Progress 07/01/04 to 06/30/08
Outputs
OUTPUTS: We have conducted experiments to assess the effect of advanced age on immune responses in horses. The results from these studies have been presented at various national scientific meetings and submitted for publication. We have also presented lectures on this topic to various audiences including veterinarians and horse owners. PARTICIPANTS: David W. Horohov; Thomas C. Chambers; Cormac Breathnach; Amanda A. Adams; Madhu Katepalli; Katharina Kohler; Cetewayo Rashid; Barry Fitzgerald; Laurie Lawrence TARGET AUDIENCES: Veterinarians and horse owners as well as scientists interested in the overall mechanisms of age-related changes in immune function. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Horses over 20 years of age constitute about 15% of the equine population and many remain actively involved in equestrian sports and in reproductive capacities as stallions and brood mares. Advancing age in horses, as with other species, is eventually associated with a decline in body condition, muscle tone and immune function, including decreased responsiveness to vaccination. Nevertheless, there are no specific recommendations regarding the vaccination of older horses and ponies. Nor is much known regarding the mechanism responsible for the age-related decline in immune function or the impact of various factors on this process. Here, we have assessed various aspects of immune function in older horses and have begun to characterize some of the factors that impact upon the age-related changes we, and others, have identified. This involved both in vitro and in vivo assessment of immune function, chromosomal structure and disease susceptibility in our population of aged horses. We have shown, for the first time, the age-related decline in immune function in horses is correlated with progressive shortening of the telomeres of equine chromosomes. However, in the oldest horses there was no relationship between overall telomere length and immune function indicating that telomere shortening alone did not account for the age-related changes in immune function. We have also shown that aged horses exhibit increased inflammatory cytokine production and that this is associated with increased adiposity. We also demonstrated that aged horses exhibit a similar susceptibility to equine influenza virus as younger, non-vaccinated horses and that vaccination of older horses with a new recombinant vaccine could protect them from infection.
Publications
- Katepalli, M.P., Adams, A.A., Lear, T.L., and Horohov, D.W. (2008). The effect of age and telomere length on immune function in the horse, Dev. Comp. Immunol., 32: 1409-1415.
- Adams, A.A., Breathnach, C.C., Katepalli, M.P., Kohler, K., and Horohov, D.W. (2008). Advanced age in horses affects divisional history of T cells and inflammatory cytokine production, Mech. Ageing Dev., 129: 656-664.
- Adams, A.A., Katepalli, M.P., Kohler, K., Reedy, S.E., Stilz, J.P., Vick, M.M., Fitzgerald, B.P., Lawrence, L.M., and Horohov, D.W. (2008). Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses, Vet. Immunol. Immunopathol., in press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Horses over 20 years of age constitute about 15% of the equine population and many remain actively involved in equestrian sports and in reproductive capacities as stallions and brood mares. Advancing age in horses, as with other species, is eventually associated with a decline in body condition, muscle tone and immune function. One of the marked changes in immune function that has been reported for the older horse is decreased responsiveness to vaccination. Nevertheless, there are no specific recommendations regarding the vaccination of older horses and ponies. It is also unclear how susceptible older horses may be to some of the infectious agents against which vaccination is routinely practiced. In particular, the relative susceptibility of the older horse to equine influenza virus is unknown. Given that most older horses have likely been exposed to equine influenza virus throughout their lives, either through vaccination or infection, or both, it is reasoned that they
retain specific immunity to this virus. However, studies in humans have clearly shown that immunity to influenza viruses wanes with age leading to increased susceptibility amongst the elderly. Indeed, it is the elderly population that is targeted for annual influenza vaccination. To address whether aged horses exhibit a similar susceptibility to equine influenza virus, we performed an infectious challenge study using a group of young and aged horses.
PARTICIPANTS: David W. Horohov, PI Thomas Chambers, co-PI Amanda Adams, graduate student Tacy Sturgill, graduate student Kristin Handke, technician Katarina Kohler, technician Cetawayo Rashid, technician
TARGET AUDIENCES: Horse owners, equine veterinarians and veterinary vaccine manufacturers.
Impacts
Our results indicated that the older horse is susceptible to this infection, though clinical signs were not as severe in the older horses as those seen in younger horses. Nevertheless, the older horses exhibited both cytokine and antibody responses similar to younger horses post-challenge and, most importantly, shed virus for several days post-infection. As such, it is clear that vaccination of the older horse to prevent equine influenza virus infection is necessary. However, given the fact that old horses do not respond well to conventional vaccines, future work will be needed to determine the optimal vaccination strategy for the older horse.
Publications
- Adams, A.A., Breathnach, C.C., Sturgill, T., Chambers, T., and Horohov, D.W. (2007). Characterization of the immunological and physiological response of aged horses to equine influenza infection. (abstract) The 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil, 2007 (ER091).
- Adams, A.A., Katepalli, M., Kohler, K., Reedy, S., Stilz, J.P., Vick, M.M., Fitzgerald, B.P., and Horohov, D.W. (2007). The contribution of body condition score and percent body fat to the inflammatory response in aged horses. (abstract) The 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil, 2007 (ER090).
- Adams, A.A., Breathnach, C.C., Sturgill, T., Chambers, T., and Horohov, D.W. (2007). Characterization of the immunological and physiological response of aged horses to equine influenza infection. Proceedings of the 88th Conference of Research Workers in Animal Disease, Chicago, Illinois, 2007 (#114).
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Progress 01/01/06 to 12/31/06
Outputs
Advanced age is associated with a low-grade, systemic inflammatory response in vivo and increased inflammatory cytokine production in vitro. One possible source of this increased inflammatory cytokine production is dysfunctional white blood cells associated with the aged immune system. In previous studies of the aged horse (≥20 yrs), our results demonstrated a significant increase in the percent of lymphocytes from old horses compared to young animals producing interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα). It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Therefore, we tested the hypothesis that increased body condition scores (BCS≥5) and percent rump fat (≥10) of aged horses are positively associated with increased in vitro
production of inflammatory cytokines, IFNγ and TNFα. Body condition scores were determined using the standard nine-point Henneke Scaling System. Rump fat thickness was determined by ultrasound measurement. PBMC were stimulated with either PMA/ionomyocin or LPS. The cells were fixed, permeabilized and intracellularly stained for IFNγ and TNFα. Samples were acquired on a FACSCaliber. Percent positive and mean fluorescent intensity (MFI) were determined using lymphocyte and monocyte gates. There was an increased percentage of lymphocytes staining for TNFα in old mares with a BCS >5 and percent rump fat >10. Likewise, IFNγ production tended to be increased in the fat mares. Furthermore, monocyte production of TNFα increased. However, LPS-stimulated monocytes from fat mares produced less TNFα compared to the thinner mares. These results indicate that body condition score and % rump fat are confounding factors when assessing age-associated
inflammatory responses. Further, differences were also apparent when PMA/ionomycin or LPS were used to stimulate the cells.
Impacts
Horses over 20 years of age constitute about 15% of the equine population and many remain actively involved in equestrian sports and reproductive capacities as stallions and brood mares. Advancing age in horses, as with other species, is eventually associated with a decline in body condition, muscle tone and immune function. Nevertheless there are no specific recommendations regarding the vaccination of older horses and ponies even though the best characterized effect of aging in horses is a reduced antibody response to vaccination. By better understanding the nature of age-related immune deficiencies in the horse we will be better able to address their unique needs in regards to vaccinations and other immune therapies. Additionally, older horses may serve as a model for aging in other species, including man. Thus, characterizing the mechanism of age-related changes in immune function in the horse may have broad applicability to veterinary and human medicine.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
As with elderly humans, the functionality of the immune system of geriatric horses appears to decline with age. Analyses of lymphocyte proliferation using thymidine incorporation assays have shown that peripheral blood mononuclear cells (PBMC) from older horses (> 20 years of age) have decreased proliferative indices compared to similar cells collected from younger horses. The significance of this effect is unknown due, in part, to the imprecision of the methodology employed. Thus, it is unclear whether decreased uptake of thymidine represents fewer cells proliferating or an overall decreased proliferative capacity of the aged PBMC. The purpose of this study was to further investigate the mechanisms of this altered response. Heparinized blood from geriatric horses (> 20 years) and young horses (4-6yrs) was collected by jugular venipuncture and the PBMC isolated using density gradient centrifugation. The PBMC were loaded with carboxyfluorescein diacetate succinimidyl
ester (CFSE) (5uM) and incubated with ConA (0, 2.5, 5, or 10ug/ml) for 96 hours. Afterwards, the cells were incubated an additional 4 hrs with PMA and ionomyocin with brefeldin A and then fixed with 2% paraformaldehyde, permeabilized and stained for interferon-gamma (IFN-g). Samples were acquired on a Becton Dickinson FACS Caliber and the data analyzed by ModFit LT 3.0. Apoptosis was determined by Annexin V staining. Analysis of the CFSE results showed that fewer PBMCs from older horses were proliferating at the low dose of ConA (2.5 ug/ml) when compared to those from young horses (P<0.05). This appeared to be due to a failure to recruit cells into the proliferative pool rather than increased cell death as there was no evidence for increased apoptosis. While the proliferative response of the older horses was diminished, IFN-g production by their PBMCs was significantly higher than those from younger horses. Closer inspection of these data indicated that different populations of cells
were mediating this response. Thus, in the older horses those cells which failed to proliferate produced IFN-g, while in the younger horse IFN-g production was by those cells undergoing cell divisions. This was likely due to there being more memory cells in the older horse PBMC population. Unlike naive cells, memory cells are capable of producing IFN-g without undergoing cell divisions. This also makes the point that the non-dividing cells in the older horses were in fact capable of responding to the mitogen by producing the cytokine and thus were not anergic. This phenomenon of decreased proliferation and increased IFN-g production is consistent with inflammaging, an age-associated increased production of pro-inflammatory cytokines with the concomitant down-regulation of specific immune responses initially described for older humans and here for horses. While this phenomenon is suspected to be associated with chronic diseases in the elderly, its role in equine health has not yet been
determined.
Impacts
Horses over 20 years of age constitute about 15% of the equine population and many remain actively involved in equestrian sports and reproductive capacities as stallions and brood mares. Advancing age in horses, as with other species, is eventually associated with a decline in body condition, muscle tone and immune function. Nevertheless there are no specific recommendations regarding the vaccination of older horses and ponies even though the best characterized effect of aging in horses is a reduced antibody response to vaccination. By better understanding the nature of age-related immune deficiencies in the horse we will be better able to address their unique needs in regards to vaccinations and other immune therapies.
Publications
- No publications reported this period
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