The immunomodulatory effect of cryopreservation in rat tracheal allotransplantation.

BACKGROUND: Cryopreservation is one solution to the problem of donor organ deficit. To investigate the effect of cryopreservation on tracheal allografts, we performed 2 experiments in rats. METHODS: In Experiment 1, we assessed second-set graft rejection. Two weeks after primary heterotopic transplantation (Group 1, fresh isografts; Group 2, fresh allografts from Lewis rats; and Group 3, cryopreserved allografts from Lewis rats; n = 5, respectively), each animal underwent secondary heterotopic grafting with isografts and allografts from Lewis and Wistar Furth rats (n = 5, respectively). Four weeks after the secondary transplantation, all grafts were retrieved for histologic analysis. In Experiment 2, we assessed the long-term results of allograft cryopreservation, without immunosuppression therapy. Six months after transplantation of fresh (Group 4) and cryopreserved (Group 5) allografts, the tracheal segments (each group, n = 5) were histologically evaluated. RESULTS: In Experiment 1, only the secondary allografts from Lewis rats in Group 2 did not maintain lumen structure and often showed dislocated or destroyed cartilage. Second-set graft rejection was specifically recognized in Group 2, but not in Group 1 or 3. In Experiment 2, the cryopreserved allografts appeared almost normal and lumen rigidity was preserved 6 months after transplantation. These allografts were superior to the fresh allografts in patency and in cartilage dislocation and mononuclear cell infiltration scores, but not in the viable chondrocyte ratio. CONCLUSIONS: We conclude that cryopreservation may produce successful long-term results because of its immunomodulatory effect on tracheal allografts.     

Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts

BACKGROUND: Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS: The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS: The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS: The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.     

Effect of cryopreservation period on rat tracheal allografts.

BACKGROUND: The effect of cryopreservation on tracheal allogenicity is still unclear. Therefore, in this study, we assessed the effect of cryopreservation period on tracheal allografts in 62 rats. METHODS: Each transplant consisted of a 3-ring segment of the trachea harvested from 8 Lewis rats, immersed in preservation solution, and cryopreserved and stored in a Bicell biofreezing vessel in a deep freezer at -80 degrees C. Six tracheal grafts without cryopreservation that underwent a heterotopically syngeneic transplantation into the omentum served as controls. Forty-eight tracheal segments were randomly assigned to 8 groups according to period of cryopreservation, which ranged from 0 to 12 months (0, 0.5 [2 weeks], 1, 2, 3, 6, 9 and 12 months). The cryopreserved grafts were then thawed and heterotopically implanted into the omentum of recipient Brown-Norway rats. After 28 days, the tracheal segments were then evaluated histologically. RESULTS: All isografts in Group 1 were intact, whereas the allografts undergoing a particularly shorter period of cryopreservation showed a more stenotic lumen. Prolonged periods of cryopreservation tended to show decreasing tendencies of viability of chondrocytes, mononuclear cell infiltration and sub-epithelial thickness, whereas all allografts showed a uniformly denuded epithelium, irrespective of the length of cryopreservation. CONCLUSIONS: A longer period of cryopreservation may help to maintain a better patency of tracheal allografts by preventing an allogeneic response. Reduced tracheal allogenicity may be associated with a decreased viability of chondrocytes by cryopreservation.     

Successful tracheal transplantation using cryopreserved allografts in a rat model.

OBJECTIVES: The purpose of this study was to determine the appropriate cryopreservation period of tracheal allografts based on morphological and immunological findings and to test the possibility of tracheal transplantation in rats using cryopreserved allografts without immunosuppression. METHODS: Morphological and immunological studies were performed to compare the differences between non-cryopreserved grafts and cryopreserved grafts. Orthotopic tracheal transplantation using cryopreserved allografts, non-cryopreserved allografts, and non-cryopreserved autografts was performed and the rejection score of each group was evaluated. RESULTS: Epithelial cells were lost when the grafts were cryopreserved for more than 20 days. Immunohistochemical staining of the trachea revealed that the MHC classII antigen was expressed on normal epithelium. These findings suggest that cryopreservation for more than 20 days decreased the antigeneicity of allografts because of epithelial desquamation. All rats that received allografts cryopreserved for more than 20 days survived until the scheduled sacrifice day. Microscopically, cryopreserved allografts that had been preserved for more than 20 days had a significantly lower rejection score than that of non-cryopreserved allografts (P < 0.05). CONCLUSIONS: We conclude that the appropriate period for cryopreservation of allografts would be 20 days or more, because cryopreservation for more than 20 days depleted epithelium, which possessed the MHC classII antigen. Therefore, a longer period of cryopreservation decreases the antigeneicity of allografts. Rat tracheal transplantations using cryopreserved allografts is possible without immunosuppression when the grafts have been cryopreserved for more than 20 days.     

Revascularization of canine cryopreserved tracheal allografts

Background. We examined the blood supply of a cryopreserved tracheal allograft and its morphohistologic changes after transplantation.

Methods. In each of 22 dogs, a five-ring tracheal segment was replaced by one of the following tracheal grafts: fresh autografts (n = 8), cryopreserved tracheal allografts (n = 8), or fresh allografts (n = 6). The cryopreserved tracheal allografts were preserved at −196°C for 60 days. No immunosuppressant was given to any of the animals. All grafts were retrieved at 1 and 12 weeks and assessed by microangiography and histology.

Results. The epithelial denudation and the revascularization of the transverse intercartilaginous arteries were recognized within 7 days as common to each of the three types of grafts. In the cryopreserved tracheal allografts, neither cartilage degradation nor graft shrinkage occurred at 7 days. However, the recanalized transverse intercartilaginous arteries completely disappeared at 12 weeks, and marked shrinkage occurred; the cartilage cells were accompanied by karyolysis and were significantly decreased in number (p < 0.05). Recanalization of the transverse intercartilaginous arteries was also demonstrated in the fresh allografts; however, necrosis abruptly occurred as a result of acute rejection responses.

Conclusions. Cryopreservation of a tracheal allograft provided sufficient reduction of the acute rejection responses, and blood supply to the cryopreserved tracheal allograft was established through the recanalized transverse intercartilaginous arteries within 7 days; however, subsequent chronic rejection responses resulted in occlusion of the transverse intercartilaginous arteries and atrophy.     

Long-term viability of articular cartilage after microsurgical whole-joint transplantation and immunosuppression with rapamycin, mycophenolate mofetil, and tacrolimus

The survival or rejection of articular cartilage in heterotopic vascularized joint transplants in rats immunosuppressed with rapamycin (SDZ RAD), mycophenolate mofetil (MMF), and tacrolimus was evaluated histologically up to 1 year after surgery. The vascularized knee joint of an ACI donor rat was transplanted to the groin of a Lewis recipient rat. Nonimmunosuppressed allografts were evaluated after 6 weeks and 3 months, and immunosuppressed allografts and control isografts were evaluated after 6 weeks, 3 months, 6 months, and 1 year. No rejection was seen in the control isografts. All allografts without immunosuppression were rejected at 6 weeks and 3 months. Eighteen of 21 knee joint transplants immunosuppressed with SDZ RAD and 17 of 22 knee joint transplants immunosuppressed with MMF were rejected between 6 weeks and 1 year. SDZ RAD and MMF caused significant side effects including compromised wound healing and bone marrow suppression culminating in weight loss and death. Eighteen of 19 knee joints immunosuppressed with tacrolimus showed no signs of rejection up to 1 year after surgery. Long-term intermittent immunosuppression with tacrolimus was significantly superior to SDZ RAD and MMF in preventing rejection of the transplanted articular cartilage of a vascularized knee joint allograft up to 1 year after surgery.     

Effects of warm ischemia and cryopreservation on cartilage viability of tracheal allografts

Background. For clinical use of a cryopreserved tracheal allograft, it is important to evaluate cartilage viability. We assessed cell viability of the cartilage in a cryopreserved tracheal allograft by measurement of Na₂³⁵SO₄ incorporation. We also investigated the effects of warm ischemic time on tracheal cartilage viability.

Methods. The tracheas from Lewis rats were harvested and preserved at different warm ischemic times from cardiac death to preservation (0, 1, 2, 4, 6, 9, and 12 hours, each group N = 8). The cartilage was labeled with 4 μCi/mL of Na₂³⁵SO₄. The specimen was hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by liquid scintillation counter. Tracheas were transplanted into Brown Norway rats.

Results. ³⁵Sulfur incorporation in the cartilage decreased as warm ischemic time increased. In addition, ³⁵Sulfur incorporation decreased from 76% to 67% after cryopreservation. Histologic examinations of the normal tracheal cartilage before preservation and after thawing were done in all the groups. After transplantation, the cartilage had severe fibrous changes, and its layer was almost nonobservable in the 9- and 12-hour groups.

Conclusions. The viability of the tracheal cartilage decreased with warm ischemic time and from 76% to 67% after cryopreservation. In the rat tracheal transplantation model, a cryopreserved tracheal allotransplant could be done safely with a graft that was cryopreserved within 6 hours of warm ischemic time.     

Integrity of airway epithelium is essential against obliterative airway disease in transplanted rat tracheas.

BACKGROUND: The pathogenesis of obliterative bronchiolitis after lung transplantation requires further elucidation. In this study we used rat trachea transplantation to examine the role of epithelium in the progression of obliterative airway disease. METHODS: Normal and denuded (i.e., epithelium removed) trachea grafts from Lewis (LEW) and Brown Norway (BN) rats were transplanted sub-cutaneously into LEW rats. Viable trachea epithelial cells (to recover epithelium) were seeded into the lumen of some of the denuded tracheas. Grafts were removed at different time-points between 2 days and 8 weeks after transplantation. Histologic analysis was performed to evaluate cellular infiltration of inflammatory cells, loss of epithelium, and obliteration of trachea lumen. RESULTS: Obliteration was found to occur in trachea transplants after loss of epithelium, caused by rejection in allografts or by enzymatic denudation in isografts. In these situations, fibroblasts started to proliferate and migrate into the lumen in the second week after transplantation. Obliteration could be prevented when epithelial integrity was restored by seeding epithelial cells; no obliteration occurred when denuded trachea isografts were seeded with epithelial cells, whereas non-seeded denuded tracheas were obliterated at Day 6 after transplantation. CONCLUSIONS: We conclude that integrity of airway epithelium is essential for rat trachea transplants to be safeguarded from obliterative airway disease. For clinical lung transplantation the results of our study suggest that protection of the integrity of airway epithelium may be important in preventing the development of obliterative bronchiolitis.     

Maximal period of cryopreservation with the Bicell biofreezing vessel for rat tracheal isografts.

OBJECTIVE: The maximal period of cryopreservation for the trachea is still unsolved. We assessed the maximal period of cryopreservation using the Bicell biofreezing vessel as an easy and cheap slow-freezing instrument for viable tracheal grafts in 95 rats. METHODS: Each isograft was harvested from 17 donor rats, immersed in the preservative solution, and stored in a Bicell device in a deep freezer at -80 degrees C. The tracheal isografts were then randomly assigned to 9 groups according to cryopreservation periods ranging from 0 to 12 months. Included in the 9 groups were 2 subgroups (n = 6 per subgroup) that were observed immediately after being thawed and 1 month after heterotopic transplantation into the omentum after being thawed. Four subgroups (n = 6 per subgroup) were added according to the cryopreservation period for 1, 3, 6, and 12 months to evaluate the graft morphology 3 months after being thawed and transplanted heterotopically. RESULTS: A prolonged period of cryopreservation had a degenerative effect on both the epithelium and cartilage. One month after transplantation, degeneration was more pronounced in the cartilage than in the epithelium, as characterized by the viable chondrocyte ratio and the epithelial score of isografts undergoing cryopreservation for more than 9 months. Three months after transplantation, the morphology of the epithelium and cartilage in isografts undergoing cryopreservation for less than 3 months was better preserved, whereas the morphology of both deteriorated in isografts undergoing cryopreservation for more than 6 months. CONCLUSIONS: We conclude that the permissible period of cryopreservation to maintain tracheal isograft viability in this simple system using a Bicell biofreezing vessel is 3 months.     

Relationship between immunological rejection and matrix GLA protein in cryopreserved vascular allografts

INTRODUCTION: Cryopreserved tissue allografts used for cardiovascular diseases become calcified as a late complication after transplantation, probably caused by immunological rejection. Recent attention has been focused on the inhibitory effect of matrix Gla protein (MGP) on ectopic vascular calcification, but the behavior of MGP in cryopreserved allografts is uncertain. In this study we examined the relationship between immunological rejection and MGP in cryopreserved rat aortic grafts after transplantation. METHODS: Cryopreserved rat aortae were isografted or allografted intraperitoneally. Fresh isografts were also tested. The grafts were retrieved 9 days after transplantation and the intragraft MGP mRNA was measured by a real-time quantitative PCR method. The effect of daily administration of FK506 on MGP mRNA levels in cryopreserved isografts and allografts after transplantation was also evaluated. RESULTS: There was no significant difference in intragraft MGP mRNA levels between fresh and cryopreserved isografts 9 days after transplantation. MGP expression levels in cryopreserved allografts were significantly lower as compared to those in cryopreserved isografts (P < .01). Daily administration of FK506 enhanced intragraft MGP mRNA (ninefold) in cryopreserved allografts (P < .01), but not in cryopreserved isografts. CONCLUSIONS: Immunological rejection is likely to inhibit MGP expression in cryopreserved vascular allografts, resulting in late-onset calcification.     

Molecular mechanism of contractile dysfunction in cardiac allograft rejection.

Alterations in the beta-adrenergic receptor adenylyl cyclase pathway are well known in heart failure. To determine if an alteration in this pathway occurs during the reversible phase of cardiac allograft rejection, we used a rat heterotopic heart transplant model. Lewis rats received either isografts or Lewis Brown Norway allografts. Cardiac grafts and native hearts were explanted 4, 5, or 6 days later. Receptor-mediated modulation of adenylyl cyclase activity was investigated using isoproterenol, forskolin, and the muscarinic and adenosine receptor agonists carbachol and R-N6-(C2-phenyl-isopropyl)-adenosine (R-PIA), respectively. Allografts demonstrated evidence of histological rejection and a significantly impaired response to forskolin and isoproterenol on all days: [table: see text] (% increase in cAMP in response to forskolin or isoproterenol +/- standard error. All results P less than 0.03 except Day 4 forskolin and Day 5 isoproterenol.) No significant difference was noted between isografts and allografts stimulated with carbachol and R-PIA. These data suggest that a primary alteration in adenylyl cyclase activity may be a component of the molecular basis of reversible contractile dysfunction in cardiac allograft rejection.     

Transplantation of the entire small bowel in inbred rats using cyclosporine

Inbred strains of rats were used to analyze unidirectional host-versus-graft disease (transplant rejection) without graft-versus-host disease in small intestinal transplants and the immunosuppressive properties of cyclosporine (CsA). Forty-six Lewis rats received heterotopic transplants of the entire small bowel in four groups: Lewis-to-Lewis isografts, without CsA; Lewis-to-Lewis isografts, with CsA (15 mg/kg/day); (Lewis X ACI)F1-to-Lewis allografts, without CsA; (Lewis X ACI)F1-to-Lewis allografts, with CsA. Small bowel rejection was associated with gross morphological changes that preceded all other findings. A histologic scoring system assessed the degree of transplant rejection. A characteristic transient weight loss was seen in animals rejecting their bowels. Glucose absorption was impaired and polyethylene glycol absorption increased during rejection. Cyclosporine inhibited all of these changes in allografted rats. It is concluded that daily administration of cyclosporine is effective in preventing the morphologic and functional changes of acute transplant rejection in intestinal allografts and does not change these parameters in transplants that are not rejecting.     

Repair of long tracheal defects with cryopreserved cartilaginous allografts.

Tracheoplasties with various autografts (cartilage, periosteum, pericardium) have been used in the treatment of long-segment tracheal stenosis. Previous studies have shown that cartilage allografts survive transplantation on a long-term basis in various sites of the body. In this study we set out to determine if cryopreserved cartilage and cryopreserved tracheal allografts would survive when used to cover tracheal defects in animals. A rectangular defect (2.8 +/- 0.3 cm long and incorporating 50% of tracheal circumference) was created in the thoracic trachea of 18 piglets. The defect was covered with the excised tracheal segment in 6 (group A, control group), with a cryopreserved tracheal allograft in 6 (group B), and with a cryopreserved cartilage allograft harvested from the scapula in 6 (group C). The allografts were cryopreserved, by a standard slow-freezing technique, at -80 degrees C for more than 21 days. All animals survived the grafting procedure and were killed after 2 months. None had signs of airway obstruction. Using the trachea above the defect as the standard, the mean sagittal narrowing of the airway in the repaired trachea was 0.4 mm in group A, 0.7 mm in group B, and 0.6 mm in group C; the coronal diameter in normal and grafted trachea was similar. The lumen of all grafts was lined by regenerating respiratory epithelium, and cilia were seen in many. Some cartilage was reabsorbed in group A and B but cartilage islands were present in all. In group A, reabsorption of cartilage was minimal. These findings suggest that segments of trachea or cartilage allografts can be cryopreserved, stored, and, subsequently, used when necessary for tracheoplasty.     

Immune function in transplanted small intestine. Total secretory IgA production and response against cholera toxin

Secretory IgA is the dominant immunoglobulin produced in the small intestine and one important component of the local defense against dietary and infectious agents present in the gut lumen. The effect of small intestine transplantation on total production of sIgA and on the response to a newly presented antigen, cholera toxin, was determined in a rat segmental heterotopic intestinal transplant model. Lewis x Brown Norway F1 (LBNF1) allografts in Lewis hosts made normal amounts of sIgA, when compared with LBNF1 Thiry-Vella loops or LBNF1 isografts. In contrast, the allografts failed to make a significant specific sIgA response when immunized with cholera toxin at days 0 and 7 following transplantation. This failure was not the result of surgical manipulation, as isografts made normal amounts of specific sIgA directed against cholera toxin. Cyclosporine immunosuppression delayed, but did not prevent, the secretion of specific antibody in isografts. This failure to respond to a new antigen may have important implications for the safety of small bowel transplantation.     

Graft protective effects of heme oxygenase 1 in mouse tracheal transplant-related obliterative bronchiolitis

BACKGROUND: Heme oxygenase (HO)-1, long believed to be a cytoprotective protein, has recently been identified as a graft survival gene. This study evaluates the role of HO-1 in a murine heterotopic tracheal allograft model for obliterative bronchiolitis. METHODS: Mice with deficient or experimentally enhanced HO-1 expression underwent subcutaneous implantation of murine tracheal isografts and allografts. Grafts were excised after 9, 16, or 21 days and evaluated by histologic examination, immunohistochemistry for HO-1 and interleukin (IL)-10 proteins, and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. To evaluate the relationships between IL-10 and HO-1, the effects of modulation of HO-1 expression on IL-10 expression were evaluated and HO-1 expression was examined in tracheal transplants from IL-10 null mice. RESULTS: Isografts demonstrated normal histology with minimal HO-1 staining, whereas allografts showed features of human airway rejection (loss of respiratory epithelium, luminal granulation tissue, lymphocytic tracheitis) with increased HO-1 staining in macrophages and mesenchymal cells. HO-1-deficient mice demonstrated a more rapid progression of the tracheal allograft injury as compared with control allografts, and this was associated with a decrease in the anti-inflammatory cytokine, IL-10. Tracheal transplants using IL-10-deficient mice also resulted in a more severe injury, and this was accompanied by a decrease in HO-1 staining. CONCLUSIONS: HO-1 protein expression is increased in murine heterotopic airway rejection, and deficiency of HO-1 accelerates the development of the obliterative bronchiolitis-like lesion. IL-10 protein expression parallels expression of HO-1, suggesting that IL-10 may participate in the genesis of HO-1's effects on the inflammatory processes triggered by allotransplantation.     

异体气管移植去抗原性的实验研究

目的探讨移植段气管去除上皮细胞和腺体细胞后异体、异位移植排斥反应的强弱。方法25只雄性SD大鼠作为供体,制备新鲜移植段气管、冷冻移植段气管和去上皮细胞移植段气管。取制备的新鲜移植段气管40个平均分为4组,分别用0、0.1、0.3和0.5mg/ml的蛋白酶溶液,4℃浸泡12h,根据镜下移植段气管上皮细胞和腺体细胞脱落情况及软骨细胞破坏程度确定蛋白酶的最佳浓度。另取30只雄性SD大鼠作受体,均分成3组,分别为:新鲜气管移植组(A组)、冷冻气管移植组(B组)及去上皮细胞移植组(C组),n=10。行左上腹旁正中切口,提出大网膜包绕各移植段气管,于21d后取出移植段气管,行组织学观察及淋巴细胞浸润测定。结果0.3mg/ml蛋白酶能去除移植段气管上皮细胞及腺体细胞,而对软骨细胞无明显损坏。异体植入大鼠腹腔的3组气管软骨均成活,血运建立,其中A组管腔内有肉芽组织,出现坏死、实变;B组有少量肉芽组织;C组管腔内无肉芽组织。A、B、C3组淋巴细胞浸润分别为29.16±2.69、15.17±2.19和11.56±0.87个/Hp,A组与B、C组比较及B组与C组比较,差异均有统计学意义(P<0.05),排斥反应强弱为:A组>B组>C组。结论0.3mg/ml蛋白酶,4℃,浸泡12h的移植段气管,能完全脱上皮细胞和腺体,对软骨细胞基本无损伤,与冷冻法比较去抗原作用更好。一期异体大网膜包裹异位移植后,血运重建且移植段气管成活。     

Cryopreservation alters antigenicity of allografts in a porcine model of transplant vasculopathy

The need for arterial grafts in coronary surgery to complement autologous vessels has generated interest in cryopreservation of small diameter allografts. We evaluated functional and histologic changes occurring in cryopreserved allografts 3 months after porcine femoral artery transplants. METHODS: Twenty recipient and 15 donor pigs included a control group of 16 fresh and 12 cryopreserved nonimplant arteries were used. Fresh (n=5) and cryopreserved (n=5) autografts were implanted to assess cryopreservation effects in the absence of rejection. Fresh allografts with or without treatment with cyclosporine (CsA) (n=6 of 8) and cryopreserved allografts with or without treatment with CsA (n=6 of 10) were performed to study the antigenicity of cryopreserved allografts. Arteries were stained with hematoxylin and eosin, Masson's trichrome, and orcein for morphometric analyses and immunostained to identify endothelial cells, smooth muscle cells, T lymphocytes, and macrophages. RESULTS: Among nonimplant arteries, cryopreservation reduced alpha-actin expression and increased the luminal area. All implanted autografts were patent. Cryopreserved autografts showed reduced alpha-actin expression and developed intimal hyperplasia compared to fresh autografts. Treatment with CsA improved the patency of fresh allografts from 0% to 83% (P <.01) and of cryopreserved allografts from 40% to 100% (P <.05). Cryopreserved allografts showed substantial intimal hyperplasia, and fresh allografts had more T lymphocyte infiltration in the intimal layer with aneurysmal dilatation. CONCLUSIONS: Cryopreservation reduces the deposition of inflammatory cells and prevents the thrombosis or aneurysmal lesions observed in fresh allografts. Therefore, cryopreservation modifies the antigenicity of vascular allografts.     

Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats

BACKGROUND: Because epithelial cells are targets of alloimmune injury leading ultimately to airway obliteration, we tested whether epithelial re-growth could prevent obliterative airway disease (OAD) in orthotopic tracheal allografts. METHODS: Brown Norway tracheal segments were orthotopically transplanted into nonimmunosuppressed Lewis rats. Allografts were removed on days 2-10 (n=13), 30 (n=4), and 60 (n=5) for histology, computerized morphometry (obliteration), and immunohistochemical detection of mononuclear cells, smooth muscle alpha-actin, and tissue phenotype. Normal tracheas, host tracheas, and heterotopically transplanted allografts served as controls. RESULTS: Orthotopic allografts removed on days 2-10 exhibited epithelial damage and re-growth and mononuclear cell infiltration. On days 30 and 60, partially ciliated cuboidal or attenuated epithelium completely covered the lumen. Although mononuclear cells declined, numerous T cells with a high CD4/CD8 ratio were found in the epithelium till day 60. Orthotopic allograft epithelium expressed donor phenotype on day 7, but recipient phenotype on days 30 and 60. Despite subepithelial alpha-actin positive myofibroblast proliferation, obliteration did not progress from day 7 to 30 and 60 (35, 30, and 33%, respectively). Although more than in normal or host tracheas, the obliteration in orthotopic allografts on days 30 and 60 was significantly less (P<0.001) than in heterotopic allografts. CONCLUSIONS: We describe, for the first time, longterm patency of fully histoincompatible orthotopic tracheal allografts in nonimmunosuppressed rats. Despite acute alloimmune injury and induction of myofibroblast proliferation, epithelial re-growth from the host limited the progression of OAD, thus emphasizing the role of epithelium in the control of airway obliteration.     

Effect of storage at 4 degrees C in a nutrient medium on antigenic properties of rat aortic valve allografts

The effects of prolonged storage at 4 degrees C in nutrient medium on the antigenic properties of aortic valve allografts are unknown. Lewis rats received heterotopic aortic valve allografts from Brown Norway donors. Valves were transplanted immediately after harvest (fresh), or after antibiotic sterilization and storage in a nutrient medium at 4 degrees C for 3, 7, 14, or 21 days. Additional rats underwent sham laparotomy (sham). All recipient rats received Brown Norway skin grafts 3 weeks after valve transplant or sham procedure. Time to skin graft rejection for all groups was as follows: fresh (n = 10), 4.5 +/- 0.9 days; sham (n = 10), 7.1 +/- 0.3 days; 3-day (n = 10), 4.9 +/- 0.3 days; 7-day (n = 10), 5.2 +/- 0.4 days; 14-day (n = 10), 6.1 +/- 0.7 days; and 21-day (n = 10), 6.0 +/- 0.6 days. Significant differences existed between the sham group and each of the transplanted groups. No significant differences existed between the fresh group and either the 3-day or 7-day groups. The difference between the fresh group and the 14-day group approached significance (0.05 less than p less than 0.10), and the difference between the fresh and 21-day groups was significant (p less than 0.05). Additional valves not used for transplantation were studied with scanning electron microscopy. The valves preserved in nutrient medium exhibited a progressive loss of endothelium as compared with fresh valves. Storage of aortic valve allografts in a nutrient medium at 4 degrees C is associated with a progressive attenuation of antigenic response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-reactivity of organs in allograft rejection. Comparison of effect of thyroid allografts on established islet allografts

The effect of allotransplantation of thyroid or islet allografts into rats with established islet allografts was studied to determine the cross-reactivity of the thyroid and islets in allograft rejection. Islets obtained from cultured neonatal rat (F344) pancreas explants were transplanted bilaterally underneath the kidney capsule of Wistar-Furth rats. After 21 days these allografts did not exhibit signs of rejection. Thyroid (half lobe) from either F344 or Brown Norway rats was transplanted underneath the capsule of the remaining kidney. Transplant of the thyroid from F344 rats resulted in immediate rejection of the islet transplant, whereas transplant of the thyroid from Brown Norway rats was without effect on the islet allograft. This indicates that the thyroid contains immunocompetent cells (cells that present antigen or induce recognition of antigen) that are capable of initiating rejection of established islet allografts. The cytotoxic T-lymphocytes that result are specific for the organ bearing the immunocompetent cells at time of transplantation.