Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study.
Abstract: The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Methods: Three adult horses with no evidence of ophthalmic or systemic disease. Methods: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. Results: No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations. Conclusions: Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from "safe" to "retinotoxic" in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.
© 2023 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.
Publication Date: 2023-04-28 PubMed ID: 37116984DOI: 10.1111/vop.13098Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research investigated the effects of the antibiotic gentamicin on certain immune cells (CD3+ T-lymphocytes) in horses, to understand if the drug could be used to treat a common eye disease (uveitis). The study found no significant effects on these immune cells, even at varying concentrations of gentamicin, suggesting it may not be an effective treatment.
Objective of the Research
- The research aimed to explore the effect of the antibiotic gentamicin on the proliferation and vitality of CD3+ T-lymphocytes (a type of white blood cell vital to the immune system) in horses.
- This was performed as an in-vitro study, using cultured cells, to further understand the potential usage of gentamicin for treatment of equine recurrent uveitis, a commonly occurring eye disease in horses.
Methodology
- The study employed peripheral blood lymphocytes from three healthy adult horses. These cells were exposed to varying concentrations of gentamicin and then stimulated to proliferate with a substance called Concanavalin A (ConA).
- Two markers were used to measure cell viability and proliferation, DAPI and CSFE, providing data on living cells and their rate of multiplication respectively.
- This experimental design was also tested using two different types of gentamicin: a preservative-free form and a proprietary brand formulation.
- Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison test to identify any significant differences in results.
Results
- The study found no significant differences in the live cell counts and rate of proliferation of the CD3+ T-lymphocytes across the different concentrations and formulations of gentamicin.
- This suggests that gentamicin, in the concentrations tested, does not affect the viability or proliferation of these immune cells in horses.
Conclusions
- The findings indicate that gentamicin, even at concentrations deemed “retinotoxic,” does not influence the behavior of equine CD3+ T-lymphocytes.
- Therefore, the study concludes that low-dose intravitreal (injected into the eye) gentamicin may not be effective in suppressing the Th1- and Th17- mediated immune response, which are thought to be involved in the development of equine recurrent uveitis.
Cite This Article
APA
Smith HL, Berglund AK, Robertson JB, Schnabel LV, McMullen RJ, Gilger BC, Oh A.
(2023).
Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study.
Vet Ophthalmol, 26(4), 347-354.
https://doi.org/10.1111/vop.13098 Publication
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Comparative Medicine Institute, North Carolina State University, North Carolina, Raleigh, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Office of Research, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Comparative Medicine Institute, North Carolina State University, North Carolina, Raleigh, USA.
- JT Vaughan Large Animal Teaching Hospital, College of Veterinary Medicine, Auburn University, Alabama, Auburn, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, North Carolina, Raleigh, USA.
MeSH Terms
- Animals
- Horses
- Gentamicins / pharmacology
- Gentamicins / therapeutic use
- Research Design
- Uveitis / drug therapy
- Uveitis / veterinary
- T-Lymphocytes
- Cell Proliferation
- Horse Diseases / drug therapy
References
This article includes 42 references
- Gilger BC, Malok E, Cutter KV, Stewart T, Horohov DW, Allen JB. Characterization of T-lymphocytes in the anterior uvea of eyes with chronic equine recurrent uveitis. Vet Immunol Immunopathol. 1999;71(1):17-28. doi:10.1016/S0165-2427(99)00082-3
- Regan DP, Aarnio MC, Davis WS, et al. Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis. Vet Ophthalmol. 2012;15(3):145-152. doi:10.1111/j.1463-5224.2011.00951.x
- Zhao Z, Granucci F, Yeh L, Schaffer PA, Cantor H. Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science. 1998;279(5355):1344-1347.
- Deeg CA, Thurau SR, Gerhards H, Ehrenhofer M, Wildner G, Kaspers B. Uveitis in horses induced by interphotoreceptor retinoid-binding protein is similar to the spontaneous disease. Eur J Immunol. 2002;32(9):2598-2606. doi:10.1002/1521-4141(200209)32:9<2598::aid-immu2598>3.0.co;2-%23
- Deeg CA, Pompetzki D, Raith AJ, et al. Identification and functional validation of novel autoantigens in equine uveitis. Mol Cell Proteomics. 2006;5(8):1462-1470. doi:10.1074/mcp.M500352-MCP200
- Jha P, Sohn J-H, Xu Q, et al. Suppression of complement regulatory proteins (CRPs) exacerbates experimental autoimmune anterior uveitis (EAAU). J Immunol. 2006;176(12):7221-7231. doi:10.4049/JIMMUNOL.176.12.7221
- Jha P, Sohn JH, Xu Q, et al. The complement system plays a critical role in the development of experimental autoimmune anterior uveitis. Invest Ophthalmol Vis Sci. 2006;47(3):1030-1038. doi:10.1167/IOVS.05-1062
- Ackermann K, Kenngott R, Settles M, Gerhards H, Maierl J, Wollanke B. In vivo biofilm formation of pathogenic leptospira spp. In the vitreous humor of horses with recurrent uveitis. Microorganisms. 2021;9(9):1915. doi:10.3390/microorganisms9091915
- Gagnon NA, Hartley C, Gilger BC. Efficacy and safety of suprachoroidal triamcinolone injection in horses with poorly responsive equine recurrent uveitis. Vet Ophthalmol. 2021;24(3):308-312. doi:10.1111/VOP.12887
- Yi NY, Davis JL, Salmon JH, Gilger BC. Ocular Distribution and Toxicity of Intravitreal Injection of Triamcinolone Acetonide in Normal Equine Eyes. Vet Ophthalmol. 2008;11:15-19.
- Douglas LC, Yi NY, Davis JL, Salmon JH, Gilger BC. Ocular toxicity and distribution of subconjunctival and intravitreal rapamycin in horses. J Vet Pharmacol Ther. 2008;31:511-516. doi:10.1111/j.1365-2885.2008.00986.x
- Gilger BC, Wilkie DA, Clode AB, et al. Long-term outcome after implantation of a suprachoroidal cyclosporine drug delivery device in horses with recurrent uveitis. Vet Ophthalmol. 2010;13(5):294-300. doi:10.1111/J.1463-5224.2010.00807.X
- Gilger BC, Salmon JH, Wilkie DA, et al. A novel bioerodible deep scleral lamellar cyclosporine implant for uveitis. Invest Ophthalmol Vis Sci. 2006;47(6):2596-2605. doi:10.1167/iovs.05-1540
- Voelter K, Vial Z, Pot SA, Spiess BM. Leptospiral antibody prevalence and surgical treatment outcome in horses with equine recurrent uveitis (ERU) in Switzerland. Vet Ophthalmol. 2020;23(4):648-658. doi:10.1111/vop.12767
- Wollanke B, Gerhards H, Schinagl C. Results of 654 trans-pars plana vitrectomies of equine eyes with recurrent uveitis-follow-up until 18u2009years after surgery. Pferdeheilkunde. 2021;37(3):204-214. doi:10.21836/PEM20210301
- Baake EIA, Von Borstel M, Rohn K, Boeve MH, Ohnesorge B. Long-term ophthalmologic examinations of eyes with equine recurrent uveitis after pars plana vitrectomy. Pferdeheilkunde. 2019;35(3):220-233. doi:10.21836/PEM20190303
- Fru00fchauf B, Ohnesorge B, Deegen E, Boevu00e9 M. Surgical management of equine recurrent uveitis with single port pars plana vitrectomy. Vet Ophthalmol. 1998;1(2-3):137-151. doi:10.1046/J.1463-5224.1998.00030.X
- Verbraeken H. Therapeutic pars plana vitrectomy for chronic uveitis: a retrospective study of the long-term results. Graefes Arch Clin Exp Ophthalmol. 1996;234(4):288-293.
- Pinard CL, Pietrement E, Macieira S, Trembley D. Intravitreal injections of gentamicin for the treatment of Leptospira-associated equine recurrent uveitis in abstracts: 36th annual meeting of the American College of Veterinary Ophthalmologists, Nashville, TN, USA, October 12-15, 2005. Vet Ophthalmol. 2005;8(6):437-450. doi:10.1111/J.1463-5224.2005.00442.X
- Fischer BM, Mcmullen RJ, Reese S, Brehm W. Intravitreal injection of low-dose gentamicin for the treatment of recurrent or persistent uveitis in horses: preliminary results. BMC Vet Res. 2019;15(1):29. doi:10.1186/s12917-018-1722-7
- Launois T, Gonz Alez Hilari LM, Barbe F, et al. Use of intravitreal injection of gentamicin in 71 horses with equine recurrent uveitis. J Equine Vet Sci. 2019;77:93-97. doi:10.1016/j.jevs.2019.02.018
- Kleinpeter A, Gu00f6pfert A, Ku00f6hler E, Brehm W. Intravitreal injection of low-dose gentamicin for the treatment of ERU-affected horses. Tierarztl Prax Ausgabe G Grosstiere-Nutztiere. 2019;47(1):25-34. doi:10.1055/a-0816-7156
- Gilger BC, Degroote RL, Deeg CA. Diseases of the uvea, uveitis, and recurrent uveitis. In: Gilger BC, ed. Equine Ophthalmology. 4th ed. Wiley; 2022:482-484 Hobo.
- Gilger BC, Reeves KA, Salmon JH. Ocular parameters related to drug delivery in the canine and equine eye: aqueous and vitreous humor volume and scleral surface area and thickness. Vet Ophthalmol. 2005;8(4):265-269. doi:10.1111/J.1463-5224.2005.00401.X
- May DR, Ericson ES, Peyman GA, Axelrod AJ. Intraocular injection of gentamicin: single injection therapy of experimental bacterial endophthalmitis. Arch Ophthalmol. 1974;91(6):487-489. doi:10.1001/archopht.1974.03900060501015
- Zachary IG, Forster RK. Experimental intravitreal gentamicin. Am J Ophthalmol. 1976;82(4):604-611. doi:10.1016/0002-9394(76)90549-3
- Mochizuki K, Torisaki M, Kawasaki K, et al. Retinal toxicity of antibiotics: evaluation by electroretinogram. Doc Ophthalmol. 1988;69(2):195-202. doi:10.1007/BF00153701
- Oum BS, D'amico DJ, Wong KW. Intravitreal antibiotic therapy with vancomycin and aminoglycoside: an experimental study of combination and repetitive injections. Arch Ophthalmol. 1989;107(7):1055. doi:10.1001/archopht.1989.01070020117043
- Conway BP, Campochiaro PA. Macular infarction after endophthalmitis treated with vitrectomy and intravitreal gentamicin. Arch Ophthalmol. 1986;104(3):367-371. doi:10.1001/archopht.1986.01050150067028
- D'amico DJ, Caspers-Velu L, Libert J, et al. Comparative toxicity of intravitreal aminoglycoside antibiotics. Am J Ophthalmol. 1985;100(2):264-275. doi:10.1016/0002-9394(85)90792-5
- Peyman GA, Paque JT, Meisels HI, Bennett TO. Postoperative endophthalmitis: a comparison of methods for treatment and prophylaxis with gentamicin. Ophthalmic Surg. 1975;6(1):15-16.
- Conway BP, Campochiaro PA, Tabatabay CA, D'amico DJ, Hanninen LA, Kenyon KR. Gentamicin toxicity in the primate retina. Arch Ophthalmol. 1989;107(1):107-112. doi:10.1001/archopht.1989.01070010109037
- Tabatabay CA, Young LHY, D'amico DJ, Kenyon KR. Immunocytochemical localization of gentamicin in the rabbit retina following intravitreal injection. Arch Ophthalmol. 1990;108(5):723-726. doi:10.1001/archopht.1990.01070070109046
- Kawasaki K, Mochizuki K, Torisaki M, et al. Electroretinographical changes due to antimicrobials. Lens Eye Toxic Res. 1990;7(3-4):693-704.
- Brown GC, Eagle RC, Shakin EP, Gruber M, Arbizio VV. Retinal toxicity of intravitreal gentamicin. Arch Ophthalmol. 1990;108(12):1740-1744. doi:10.1001/archopht.1990.01070140094037
- Kawasaki K, Ohnogi J. Nontoxic concentration of kanamycin and gentamicin for intravitreal use-evaluated by in vitro ERG. Doc Ophthalmol. 1988;69(4):331-340. doi:10.1007/BF00162747
- Radcliffe CH, Flaminio MJBF, Fortier LA. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem Cells Dev. 2010;19(2):269-281. doi:10.1089/SCD.2009.0091/FORMAT/EPUB
- Berglund AK, Long JM, Robertson JB, Schnabel LV. TGF-u03b22 reduces the cell-mediated immunogenicity of equine MHC-mismatched bone marrow-derived mesenchymal stem cells without altering immunomodulatory properties. Front Cell Dev Biol. 2021;9:1-10. doi:10.3389/fcell.2021.628382
- Gerding JC, Gilger BC. Prognosis and impact of equine recurrent uveitis. Equine Vet J. 2016;48(3):290-298. doi:10.1111/EVJ.12451
- Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15(2):167-193. doi:10.1128/CMR.15.2.167-193.2002
- Deeg CA, Ehrenhofer M, Thurau SR, Reese S, Wildner G, Kaspers B. Immunopathology of recurrent uveitis in spontaneously diseased horses. Exp Eye Res. 2002;75(2):127-133. doi:10.1006/exer.2002.2011
- Deeg CA, Hauck SM, Amann B, Kremmer E, Stangassinger M, Ueffing M. Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis. Mol Immunol. 2007;44(13):3291-3296. doi:10.1016/j.molimm.2007.02.027