- Email: [email protected]
Post-Transplant Immune Tolerance in Rats Following Lymphocyte Injection Into the Anterior Chamber of the Eye
Post-Transplant Immune Tolerance in Rats Following Lymphocyte Injection Into the Anterior Chamber of the Eye Recep Erçin Sönmeza,*, Mehmet Ilhanb, and Cemalettin Ertekinb a Department of General Surgery, Istanbul Medeniyet University, Istanbul, Turkey; and bDepartment of General Surgery, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey
ABSTRACT Objective. We aimed to compare the clinical and histopathological results of skin graft transplants between rats that had been injected with lymphocytes into the anterior chamber of the eye with those that had not. Methods. A total of 16 Wistar albino, male rats were included in the study. Subjects were divided into 2 groups, namely a test group and a control group. Lymphocyte suspensions derived from the subjects of the control group were injected into the anterior chamber of the eye of each opposing subject of the test group. Also, an identical volume of physiological saline was injected into the anterior chamber of each subject in the control group to prevent bias. One week after this procedure, circular skin grafts of 1 cm in diameter were transplanted within the opposing groups. After a period of 1 week, transplanted graft tissues were excised to compare tissue healing. Results. The occurence of granulation and reepithelialization was more evident in the test group (96% and 33%, respectively, vs 80% and 17% for the control group, respectively). On the other hand, it was determined that acute inflammation was more intense in the control group (77% vs 50% for the test group). Conclusion. We had created immune tolerance in rats through anterior chamber lymphocyte injection, which slowed down the rejection process. If this can be successfully implemented in practice, survival for transplant patients without long-term rejection will move closer to becoming a reality.
A
HUMAN has several special anatomic regions that are inherently isolated from the immune system. These include the blood brain barrier, the placenta-fetus, the testes, and the anterior chamber of the eye. Due to their inherent immunologic and anatomic structures, they prevent the formation of an inflammatory response by gaining tolerance when exposed to antigens [1e4]. The concept of anterior chamber-associated immune deviation (ACAID), or “immune deviation associated with the anterior chamber of the eye,” is defined as the suppression of cellular and humoral immunoreactivity due to the presence of molecules such as interleukin 4, interleukin 10, interleukin 13, melanocyte-stimulating hormone (MSH), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), somatostatin, and tumor necrosis factor beta in the anterior chamber, the expression of Fas-Fas ligand, and the expression of T-reg 1e2 when the antigen is presented [5e10]. 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.064
2132
Alloantigens captured by the antigen-presenting cells are initially delivered to the thymus and then to the spleen after exiting the anterior chamber of the eye. Regulatory T cells, which have anti-inflammatory properties, become active after mutual interactions with other immune system components. These elements then suppress the T helper cells, which are responsible for the antigen-sensitive systemic response [11,12]. We planned on 2 different groups of rats of equal numbers, comprising a control group and a test group. The
*Address correspondence to Recep Erçin Sönmez, MD, Istanbul Medeniyet University, Department of General Surgery, itim Mah. Dr Erkin Cad. Kadıköy/_Istanbul 34722, Turkey. Tel: Eg 00905414454063. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 2132e2135 (2019)
POST-TRANSPLANT IMMUNE TOLERANCE IN RATS
lymphocyte derivates obtained after centrifugation from the control group of rats were injected into the anterior chamber of the eye of the test group rats. On the 7th day after this procedure, split-thickness skin graft tissues taken from each group were transplanted in the reciprocal group. Our 2 groups of results were compared clinically and histopathologically. The main objective was to protect graft survival through acquired immune tolerance that was created in vivo.
2133 of physiological saline into the anterior chamber of the eye without lymphocyte injection. One week after the operation, transplanted graft tissues were excised, and the healing process was histopathologically and clinically compared.
Ethical Approval All applicable international (Animal Research: Reporting of In Vivo Experiments guidelines), national, and institutional guidelines for the care and use of animals were followed. Animal experiments were approved by the Istanbul University Animal Experiments Local Ethics Committee (No: 35980450-050.01.04).
MATERIAL AND METHODS The average weight of the Wistar albino, male rats included in the study was 200 to 250 g (mean 225 g). The animals were maintained on a typical rat diet at room temperature with free access to tap water. Regular daily checks were conducted in the morning and evening. A total of 2 groups comprising test and control subjects were formed. Eight subgroups were labeled as reciprocal graft transplants (A1-A2, B1-B2, C1-C2, D1-D2, E1-E2, F1-F2, G1-G2, H1-H2).
Lymphocyte Isolation In Vivo Conditions Using aseptic techniques, 3 mL of Ficoll-Paque PLUS solution was withdrawn into a centrifuge tube. A 1 mL diluted blood sample taken from the dorsal vein of the tails of each rat in the control group was added to the Ficoll-Paque PLUS solution. It was then centrifuged at 400 rpm for 30 to 40 minutes at a temperature of 18 C to 20 C. Following the completion of the centrifugation process, the upper layer was carefully removed with a clean Pasteur pipette, leaving the middle layer of lymphocytes. Using a second, clean Pasteur pipette, the lymphocyte layer was transferred to an empty centrifuge tube. A minimum of 1.5 mL of balanced salt solution was added to the test tube containing the lymphocytes. The contents were carefully mixed using the Pasteur pipette. The mixture was then centrifuged at 60 to 100 rpm for 10 minutes at 18 C to 20 C. At the end of the procedure, the supernatant was separated. The remaining lymphocytes were mixed with approximately 1.5 to 2 mL of physiological saline using a Pasteur pipette. The lymphocyte solution was centrifuged at 60 to 100 rpm for 10 minutes at a temperature of 18 C to 20 C. At the end of the process, the supernatant was separated. The isolated lymphocytes were then ready for application. Lymphocytes, obtained in vivo by centrifugation of 1 mL serum samples from rats of the control group in Ficoll-Paque PLUS medium, were injected into the anterior chamber of the eye of the rats of the test group.
Surgical Procedure General anesthesia was provided by administering ketamine 50 mg/kg/ip, xylazine 5 mg/kg/ip to the rats, and feed intake was halted 12 hours before the experiment. The subjects’ back region, where the incision was to be applied, was shaved and cleaned with 10% polyvinylprolidone iodine antiseptic solution before the transplantation procedure. Approximately 1 cm of excised split-thickness skin graft tissue taken from the rats of the control group was transplanted to the rats of the test group in the form of a 1 cm diameter disc, using 5-0 Prolene sutures with a separate single technique. In the control group, skin graft transplantation was performed from the corresponding test group, following the injection of an identical volume
RESULTS
The mean follow-up period was 2 weeks. There was no evidence of infection at the wound site (malodorous discharge, redness, swelling, wound disintegration) within the follow-up period of the subjects. During the days following transplantation, graft color changes were observed in both control and test groups. One week postoperation, it was determined that the transplanted graft tissue of the control group was darker, harder, and more lifeless than the test group. On the contrary, that of the test group was found to be softer and more vigorous, although the body appeared to be paler than its skin color. Microscopic Evaluation of Lymphocytes
Under the light microscope, an average of 2 100,000 cells were observed in 2 microliters under 10X magnification. In flow cytometry, 95% of the lymphocytes were detected as the result of the analysis. The lymphocyte derivates obtained from the subjects in the control group had similar ratios. Histologic Findings
Under the light microscope, the formation of reepithelialization and neovascularization appeared to be more pronounced in the test group (Fig 1A). In the control group, more lymphocytes were detected on average, thus indicating the activity of acute inflammation (Fig 1B). Abramov’s histologic, wound healing, scoring system scale was modified to calculate the score, calculated as a percentage of the total score (maximum 3 points for each criterion). Average scores were calculated for the test group: acute inflammation 50%, granulation texture 96%, granulation texture and fibroblast maturation 96%, collagenization 59%, neovascularization 96%, and reepithelialization 33%. The mean scores for the histopathological evaluation for the control group were acute inflammation 77%, granulation texture 80%, granulation texture and fibroblast maturation 83%, collagenization 53%, neovascularization 93%, and reepithelialization 17%. DISCUSSION
The most important factor determining graft survival in transplantation immunology is preservation of immunologic tolerance. In the absence of immunosuppression in transplantation performed between individuals with a different
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SÖNMEZ, ILHAN, AND ERTEKIN
Fig 1. (A) Reepithelization with collagen deposition in the dermis (marked with red arrow) and formation of vessels showing neovascularization in the test group (marked with black arrows) (400X magnification, hematoxylin eosin staining). (B) Lymphocyte clusters showing acute inflammation in the control group (marked with black arrows) (400X magnification, hematoxylin eosin staining).
genetic make-up, rejection and tissue damage is an inevitable result [13e16]. Recently, cell-based therapies for inducing tolerance have begun to appear in the literature. Induction studies of mesenchymal stem cells, regulatory myeloid cells, regulatory T cells, and other cell types have been attempted. Although a definitive conclusion has not been reached, the most recent data is promising for the future [17e23]. After the feature of the anterior chamber of the eye was revealed, work in this area has begun and has developed, especially in the last few years. In recent publications, it is also stated that the creation of antigen specific tolerance is the desired target in transplantation immunology and, therefore, in surgery [24,25]. We tried to create an immune tolerance model in 16 male, Wistar albino rats. We identified control and test groups separately. We aimed to induce the ACAID system following the introduction of lymphocytes into the anterior chamber of the eye. According to the results of our findings, it was determined that the amount of granulation tissue, fibroblast maturation, and reepithelization were more prominent in the test group. Whereas in the control group, acute inflammation was more intense. To interpret these observations, the granulation and reepithelialization in the transplanted tissue was found to be greater in the test group, in which the ACAID system was induced after lymphocyte injection, because of the tolerance gained against the control group. These ratios were found to be lower in the subjects of the control group. Although rejection was observed in both groups, this result showed that tissue rejection developed earlier and faster in the subjects that had not achieved tolerance after ACAID induction. Similarly, in the histopathologic evaluation of the control group, the lymphocyte clusters provided an idea of the intensity of the acute inflammatory process, which is in excess compared to the test group. Rapid immune response against the transplanted graft resulted in tissue damage and accompanying rejection. Besides that, no significant difference was observed in our study when the collagenization and neovascularization scores were compared among themselves. In
this respect, more meaningful results can be obtained with a greater number of subject groups. CONCLUSIONS
The desire is to enhance graft survival using the body’s immune tolerance. We, therefore, attempted to create immune tolerance drawing inspiration from this idea. Although our number of subjects was small, we think that the results we achieved will lead to larger patient populations and specialized studies. If this model could successfully be applied in routine clinics, it would be beyond imagination to have long-term, non-rejection graft survival and low morbidity and mortality rates in transplant surgery. REFERENCES [1] Streilein JW, Niederkorn JY. Induction of anterior chamberassociated immune deviation requires an intact, functional spleen. Ocul Immunol Inflamm 2007;15:187e94. [2] Shechter R, London A, Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol 2013;13:206e18. [3] Routy JP, Routy B, Graziani GM, Mehraj V. The kynurenine pathway is a double-edged sword in immune-privileged sites and in cancer: implications for immunotherapy. Int J Tryptophan Res 2016;9:67e77. [4] Ichiryu N, Fairchild PJ. Immune privilege of stem cells. Methods Mol Biol 2013;1029:1e16. [5] Stein-Streilein J, Streilein JW. Anterior chamber associated immune deviation (ACAID): regulation, biological relevance, and implications for therapy. Intern Rev Immunol 2002;21:123e52. [6] Vendomèle J, Khebizi Q, Fisson S. Cellular and molecular mechanisms of anterior chamber-associated immune deviation (ACAID): what we have learned from knockout mice. Front Immunol 2017;8:1686. [7] D’orazıo TJ, Nıederkorn JY. Splenic B cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (ACAID). Immunology 1998;95:47e55. [8] Farooq SM, Elkhatib WF, Ashour HM. The in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in C57BL/6 mice. Brain Behav Immun 2014;42: 118e22.
POST-TRANSPLANT IMMUNE TOLERANCE IN RATS [9] Zhang Y, Zhang M, Zhao S, Li X, Jia Z, Zhang L, et al. Effects of human umbilical cord-derived mesenchymal stem cells on anterior chamber-associated immune deviation. Int Immunopharmacol 2013;15:114e20. [10] Treacy O, Fahy G, Ritter T, O’Flynn L. Corneal immunosuppressive mechanisms, anterior chamber-associated immune deviation (ACAID) and their role in allograft rejection. Methods Mol Biol 2016;1371:205e14. [11] Xing Y, Hogquist KA. T-Cell tolerance: central and peripheral. Cold Spring Harb Perspect Biol 2012;1(6). [12] Camelo S, Kezic J, McMenamin PG. Anterior chamberassociated immune deviation: a review of the anatomical evidence for the afferent arm of this unusual experimental model of ocular immune responses. Clin Exp Ophthalmol 2005;33:426e32. [13] Abramov Y, Golden B, Sullivan M, Botros SM, Miller JJR, Alshahrour A, et al. Histologic characterization of vaginal vs. abdominal surgical wound healing in a rabbit model. Wound Repair Regen 2007;15:80e6. [14] Schulz-Juergensen S, Marischen L, Wesch D, Oberg HH, Fändrich F, Kabelitz D, et al. Markers of operational immune tolerance after pediatric liver transplantation in patients under immunosuppression. Pediatr Transplant 2013;17:348e54. [15] Wu H, Noordmans GA, O’Brien MR, Ma J, Zhao CY, Zhang GY, et al. Absence of MyD88 signaling induces donor-specific kidney allograft tolerance. J Am Soc Nephrol 2012;23:1701e16. [16] Miller ML, Daniels MD, Wang T, Chen J, Young J, Xu J, et al. Spontaneous restoration of transplantation tolerance after acute rejection. Nat Commun 2015;6:7566.
2135 [17] Hippen KL, Merkel SC, Schirm DK, Sieben CM, Sumstad D, Kadidlo DM, et al. Massive ex vivo expansion of human natural regulatory T cells (Tregs) with minimal loss of in vivo functional activity. Sci Transl Med 2011;3:83ra41. [18] Leventhal J, Miller J, Abecassis M, Tollerud DJ, Ildstad ST. Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance. Clin Pharmacol Ther 2013;93:36e45. [19] Casiraghi F, Remuzzi G, Perico N. Mesenchymal stromal cells to promote kidney transplantation tolerance. Curr Opin Organ Transplant 2014;19:47e53. [20] Souza-Moreira L, Delgado-Maroto V, Delgado M. Potential applications of vasoactive intestinal Peptide-based therapies on transplantation. Endocr Metab Immune Disord Drug Targets 2012;12:333e43. [21] Sicard A, Koenig A, Morelon E, Defrance T, Thaunat O. Cell therapy to induce allograft tolerance: time to switch to plan B? Front Immunol 2015;6:149. [22] Choi YS, Jeong JA, Lim DS. Mesenchymal stem cellmediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect. Immunol Invest 2012;41:214e29. [23] Dangi A, Yu S, Luo X. Apoptotic cell-based therapies for promoting transplantation tolerance. Curr Opin Organ Transplant 2018;23:552e8. [24] Kirk AD, Turgeon NA, Iwakoshi NN. B cells and transplantation tolerance. Nat Rev Nephrol 2010;6:584e93. [25] Vanhove B. Monoclonal antibodies in organ transplantation. Med Sci 2009;25:1121e5.
ABSTRACT Objective. We aimed to compare the clinical and histopathological results of skin graft transplants between rats that had been injected with lymphocytes into the anterior chamber of the eye with those that had not. Methods. A total of 16 Wistar albino, male rats were included in the study. Subjects were divided into 2 groups, namely a test group and a control group. Lymphocyte suspensions derived from the subjects of the control group were injected into the anterior chamber of the eye of each opposing subject of the test group. Also, an identical volume of physiological saline was injected into the anterior chamber of each subject in the control group to prevent bias. One week after this procedure, circular skin grafts of 1 cm in diameter were transplanted within the opposing groups. After a period of 1 week, transplanted graft tissues were excised to compare tissue healing. Results. The occurence of granulation and reepithelialization was more evident in the test group (96% and 33%, respectively, vs 80% and 17% for the control group, respectively). On the other hand, it was determined that acute inflammation was more intense in the control group (77% vs 50% for the test group). Conclusion. We had created immune tolerance in rats through anterior chamber lymphocyte injection, which slowed down the rejection process. If this can be successfully implemented in practice, survival for transplant patients without long-term rejection will move closer to becoming a reality.
A
HUMAN has several special anatomic regions that are inherently isolated from the immune system. These include the blood brain barrier, the placenta-fetus, the testes, and the anterior chamber of the eye. Due to their inherent immunologic and anatomic structures, they prevent the formation of an inflammatory response by gaining tolerance when exposed to antigens [1e4]. The concept of anterior chamber-associated immune deviation (ACAID), or “immune deviation associated with the anterior chamber of the eye,” is defined as the suppression of cellular and humoral immunoreactivity due to the presence of molecules such as interleukin 4, interleukin 10, interleukin 13, melanocyte-stimulating hormone (MSH), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), somatostatin, and tumor necrosis factor beta in the anterior chamber, the expression of Fas-Fas ligand, and the expression of T-reg 1e2 when the antigen is presented [5e10]. 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.064
2132
Alloantigens captured by the antigen-presenting cells are initially delivered to the thymus and then to the spleen after exiting the anterior chamber of the eye. Regulatory T cells, which have anti-inflammatory properties, become active after mutual interactions with other immune system components. These elements then suppress the T helper cells, which are responsible for the antigen-sensitive systemic response [11,12]. We planned on 2 different groups of rats of equal numbers, comprising a control group and a test group. The
*Address correspondence to Recep Erçin Sönmez, MD, Istanbul Medeniyet University, Department of General Surgery, itim Mah. Dr Erkin Cad. Kadıköy/_Istanbul 34722, Turkey. Tel: Eg 00905414454063. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 2132e2135 (2019)
POST-TRANSPLANT IMMUNE TOLERANCE IN RATS
lymphocyte derivates obtained after centrifugation from the control group of rats were injected into the anterior chamber of the eye of the test group rats. On the 7th day after this procedure, split-thickness skin graft tissues taken from each group were transplanted in the reciprocal group. Our 2 groups of results were compared clinically and histopathologically. The main objective was to protect graft survival through acquired immune tolerance that was created in vivo.
2133 of physiological saline into the anterior chamber of the eye without lymphocyte injection. One week after the operation, transplanted graft tissues were excised, and the healing process was histopathologically and clinically compared.
Ethical Approval All applicable international (Animal Research: Reporting of In Vivo Experiments guidelines), national, and institutional guidelines for the care and use of animals were followed. Animal experiments were approved by the Istanbul University Animal Experiments Local Ethics Committee (No: 35980450-050.01.04).
MATERIAL AND METHODS The average weight of the Wistar albino, male rats included in the study was 200 to 250 g (mean 225 g). The animals were maintained on a typical rat diet at room temperature with free access to tap water. Regular daily checks were conducted in the morning and evening. A total of 2 groups comprising test and control subjects were formed. Eight subgroups were labeled as reciprocal graft transplants (A1-A2, B1-B2, C1-C2, D1-D2, E1-E2, F1-F2, G1-G2, H1-H2).
Lymphocyte Isolation In Vivo Conditions Using aseptic techniques, 3 mL of Ficoll-Paque PLUS solution was withdrawn into a centrifuge tube. A 1 mL diluted blood sample taken from the dorsal vein of the tails of each rat in the control group was added to the Ficoll-Paque PLUS solution. It was then centrifuged at 400 rpm for 30 to 40 minutes at a temperature of 18 C to 20 C. Following the completion of the centrifugation process, the upper layer was carefully removed with a clean Pasteur pipette, leaving the middle layer of lymphocytes. Using a second, clean Pasteur pipette, the lymphocyte layer was transferred to an empty centrifuge tube. A minimum of 1.5 mL of balanced salt solution was added to the test tube containing the lymphocytes. The contents were carefully mixed using the Pasteur pipette. The mixture was then centrifuged at 60 to 100 rpm for 10 minutes at 18 C to 20 C. At the end of the procedure, the supernatant was separated. The remaining lymphocytes were mixed with approximately 1.5 to 2 mL of physiological saline using a Pasteur pipette. The lymphocyte solution was centrifuged at 60 to 100 rpm for 10 minutes at a temperature of 18 C to 20 C. At the end of the process, the supernatant was separated. The isolated lymphocytes were then ready for application. Lymphocytes, obtained in vivo by centrifugation of 1 mL serum samples from rats of the control group in Ficoll-Paque PLUS medium, were injected into the anterior chamber of the eye of the rats of the test group.
Surgical Procedure General anesthesia was provided by administering ketamine 50 mg/kg/ip, xylazine 5 mg/kg/ip to the rats, and feed intake was halted 12 hours before the experiment. The subjects’ back region, where the incision was to be applied, was shaved and cleaned with 10% polyvinylprolidone iodine antiseptic solution before the transplantation procedure. Approximately 1 cm of excised split-thickness skin graft tissue taken from the rats of the control group was transplanted to the rats of the test group in the form of a 1 cm diameter disc, using 5-0 Prolene sutures with a separate single technique. In the control group, skin graft transplantation was performed from the corresponding test group, following the injection of an identical volume
RESULTS
The mean follow-up period was 2 weeks. There was no evidence of infection at the wound site (malodorous discharge, redness, swelling, wound disintegration) within the follow-up period of the subjects. During the days following transplantation, graft color changes were observed in both control and test groups. One week postoperation, it was determined that the transplanted graft tissue of the control group was darker, harder, and more lifeless than the test group. On the contrary, that of the test group was found to be softer and more vigorous, although the body appeared to be paler than its skin color. Microscopic Evaluation of Lymphocytes
Under the light microscope, an average of 2 100,000 cells were observed in 2 microliters under 10X magnification. In flow cytometry, 95% of the lymphocytes were detected as the result of the analysis. The lymphocyte derivates obtained from the subjects in the control group had similar ratios. Histologic Findings
Under the light microscope, the formation of reepithelialization and neovascularization appeared to be more pronounced in the test group (Fig 1A). In the control group, more lymphocytes were detected on average, thus indicating the activity of acute inflammation (Fig 1B). Abramov’s histologic, wound healing, scoring system scale was modified to calculate the score, calculated as a percentage of the total score (maximum 3 points for each criterion). Average scores were calculated for the test group: acute inflammation 50%, granulation texture 96%, granulation texture and fibroblast maturation 96%, collagenization 59%, neovascularization 96%, and reepithelialization 33%. The mean scores for the histopathological evaluation for the control group were acute inflammation 77%, granulation texture 80%, granulation texture and fibroblast maturation 83%, collagenization 53%, neovascularization 93%, and reepithelialization 17%. DISCUSSION
The most important factor determining graft survival in transplantation immunology is preservation of immunologic tolerance. In the absence of immunosuppression in transplantation performed between individuals with a different
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SÖNMEZ, ILHAN, AND ERTEKIN
Fig 1. (A) Reepithelization with collagen deposition in the dermis (marked with red arrow) and formation of vessels showing neovascularization in the test group (marked with black arrows) (400X magnification, hematoxylin eosin staining). (B) Lymphocyte clusters showing acute inflammation in the control group (marked with black arrows) (400X magnification, hematoxylin eosin staining).
genetic make-up, rejection and tissue damage is an inevitable result [13e16]. Recently, cell-based therapies for inducing tolerance have begun to appear in the literature. Induction studies of mesenchymal stem cells, regulatory myeloid cells, regulatory T cells, and other cell types have been attempted. Although a definitive conclusion has not been reached, the most recent data is promising for the future [17e23]. After the feature of the anterior chamber of the eye was revealed, work in this area has begun and has developed, especially in the last few years. In recent publications, it is also stated that the creation of antigen specific tolerance is the desired target in transplantation immunology and, therefore, in surgery [24,25]. We tried to create an immune tolerance model in 16 male, Wistar albino rats. We identified control and test groups separately. We aimed to induce the ACAID system following the introduction of lymphocytes into the anterior chamber of the eye. According to the results of our findings, it was determined that the amount of granulation tissue, fibroblast maturation, and reepithelization were more prominent in the test group. Whereas in the control group, acute inflammation was more intense. To interpret these observations, the granulation and reepithelialization in the transplanted tissue was found to be greater in the test group, in which the ACAID system was induced after lymphocyte injection, because of the tolerance gained against the control group. These ratios were found to be lower in the subjects of the control group. Although rejection was observed in both groups, this result showed that tissue rejection developed earlier and faster in the subjects that had not achieved tolerance after ACAID induction. Similarly, in the histopathologic evaluation of the control group, the lymphocyte clusters provided an idea of the intensity of the acute inflammatory process, which is in excess compared to the test group. Rapid immune response against the transplanted graft resulted in tissue damage and accompanying rejection. Besides that, no significant difference was observed in our study when the collagenization and neovascularization scores were compared among themselves. In
this respect, more meaningful results can be obtained with a greater number of subject groups. CONCLUSIONS
The desire is to enhance graft survival using the body’s immune tolerance. We, therefore, attempted to create immune tolerance drawing inspiration from this idea. Although our number of subjects was small, we think that the results we achieved will lead to larger patient populations and specialized studies. If this model could successfully be applied in routine clinics, it would be beyond imagination to have long-term, non-rejection graft survival and low morbidity and mortality rates in transplant surgery. REFERENCES [1] Streilein JW, Niederkorn JY. Induction of anterior chamberassociated immune deviation requires an intact, functional spleen. Ocul Immunol Inflamm 2007;15:187e94. [2] Shechter R, London A, Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol 2013;13:206e18. [3] Routy JP, Routy B, Graziani GM, Mehraj V. The kynurenine pathway is a double-edged sword in immune-privileged sites and in cancer: implications for immunotherapy. Int J Tryptophan Res 2016;9:67e77. [4] Ichiryu N, Fairchild PJ. Immune privilege of stem cells. Methods Mol Biol 2013;1029:1e16. [5] Stein-Streilein J, Streilein JW. Anterior chamber associated immune deviation (ACAID): regulation, biological relevance, and implications for therapy. Intern Rev Immunol 2002;21:123e52. [6] Vendomèle J, Khebizi Q, Fisson S. Cellular and molecular mechanisms of anterior chamber-associated immune deviation (ACAID): what we have learned from knockout mice. Front Immunol 2017;8:1686. [7] D’orazıo TJ, Nıederkorn JY. Splenic B cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (ACAID). Immunology 1998;95:47e55. [8] Farooq SM, Elkhatib WF, Ashour HM. The in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in C57BL/6 mice. Brain Behav Immun 2014;42: 118e22.
POST-TRANSPLANT IMMUNE TOLERANCE IN RATS [9] Zhang Y, Zhang M, Zhao S, Li X, Jia Z, Zhang L, et al. Effects of human umbilical cord-derived mesenchymal stem cells on anterior chamber-associated immune deviation. Int Immunopharmacol 2013;15:114e20. [10] Treacy O, Fahy G, Ritter T, O’Flynn L. Corneal immunosuppressive mechanisms, anterior chamber-associated immune deviation (ACAID) and their role in allograft rejection. Methods Mol Biol 2016;1371:205e14. [11] Xing Y, Hogquist KA. T-Cell tolerance: central and peripheral. Cold Spring Harb Perspect Biol 2012;1(6). [12] Camelo S, Kezic J, McMenamin PG. Anterior chamberassociated immune deviation: a review of the anatomical evidence for the afferent arm of this unusual experimental model of ocular immune responses. Clin Exp Ophthalmol 2005;33:426e32. [13] Abramov Y, Golden B, Sullivan M, Botros SM, Miller JJR, Alshahrour A, et al. Histologic characterization of vaginal vs. abdominal surgical wound healing in a rabbit model. Wound Repair Regen 2007;15:80e6. [14] Schulz-Juergensen S, Marischen L, Wesch D, Oberg HH, Fändrich F, Kabelitz D, et al. Markers of operational immune tolerance after pediatric liver transplantation in patients under immunosuppression. Pediatr Transplant 2013;17:348e54. [15] Wu H, Noordmans GA, O’Brien MR, Ma J, Zhao CY, Zhang GY, et al. Absence of MyD88 signaling induces donor-specific kidney allograft tolerance. J Am Soc Nephrol 2012;23:1701e16. [16] Miller ML, Daniels MD, Wang T, Chen J, Young J, Xu J, et al. Spontaneous restoration of transplantation tolerance after acute rejection. Nat Commun 2015;6:7566.
2135 [17] Hippen KL, Merkel SC, Schirm DK, Sieben CM, Sumstad D, Kadidlo DM, et al. Massive ex vivo expansion of human natural regulatory T cells (Tregs) with minimal loss of in vivo functional activity. Sci Transl Med 2011;3:83ra41. [18] Leventhal J, Miller J, Abecassis M, Tollerud DJ, Ildstad ST. Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance. Clin Pharmacol Ther 2013;93:36e45. [19] Casiraghi F, Remuzzi G, Perico N. Mesenchymal stromal cells to promote kidney transplantation tolerance. Curr Opin Organ Transplant 2014;19:47e53. [20] Souza-Moreira L, Delgado-Maroto V, Delgado M. Potential applications of vasoactive intestinal Peptide-based therapies on transplantation. Endocr Metab Immune Disord Drug Targets 2012;12:333e43. [21] Sicard A, Koenig A, Morelon E, Defrance T, Thaunat O. Cell therapy to induce allograft tolerance: time to switch to plan B? Front Immunol 2015;6:149. [22] Choi YS, Jeong JA, Lim DS. Mesenchymal stem cellmediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect. Immunol Invest 2012;41:214e29. [23] Dangi A, Yu S, Luo X. Apoptotic cell-based therapies for promoting transplantation tolerance. Curr Opin Organ Transplant 2018;23:552e8. [24] Kirk AD, Turgeon NA, Iwakoshi NN. B cells and transplantation tolerance. Nat Rev Nephrol 2010;6:584e93. [25] Vanhove B. Monoclonal antibodies in organ transplantation. Med Sci 2009;25:1121e5.