (i.e., IL-4) cytokines as well as pro-inflammatory cytokines produced by macrophages. Since an immunological response is being induced in the HG by the Newcastle Disease/Infectious Bronchitis vaccination, other cytokines
of accessory cells and T cells should be induced and reflect the relative contribution of each in the initial inflammatory phase of the response followed by the engagement of T cells leading to adaptive aspects of the immune response.
Impacts
The inflammatory responses associated with induction of immune responses would appear to be an important indicator of vaccine efficacy. That is to say that various mediators of inflammation are induced during the early stages of induced immunity. The effective and regulated control of these inflammatory reactions would be very important to determining the overall potency of the vaccine to then allow for a persistent adaptive response whether it be humoral or cell-meidated or a combination of both. Therefore, continued evaluation of cytokine and immune mediator induction will provide for more effective development of efficaious vaccines.
Publications
- Herlong, J.L., 2005. Hematopoietic Prostaglandin D2 Synthase Expression in a Retrovirally Transformed Avian T-Cell Line. M.S. Thesis. Clemson University, Clemson, SC.
- Herlong, J.L. and T.R. Scott, 2006. Positioning of prostanoids of the D and J series in the immunopathogenic scheme. Immunology Letters 102:121-131.
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Progress 01/01/04 to 12/31/04
Outputs
Continued work on this project has led to the discovery of an important enzyme being expressed in both Harderian glands and a T-cell line of chickens. The enzyme is hematopoietic prostaglandin D2 synthase, which is important in the conversion of PGH2 to PGD2. PGD2 is a potent inflammatory meidator and binds to receptors on mucosal surfaces as well as on T cells, eosinophils and basophils. Our lab has identified H-PGDS by proteomics analysis and have sequenced cDNA produced from mRNA recovered from both the HG and T-cell line. The sequence is 99% homologous with a splenic form of the enzyme sequenced previously in chickens. Our lab has also examined the expression of mRNA for H-PGDS in the T-cell line through real-time RT-PCR. The enzyme apprears to be constitutively expressed with little if any alteration by stimulates or inhibitors of second messenger systems.
Impacts
The identification of H-PGDS has now allowed our lab to examine the expression, and ultimately, the activity of this enzyme, which is important in inflammatory responses. Known agents of the envirnoment that would influence the expression and activity of this enzyme system can be understood in terms of the alteration of reactivity that would compromise immune reactivity.
Publications
- Scott, T.R., A.R. Messersmith, W.J. McCrary, J.L. Herlong, and S.C.Burgess, 2005. Cloning and sequencing of hematopoietic prostaglandin D2 synthase in the chicken Harderian gland. Vet. Immuno. Immunopathol. In review.
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Progress 01/01/03 to 12/31/03
Outputs
Work on this project continued with examining the effects of fumonisin B1 on C1 Vick T cells in vitro. This T cell line has been found to be a good model for studying the toxins effects on an immunological cell. Experimentation conducted has involved 1) detection of IL-2R expression, 2) detection of beta1 integrin molecules, and 3) expression of hematopoietic prostaglandin D2 synthase. Cells exposed to the toxin for 1, 2, and 3 days of culture had reduced levels of cell proliferation as detected by the MTT assay. The largest degree of inhibition was observed after 3 days of culture. Cells in subsequent studies were treated with the toxin for 2 and 3 days and the cells were stained with a monoclonal antibody against IL-2R. The detection of IL-2R on 2-day treated cells was variable, while the detection of IL-2R after 3 days of treated resulted in a significant decrease in IL-2R presence as determined by flow cytometry. This effect is believed to be associated with
interruptions of the ceramide pathway, which is affected by fumonisins. In conjunction with the detection of IL-2R on the C1 cells, other studies are being carried out to examine the extend of beta1 integrin presences on these cells. These cells grow as aggregates in suspension culture; therefore, it is presumed that cell adhesion molecules are participating in the aggregation of the cells. T cells are known to express beta1 integrins, as well as others, so available monoclonal antibodies for alpha 5, 6, and 7 integrin have been used in preliminary studies to determine the possible presence of these on C1 cells. Results indicated that these integrins are found on the cells and possibly contribute to the large aggregate nature of these cultured cells. Fumonisin B1 treatment reduces the size of these cell aggregates and possibly integrin expression. Studies are on-going to make this determination. It is unknown if the C1 Vick cell line is a TH1 or TH2 population. At this point, there
are no cytokines genes identified to help make the determination of helper sub-population identity. However, the literature supports the role of hematopoietic prostaglandin D2 synthase as an indicator of TH2 cells. We have recently isolated mRNA from a chicken tissue that expresses this enzyme. By RT-PCR of mRNA from the C1 Vick cell line, cDNA for PGD2 synthase has been produced and the sequence will be verified. Future studies with fumonisin B1 exposure will involve examining the effects, if any on this enzyme associated with C1 cells.
Impacts
With the continued identification of genes in immune cells, examinations of toxin effects on specific sets of cells will be imporved. This will allow for targeted studies of toxin effects on immune cell reactions, which can then be extended to other specific tissues of the body. It is expected that detection systems for toxin exposure will improve with more incorporation of molecular biological techniques.
Publications
- Scott, T.R., 2004. Our current understanding of humoral immunity of poultry. Poultry Sci. In press.
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Progress 01/01/02 to 12/31/02
Outputs
Work with the cell line that is being characterized for its response to fumonisin B1 exposure is ongoing. The preliminary studies with the line (C1 Vick chicken T-cell line) have established that this line produces IL-2 (as revealed by Western blotting) and expresses the IL-2R on its surface. Furthermore, stimulation of the cell line with PMA, leads to activation of second messenger signal transduction elements like NF-kappa-B, NFAT, and AP-1. Experiments are being conducted to examine the effect of fumonisin B1 on the activation of these cells. In additional, the role of ceramide in this activation process well be assessed.
Impacts
It is expected that the results of studies completed will provide information of the overall damaging effect of this particular mycotoxin the health of poultry. Many of the causes of health problems in poultry are exacerbated by the presence of mycotoxins in the diet. A deeper understanding of the ramifications of this exposure on the immune system will help provide better choices for animal health care.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Fumonisin B1 (FB1) is a toxic secondary metabolite produced by Fusarium spp. and is found worldwide as a contaminant of corn. The results obtained from this study with chickens indicate that FB1 was toxic to lymphocytes collected from the spleen, but stimulatory to thymic cells. There was no effect of FB1 on peripheral blood lymphocyte viability in this study. When evaluating the effect of FB1 on mitogenic response, the data showed neither spleen cells nor PBL stimulated with LPS were affected by FB1 exposure up to 48 hours. However, after 72 hours of FB1 exposure of 10 ug/ml or greater, cells from the spleen did show a decrease in response. Furthermore, FB1 did not have an affect on mitogenesis of PBL and thymus cells exposed to Con A, but caused a significant decrease in mitogenesis of spleen cells after 24 hr exposure. The effects of FB1 on macrophage viability, mitogenic response, phagocytic ability and nitric oxide (NO) production were determined. FB1 decreased
macrophage enzymatic activity and phagocytic response for spleen and peritoneal macrophages. FB1 at the highest concentration used, 50 ug/ml, caused an increase in NO production 2x that of controls. Finally, the apoptotic effects of FB1 on avian immune cells were determined. Both spleen and thymus cells exposed to FB1 had DNA laddering patterns characteristic of apoptosis. In addition, preliminary results indicate that caspase-3 activity within the thymus increased with FB1 exposure, yet decreased in spleen cells. For the studies with horses, a method was developed for increasing follicular growth in mares utilzing domperidone. The drug treatment was used for both transitional mares to bring them into estrus earlier and for mares that did not develop follicles during the normal breeding season. Studied the effect of endophyte fescue (E+) on cardiorespiratory and thermoregulatory systems in the horse. After exercise, E+ prolonged the time for body temperature to return to normal,
increased water consumption, and respiration rate. Data were collected on the use of domperidone for increasing intestinal motility in post-gastric surgery and in reflux gastritis. Also, domperidone was used to treat agalactia and low-level mick production. Data were collected that showed a relationship (P<.001) between the presence of poison hemlock and "Mare Reproductive Loss Syndrome" in Kentucky pastures. U.S. Patent No. 6,224,895 B1 (Method for Promoting Ovulation, Parturition, and Lactation in Mammals) was approved.
Impacts
Research with FB1 is helping to develop a better understanding of the role of this toxin on immunity of animals. The potential losses in the animal industry to toxin exposure are related to compromising the immune system. Work with toxin effects on horses is resulting in therapeutic treatment of severe toxicosis with drugs like domperidone. This work has and will be benefical to the horse industry to eleviate costly loss of valuable animals.
Publications
- Cross, D.L. and C.S. Adams, 2001. Efficacy of equidone for treatment of fescue toxicosis. Proc. N. Amer. Vet. Conf. Large Animal pp. 90-91.
- Cross, D.L. and S.L. Gray, 2001. Other uses of equidone (domperidone) for large and small animals. Proc. N. Amer. Vet. Conf. Large Animal p. 92.
- King, S.S., M.L. Mallar, L.G. Nequin, J.F. Roser, K.L. Jones, and D.L. Cross, 2001. Evidence against dopaminergic influence over the fall transition. Proc. Equine Nutr. Phys. Soc. p. 298.
- Campbell, C.E., D.L. Cross, W.C. Bridges, and T. Gimenez, 2001. Determination of an effective domperidone dosage for treatment of fescue toxicosis in gravid mares. Proc. Equine Nutr. Phys. Soc. p. 302.
- Henderson, J.A., T. Gimenez, and D.L. Cross, 2001. Enviromental influences on triiodothyronine production by the mammary gland in the lactating mare. Proc. Equine Nutr. Phys. Soc. pp. 306-311.
- Wright, R.G., B. Boyce, T. Van Dreumel, M.J. Hazlett, and D.L. Cross, 2001. Ergot alkaloid toxicity in foaling mares associated with eating cereal rye straw. Proc. Equine Nut. Phys. Soc. pp. 312-313.
- Cray, S.L., D.L. Cross, T. Gimenez, P.D. McMillan, and P. Evans, 2001. Factors associated with mare reproductive loss syndrome in Central Kentucky and surrounding areas. Proc. SEAR-IEG-8 pp. 46-49.
- Cross, D.L., 2001. Symptoms and treatments of fescue toxicosis. Equine and Bovine p. 7.
- Boone, B.J., 2001. Effects of fumonisin B1 on avian immune responses. Ph.D. Dissertation. Clemson University. Clemson, SC 29634
- Brendemuchl, J.P., and D.L. Cross, 2001. Influence of the dopamine agonist domperidone on the vernal transition in seasonally anestrous mares. J. Reprod. Fert. 56:185-193.
- Vivrette, S., M.E. Stebbins, O. Martin, K. Dooley, and D.L. Cross, 2001. Cardiorespiratory and thermoregulatory effects of endophyte-infected fescue in exercising horses. J. Equine Vet. Sci. 21:65-67.
- Cross, D.L., 2001. Toxic effects of Neotyphodium coenophialuum in cattle and horses. Proc. 4th Int. Neotyphodium/Grass Interactions Symp. pp. 219-235. Soest, Germany. H.P. Volker and P.D. Dapprich, eds.
- Cross, D.L. and T. Gimenez, 2001. Fescue toxicosis in cattle. Proc. N. Amer. Vet. Conf. Large Animal pp. 87-89.
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