Antigenicity of fresh and cryopreserved rat valve allografts

Aortic valve allografts have demonstrated excellent clinical performance, but the importance of antigenic differences between donor and recipient is largely unknown. To determine the antigenicity of aortic valve grafts, rat aortic valves with a short portion of thoracic aorta were transplanted into the abdominal aorta of recipient rats. Valves were used immediately after harvest (fresh) or following cryopreservation. Three weeks after this procedure, the recipient rats received a skin graft from a rat of a strain syngeneic to that of the aortic valve donor. Additional groups of rats were subjected to sham operation (sham) followed three weeks later by skin grafting. Recipient rats were of the Lewis strain. Donor rats were of the Lewis, F344 (weakly allogeneic, RT1-compatible, non-RT1-incompatible), LBN F1 (moderately allogeneic, one-haplotype-identical and one-haplotype-incompatible at both the RT1 and non-RT1 loci), or BN (strongly allogeneic, RT1 and non-RT1-incompatible) strain. Time to skin graft rejection was measured. Among rats receiving the F344 grafts, the time to skin graft rejection (mean +/- SD) was sham: 9.1 +/- 1.0 days, fresh: 7.1 +/- 1.2 days, cryopreserved: 6.9 +/- 0.7 days. Among rats receiving the LBN F1 grafts, the corresponding times were sham: 7.8 +/- 0.8 days, fresh: 5.6 +/- 0.5 days, cryopreserved: 5.4 +/- 0.5 days. Among rats receiving the BN grafts, the corresponding times were sham: 7.1 +/- 0.3, fresh: 4.5 +/- 1.0 days, and cryopreserved: 4.3 +/- 0.7 days. Significant differences (P less than 0.05) existed between sham and fresh and between sham and cryopreserved, but not between fresh and cryopreserved. Significant differences (P less than 0.05) also existed between each histocompatibility grouping. It is concluded that aortic valve allografts in rats are antigenic and produce recipient sensitization. Cryopreservation does not diminish this sensitization. The degree of antigenicity is related to the degree of histoincompatibility between donor and recipient. Both RT1 and non-RT1 antigens appear to play a role in this process.     

Preparation of venous allografts. A comparison of techniques.

In order to evaluate alternate techniques of preparing veins for use as homografts, 102 femoral veins were harvested from adult mongrel dogs. The veins were treated in four different ways, then transplanted into recipient animals bypassing their ligated femoral arteries. Group I--24 veins (6 cm each) were harvested and immediately transplanted. Group II--24 veins were stored in 15% dimethyl sulfoxide (DMSO) solution at -120 C for 21 days prior to transplantation. Group III--26 veins were stored for 21 days in plasminate solution at -60 C prior to use as allografts. Group IV--28 veins were stored in 0.5% gluteraldehyde solution for 21 days prior to implantation. Animals were randomly sacrificed at 1-month, 2-month, 6-month, and 12-month intervals. Patency of the transplant was determined weekly by ultrasound. Specimens were sent for light and scanning electron microscopy at the time of harvest, prior to implantation, and at sacrifice. Endothelial damage was graded on a scale of 0-16. Veins in Group II had a significantly higher patency rate (68% at 1 year) than Group III (35%) and Group IV (11%) (p less than 0.05). The intimal layer of all patent vessels was replaced by an organized mural thrombus. Partial endothelialization of the luminal surface was most prevalent in Group II. Intimal damage related to storage technique was significant in Group III (p less than 0.01). At sacrifice, severe endothelial damage was present in all groups (p less than 0.01). In conclusion, veins stored in 15% dimethylsulphoxide (DMSO) solution at -120 C have immunologic and physical characteristics that yield patency rates acceptable for clinical use when autogenous tissue is not available.     

Immunosuppressive treatment of venous allografts

In order to evaluate whether temporary immunosuppressive therapy is able to improve the results obtained with viable venous allografts and achieve better results than with synthetic grafts, 142 arterial reconstructions were performed in mongrel dogs bypassing their ligated femoral arteries. Histological as well as immunological studies were performed and patency determined by weekly palpation and regular angiography. The 6-month cumulative patency rates were: Group I: synthetic grafts (a) Dacron: 48%, (b) plasma-TFE: 53%. Group II: fresh grafts (a) autografts: 100%, (b) allografts: 37%, (c) allografts treated with cyclosporin 4 mg kg-1 daily for 1 month: 74% (100% after 1 month). Group III: grafts preserved in Hanks' solution with 15% DMSO at -160 degrees C for 1 month (a) autografts: 77%, (b) allografts: 35%, (c) allografts treated with methylprednisolone 1 mg kg-1 daily: 38%, (e) allografts treated with cyclosporin and methylprednisolone: 83%. Group IV: human saphenous veins implanted as xenografts and treated with cyclosporin and methylprednisolone: 18%. Immunosuppressive therapy with cyclosporin seems to be able to prevent early thromboses due to rejection seen after implantation of viable fresh or cryopreserved venous allografts, and the results are significantly better than those obtained with synthetic grafts. Tissue matching might further improve these results. This study suggests that cryopreserved venous allografts could be used for the creation of a vein-bank and their use, in combination with tissue typing and temporary immunosuppressive therapy may be warranted for arterial reconstructions when autologous saphenous vein is not available.     

Microsurgical application of freeze-dried venous allografts

Freeze-dried venous interposition allografts with an internal diameter of 1.0 mm and a length of 1.0 cm were placed into the femoral arteries of 17 Sprague-Dawley albino male rats in order to investigate patency and host tissue response. The immediate patency rate was 88%. The epigastric island flap was monitored as a sign of patency in the early postoperative period (defined as 1 to 3 days). Twelve of the 17 subjects were observed for 2 months, at which point 66% (8/12) remained patent. There were two aneurysmal dilatations. Histopathological studies and transmission electron microscopy demonstrated that freeze-dried veins undergo complete remodeling in vivo by a normal reparative process and that they do not induce a cellular immune response in the host.     

Transplantation of cryopreserved canine venous allografts

Local vascular reconstructions frequently require the use of vein grafts to bridge arterial or venous defects. Most previous studies on the use of cryopreserved veins have used relatively large caliber vessels. There have been few studies on the effectiveness of cryopreserved micro- or small-venous allografts. Here, we tested two types of cryopreserved venous allografts: (1) 1.5- to 1.9-mm diameter microvenous grafts (MVG); and (2) 4- to 5-mm diameter small venous grafts (SVG). Cryopreserved MVG allografts were placed into saphenous arteries of six experimental dogs and SVG cryopreserved allografts were placed into femoral arteries of six experimental dogs for 3 to 6 weeks. Two fresh MVG autografts were also transplanted into experimental dogs as controls and autografts were transferred to the contralateral side in SVG dogs as controls. None of the six cryopreserved MVG grafts retained patency but three/six cryopreserved SVG allografts were patent at harvest. Histological examination of grfts revealed control autografts were undergoing arterialization with an intact intima. Experimental cryopreserved allografts showed extensive medial fibrosis, significant lymphocytic infiltrates, and sporadic areas of intact intima for both patent and nonpatent grafts.     

Cold-stored venous allografts in the treatment of critical limb ischaemia.

OBJECTIVES: To assess the outcome of cold-stored venous allografts in critically ischemic limbs in patients with no ipsilateral autogenous greater saphenous vein. DESIGN: A non-randomised, retrospective, single-center study. METHODS: From September 2000 to June 2006, 46 cold-stored venous allografts obtained during multiorgan harvest were implanted into 44 critically ischaemic limbs of 43 patients. The indication for reconstructions was rest pain (24%) or tissue lost (76%). Sixty-seven percent of procedures were performed as secondary reconstructions, and 61% of veins were anastomosed to tibial or pedal arteries. Thirty-seven percent of patients received prednisone, and 46% tacrolimus as postoperative immunosuppressive therapy. Mean patient follow-up period was 13.3 months (range 1 week to 60 months). RESULTS: The secondary patency rate for the cohort was 83+/-5.6% at 1 month, 64+/-8.2% at 6 months, 57+/-10.0% at 12 months and 46+/-10.7% at 24 months. Limb salvage rate was 96+/-3.1% at 1 month, 78+/-6.9% at 6 months, 71+/-8.1% at 12 months and 50+/-11.8% at 24 months. CONCLUSION: Cold-stored venous allografts are an alternative conduit for limb salvage procedures when ipsilateral autogenous vein is unavailable.     

The preservation of the mechanical properties of venous allografts by freezing.

Stored venous allografts are potential alternatives to autogenous materials when the latter are unavailable for arterial replacement. Among the criteria necessary for utilization would be the preservation of hemodynamically favorable elastic properties. A study was undertaken to assess the effects of frozen storage on venous compliance (percentage volume change/mm Hg) and elastic modulus (dyn/cm² × 10⁸) of 40 canine jugular vein segments. The veins were allotted to two experimental groups. Five from each group acted as controls. Fifteen vein segments were frozen immediately with liquid N₂ (?196°C) and stored at ?70°C (unprotected). Another fifteen were pre-treated with 15% DMSO and then frozen (DMSO). The veins were thawed by immersion in Hanks' balanced salt solution (37°C) after either 1 or 24 hr, or 1 week. Compliance and elastic modulus were calculated from the volumetric responses to pressure of the vein segments mounted in a special saline-filled cylinder. There was a modest but statistically insignificant increase in compliance from initial control values of 1.9 ± 0.68 (unprotected) and 1.3 ± 0.44 (DMSO-treated) to 2.16 ± 0.29 and 2.04 ± 0.70, respectively, at 1 week. The elastic moduli of the control groups (16.3 ± 4.5, unprotected; 22.6 ± 4.5, DMSO) were also indistinguishable from the frozen groups (13.5 ± 2.3, unprotected; 15.4 ± 3.9, DMSO). Short-term freezing preserves the important functional elastic properties of veins and this preservation is independent of the use of DMSO.     

Portal versus systemic venous drainage for small-bowel allografts

The effect of portal venous drainage (PV-D) on the survival of accessory small-bowel allografts was studied in a rat model. In the LBN-F1----LEW combination (rejection reaction only), grafts with caval venous drainage (CV-D) were rejected acutely (mean 11.8 days) and those with PV-D were rejected chronically (mean, 22.8 days). In the LEW----LBN-F1 combination, the survival of recipients subject perforce to a fatal graft-versus-host reaction was not influenced by the type of venous drainage used (CV-D: 14.1 days; PV-D: 14.8 days). These findings suggest that the delaying influence of the liver on the rejection process is not due to unspecific filtration of antigens but must involve their specific recognition or alteration. In the BN----LEW combination, in which rejection and the graft-versus-host reaction occur simultaneously, the type of venous drainage used did not influence graft survival (CV-D: 16.2 days; PV-D: 15.5 days), nor did it modify the rejection process. This indicates that the liver has a minor effect on the rejection process, sufficient to inhibit the rejection of semiallogeneic LBN-F1 grafts but insufficient to alter the rejection of allogeneic BN grafts by LEW recipients. With CV-D, LBN-F1 grafts were rejected as rapidly as were BN grafts. This unexpected finding may be explained by the fact that in the LBN-F1----LEW combination only rejection occurs, unimpeded by a graft-versus-host reaction, which is known to impair the immune system of the recipient; in the BN----LEW combination, however, the graft-versus-host reaction temporarily opposes the rejection reaction, thereby allowing prolonged graft survival.     

Factors affecting patency of venous allografts in miniature swine

In immunologically defined National Institutes of Health miniswine, a segment of internal jugular vein was anastomosed to the carotid artery as an interposition graft. Patency of swine major histocompatibility complex matched, one haplotype mismatched, and complete mismatched veins was 9.8, 6.3, and 3.0 weeks respectively (p = 0.009). More than 90% of mismatched and 20% of matched allografts developed a positive crossmatch before occlusion (p = 0.006). The mixed lymphocyte response did not predict graft occlusion. Treatment of 10 swine with cyclosporine (10 mg/kg/day) did not significantly improve patency for one haplotype mismatched grafts. In haplotype mismatched veins, cryopreserved grafts occluded more rapidly than noncryopreserved grafts: mean 2.4 versus 6.3 weeks, respectively (p = 0.002). In all cryopreserved vein grafts, alloantibody appeared at or after graft occlusion rather than before occlusion as seen with fresh allografts (p = 0.046). The mean patency of cryopreserved versus fresh autografts was 3.3 and greater than 32 weeks, respectively (p = 0.004). In summary, these results indicate that (1) allograft patency is related to the degree of swine major histocompatibility complex match and development of cytotoxic alloantibodies; (2) moderate-dose cyclosporine does not prolong allograft patency nor suppress development of antibody; (3) cryopreservation may accelerate graft occlusion through nonimmunologic mechanisms.     

A new technique for venous anastomosis of pancreatic allografts

Venous thrombosis is still a frequent cause of graft loss after pancreas transplantation where the portal vein is used for revascularisation. It is known that an increase in velocity of venous flow decreases the incidence of thrombosis. According to the equation of continuity, the flow velocity in the portal vein should decrease to 25% as compared with that in the splenic vein. Based on the rheological considerations we started to use the superior mesenteric vein, the diameter of which is similar to that of the splenic vein, for revascularisation of pancreas transplants after sewing the portal vein closed at its origin. This technique has been applied in 12 consecutive pancreatic transplants. Two patients died of a cerebrovascular accident and myocardial infarction, respectively. The only single pancreatic graft was lost due to rejection. The remaining 9 patients are alive and well between 2 and 23 months with normally functioning grafts. Another advantage of this technique is that a full-length portal vein can be left with the liver graft in the event of simultaneous liver and pancreas procurement.     

A multicenter, phase I evaluation of cryopreserved venous valve allografts for the treatment of chronic deep venous insufficiency.

PURPOSE: A phase I feasibility study was conducted to determine whether cryopreserved venous valved segments would remain patent/competent in a short-term period (6 months). METHODS: The target group consisted of 10 patients (C(4-6), E, A(D), P(R)). The exclusion criteria included untreated superficial/perforator venous disease, significant venous or arterial obstruction, hypercoagulability or coagulopathy, and significant preexisting medical conditions. Required preoperative tests were venous duplex, ascending/descending venography, and a physiologic study (eg, APG, blood typing, an ankle/brachial index, and if post-thrombotic, a hypercoagulability work-up). A single-valve transplant was placed below all reflux, aided by anticoagulation with or without a distal arteriovenous fistula. Postoperative assessment included duplex scanning/clinical examination (at 1, 3, and 6 months), descending venogram (at 1 month), and physiologic study (at 1 and 6 months). The primary end point was valve patency/competence, with clinical outcome as a secondary end point. Adverse events were recorded. RESULTS: After eliminating protocol violations, nine patients with superficial femoral (5) or popliteal (4) vein valve transplants were studied. Six-month actuarial results show a patency rate of 67% +/- 16% and 78% +/- 13%, respectively, a primary and secondary competency rate of 56% +/- 17% and 67% +/- 16%, respectively, and a 100% patient survival rate. Clinical outcome averaged 1.1, with healing and/or freedom from ulcer recurrence, in six of nine patients. A postoperative risk of seroma formation (3) and cellulitis (1) exists. CONCLUSION: In patients with few remaining therapeutic options, one can achieve a 6-month assisted patency and competency rate of 78% and 67%, respectively, with an improved clinical outcome.     

Prolonged preservation of venous allografts for vascular access in hemodialysis

The technique of deep-freezing in liquid nitrogen of stripped saphenous veins permitted their long-term preservation, and the subsequent creation of a "bank" of venous allografts, used for arteriovenous internal shunts for hemodialysis. Histological, histochemical and immunological tests, and first clinical results have been favorable.     

Successful surgical salvage of pancreas allografts after complete venous thrombosis

BACKGROUND: Complete venous thrombosis of the pancreas after simultaneous pancreas-kidney (SPK) transplantation usually results in graft loss. We describe a technique that allows salvage of the graft after complete venous thrombosis. METHODS: A total of 150 patients with insulin dependent diabetes mellitus/end stage renal disease underwent SPK over the past decade at the University of Miami. Of these, three patients developed complete venous thrombosis after induction therapy with antiinterleukin-2R antibody and i.v. tacrolimus. These three patients underwent surgical thrombectomy followed by heparinization and oral anticoagulation. The splenic vein was opened distally at the tail of the pancreas and the superior mesenteric vein at the level of the mesentery or head of the pancreas. Thrombectomy was performed with a Fogarty catheter. The portal anastomosis was not opened or manipulated. The arterial "Y" graft was not clamped and the right iliac vein was controlled proximally with a double wrapped vessel-loop to contain possible thrombus. In one patient, the partially thrombosed splenic artery was opened at the tail of the pancreas and thrombectomy was performed in the same fashion. There were no apparent technical problems. A pancreatic biopsy was not performed, nor was acute rejection treated empirically. RESULTS: Intraoperative and serial Doppler ultrasound showed good flow through the allograft. In all three patients the exocrine and endocrine function of the pancreas was preserved with a mean follow-up of 15 months. CONCLUSIONS: The described surgical thrombectomy followed by systemic anticoagulation may be useful in the salvage of the allograft pancreas in case of complete venous thrombosis.     

Portal venous ultraviolet B-irradiated donor alloantigen prevents rejection in circumferential rat tracheal allografts.

BACKGROUND: Before tracheal transplantation can be considered as a method of reconstruction in patients with extensive circumferential tracheal defects, we must achieve a state of nontoxic, donor-specific tolerance so that the risks of such a transplant do not outweigh the benefits. OBJECTIVE: Our objective was to determine whether a single intraportal injection of modified donor alloantigen achieves donor-specific immunosuppression for major histocompatibility complex-mismatched rat tracheal allografts. STUDY DESIGN: Buffalo (recipient) rats were pretreated with either a single portal-vein administration of ultraviolet B (UVB)-irradiated donor splenocytes (n = 4) or an intraportal inoculation of nonirradiated donor splenocytes (n = 4). Major histocompatibility complex-mismatched Lewis (donor) tracheal allograft segments were then grafted into treatment groups 7 days after donor-cell pretreatment. Tracheal rejection was assessed by histologic analysis, mucosal cilia motility, and in vitro immunologic assessment. RESULTS: The UVB-treated group demonstrated no acute or chronic rejection as well as complete functional recovery. In vitro immunologic assessment demonstrated a donor-specific hyporesponsiveness and donor allospecificity. Untreated animals and those receiving nonirradiated donor splenocytes showed acute rejection of their tracheal allografts. CONCLUSION: Recipient pretreatment with intraportally administered UVB-irradiated donor splenocytes prevents rejection of circumferential rat tracheal allograft segments by inducing a donor-specific immune hyporesponsiveness.