Tracheal Allografts Research Articles

Donor antigen–presenting cells are important in the development of obliterative airway disease

Objective: Obliterative airway disease, which resembles obliterative bronchiolitis histologically, develops in murine heterotopic tracheal allografts. Chimeric tracheas were used to examine whether donor-type antigen-presenting cells are important in the development of obliterative airway disease. To separate the contributions of CD4+ and CD8+ direct pathways, we transplanted tracheas from knockout mice lacking major histocompatibility complex (MHC) class I or II antigens. Methods: Chimeric tracheas were created via bone marrow transplantation in fully MHC-mismatched combinations. Tracheas from naive B6, autologously reconstituted B6, chimeric B6 bearing recipient-type C3H antigen-presenting cells, MHC class I knockout B6 (B6I–), MHC class II knockout B6 (B6II–), or C3H mice were transplanted into C3H recipients. The tracheas were harvested at days 14 and 28. Results: At day 28, isografts showed no occlusion, normal respiratory epithelium, and minimal infiltrates. Naive or autologously reconstituted B6, B6I–, and B6II– tracheas showed minimal occlusion at day 14 but contained intraepithelial infiltrates. By day 28, the naive or autologously reconstituted B6 tracheas had occlusion of 69.5% ± 11.6% (mean ± standard error of the mean), and in comparison, B6I– and B6II– tracheas had occlusions of 53.0% ± 16.3% and 52.2% ± 15.9%, respectively (P = .20, .19). In chimeric B6 tracheas, minimal occlusion was seen at day 14 and remained 33.6% ± 16.2% (P = .039) at day 28. Subtle epithelial changes and minimal infiltrates were seen. Conclusions: Obliterative airway disease appears to involve donor-type antigen-presenting cells and develops in the absence of either MHC class I or II antigens. These findings suggest that either CD8+ or CD4+ direct allorecognition is important in the development of obliterative airway disease. (J Thorac Cardiovasc Surg 2000;120:1070-7)

Assessment of cartilage viability in the cryopreserved tracheal allograft by measurement of Na 235SO 4 incorporation

Assessment of cartilage viability in the cryopreserved tracheal allograft by measurement of Na 235SO 4 incorporation

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

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. 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. 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. 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.

A new tracheal bioartificial organ: evaluation of a tracheal allograft with minimal antigenicity after treatment by detergent.

The aim of this study was to reduce the antigenicity of tracheal allografts by detergent treatment. We attempted to apply these grafts to tracheal immunosuppressant-free allotransplantation. Fresh tracheal grafts were harvested from donor beagle dogs and treated with a detergent at 4 degrees C for 48 hours. By using treated grafts, we have performed tracheal immunosuppressant-free transplantation in six dogs at an intrathoracic five-ring defect. Implanted grafts were covered with an omental pedicle. In five of the six grafts, complete removal of the epithelium and mixed glands was recognized with both light microscopy and scanning electron microscopy. The appearance of the cartilage cells in the grafts was similar to those in fresh trachea. Five dogs that received detergent treated grafts survived uneventfully. The grafts had been incorporated by the host trachea without stenosis. On the one tracheal graft in which removal of the epithelium was incomplete (noted after implantation), moderate stenosis occurred 1 month after placement. These results suggest that removal of the tracheal epithelium and mixed glands can remarkably reduce tracheal antigenicity. A tracheal graft that has its epithelium and mixed glands removed can be used in tracheal immunosuppressant-free allotransplantation.

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.

Blockade of complement inhibits obliterative bronchiolitis in rat tracheal allografts.

The role of complement activation in the development of obliterative bronchiolitis, a manifestation of chronic lung allograft rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft complement components C3 and C5b-9 (membrane attack complex) as well as IgM and IgG deposits were demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. A 7-d treatment with recombinant human soluble complement receptor type 1 (sCR1; 20 mg/kg/d, intraperitoneal), an inhibitor of both the classic and alternative complement pathways, significantly decreased epithelial necrosis and intragraft neutrophil infiltration, and reduced obliterative changes by 40%. Immunohistochemical analysis of the grafts showed that sCR1 treatment significantly decreased early C5b-9 and IgG deposits, neutrophil chemoattractant IL-8 immunoreactivity, and ICAM-1 expression. Treatment with sCR1 was associated with increased staining for Th2 cytokines, in particular IL-10, with concomitant downregulation of IL-2 and TNF-alpha immunoreactivity. In contrast, sCR1 treatment did not affect the number of graft-infiltrating CD4(+) and CD8(+) T cells, CD45(+) B cells, ED1(+) and ED3(+) macrophages, or immune activation with expression of IL-2Ralpha or MHC class II. In conclusion, this is the first study to demonstrate that blockade of complement activation attenuates the development of OB and suggests that in addition to T cell-driven responses, humoral and antigen-independent immune responses also operate in the disease process. A blockade of complement activation renders the chemokine milieu unattractive to neutrophils and also modulates the alloimmune response toward Th2 cytokines, which may have an antiproliferative role in fibroproliferative disorders.

Role of platelet-derived growth factor in obliterative bronchiolitis (chronic rejection) in the rat.

The role of platelet-derived growth factor (PDGF) in the development of obliterative bronchiolitis (OB) as a manifestation of chronic rejection was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft PDGF-Ralpha and -Rbeta mRNA expression, and in PDGF-AA and -Ralpha immunoreactivity, was demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. Treatment with CGP 53716, a protein-tyrosine kinase inhibitor selective for PDGF receptor, alone and in combination with suboptimal doses of cyclosporin A, significantly reduced myofibroproliferation and the degree of OB by more than 50%. CGP 53716 did not affect airway wall inflammatory cell proliferation, the number of graft-infiltrating CD4(+) or CD8(+) T cells, ED3(+) macrophages, or the level of immune activation determined as IL-2R and MHC class II expression. This study suggests a regulatory role for PDGF, especially for PDGF-AA and -Ralpha, in the development of obliterative bronchiolitis in this model, and demonstrates that inhibition of PDGF receptor protein-tyrosine kinase activation prevents these obliterative changes. Thus, receptor protein-tyrosine kinase inhibitors may provide a novel therapeutic strategy for the prevention of chronic rejection.

Tracheal allograft reconstruction: the total North American and worldwide pediatric experiences

Background. We reviewed both the North American and the total worldwide pediatric experience with tracheal allograft reconstruction as treatment for patients with long segment and recurrent tracheal stenosis. Methods. The stenosed tracheal segment is opened to widely patent segments. The anterior cartilage is resected and the posterior trachealis muscle or tracheal wall remains. A temporary silastic intraluminal stent is placed and absorbable sutures secure the chemically preserved cadaveric trachea. After initial success with this technique in Europe, several North American centers have now performed the procedure. The cumulative North American experience includes 6 patients (3 adults and 3 children). Worldwide, more than 100 adults and 31 children, aged 5 months to 18 years, with severe long segment tracheal stenosis have undergone tracheal allograft reconstruction. Results. In North America, 5 of 6 patients have survived, with one early death due to bleeding from a tracheal-innominate artery fistula in a previously irradiated neck. Worldwide, 26 children survived (26 of 31 = 84%) with follow-up from 5 months to 14 years. Only 1 of 26 pediatric survivors (1 of 26 = 3.8%) had a tracheostomy. Conclusions. Tracheal allograft reconstruction demonstrates encouraging short- to medium-term results for patients with complex tracheal stenosis. Allograft luminal epithelialization supports the expectation of good long-term results.

The effects of early and delayed immunosuppression in experimental tracheal transplantation with omentopexy

Background/Purpose : Best results in experimental tracheal allotransplantation are obtained when metachronous revascularization by omentopexy and immunosuppression are used. Nevertheless, this method of revascularization implies in a 4-day period of ischemia to the graft. The aim of this study was to assess the influence of the 4-day period of ischemia on host sensitization as well as the effect of early or delayed immunosuppression on the outcome of the grafts. Methods : Thirty rabbits were submitted to tracheal allotransplantation and divided according to position of the graft (orthotopic or heterotopic transplants) and the initiation of immunosuppression (early or delayed). The quality of the revascularization was evaluated by the identification of Indian ink, perfused through the abdominal aorta, inside the submucosal vessels. The outcome of the grafts was evaluated by histological analysis according to a semiquantitative scale of alterations. Results : Grafts were better revascularized in heterotopic position. Grafts with late immunosuppression presented good outcome only when heterotopically positioned. No significant differences were observed in grafts placed heterotopically or orthotopically when immunosuppression was initiated early after the transplant. Conclusions : Transient ischemia produced by metachronous revascularization is not the single factor responsible for the histological alterations observed in tracheal allografts. These alterations probably also are produced by the activation of immune responses. This activation is more intense in more ischemic grafts, but can be suppressed by early administration of immunosuppression.

Cytomegalovirus infection and cardiac allograft vasculopathy

There is a wealth of clinical and experimental evidence indicating the interaction of cytomegalovirus (CMV) infection and rejection in cardiac and other solid organ allografts. A plausible explanation for this association comes from data showing that therapy with biologicals, sepsis, and rejection, all lead to the release of TNF-alpha which, upon binding to its receptor, activates NF-kB. TNF-alpha is also able to stimulate the activity of the CMV-IE enhancer/promoter region. CMV infection of several cell lines leads to NF-kB activation. NF-kB binding sites are present in regulatory regions of various cellular and viral genes, including the IE enhancer region of CMV. In a reciprocal situation, CMV infection, most likely via gamma-interferon, leads to upregulation of MHC antigens in the transplant and, thereby, to increased transplant immunogenicity. Thus, a vicious circle is induced. We have investigated in detail the pathobiology of CMV and allograft vasculopathy (chronic rejection) in experimental animals, using aortic and cardiac allografts as well as a trachea model. The results may be summarized as follows: Infection of the recipient with rat CMV results in an early inflammatory response in the aortic and cardiac allograft vascular adventitia and intima (endothelialitis) and in the airway wall of tracheal allografts. This early inflammatory response leads to enhanced intimal thickness in aortic and cardiac allografts and enhanced luminal occlusion of tracheal allografts. Timewise, this coincides with early activation of intragraft inflammatory leukocytes and increased mRNA of various growth factors and cytokines. When the recipients receive gancyclovir, the enhanced intimal response in aortic and cardiac allografts and luminal occlusion in tracheal allografts is entirely abolished. Gancyclovir treatment dramatically reduces the inflammatory response in the allograft, and thereby growth factor synthesis in response to injury. However, gancyclovir does not prevent the expression of IE antigen of CMV, suggested to inactivate tumor suppressor protein p53 predisposing smooth muscle cells to increased growth. Taken together, the effect of CMV infection on cardiac allograft dysfunction is bidirectional and biphasic. The bidirectional nature emerges from the observations that acute CMV infection may accelerate acute rejection, and, on the other hand, acute alloimmune response-associated cytokine response may activate latent CMV infection. The biphasic effect of CMV on allograft dysfunction refers to its early and late detrimental effects, i.e. during the time of acute and chronic rejection. These two effects of CMV on allograft dysfunction emphasize the need for precise diagnosis of CMV infection in transplant recipients and pre-emptive or prophylactic anti-viral therapy. The benefits of this strategy may not be evident during the early post-transplant period, but 5-10 years after transplantation they manifest as better graft survival.

Administration of exogenous interleukin-2 enhances obliterative airway disease in cyclosporine-treated rats following tracheal allografts

Administration of exogenous interleukin-2 enhances obliterative airway disease in cyclosporine-treated rats following tracheal allografts

Epithelial re-growth prevents luminal occlusion in orthotopic tracheal allografts despite acute rejection and proliferation of α-actin positive cells

Epithelial re-growth prevents luminal occlusion in orthotopic tracheal allografts despite acute rejection and proliferation of α-actin positive cells

Epithelial regeneration and preservation of tracheal cartilage after tracheal replacement with cryopreserved allograft in the rat

Objective: We investigated the origin of the epithelium in transplanted cryopreserved tracheal allografts in rats and tried to clarify the mechanism by which immunogenicity is reduced in this procedure. Methods: Tracheal transplantation was performed with PVG rats (allele at the RT1 locus: c) used as donors and ACI rats (allele at the RT1 locus: a) as recipients. After resection of a 5-ring segment of the cervical trachea of an ACI rat, the trachea was reconstructed with the cryopreserved tracheal segment of a PVG rat ( n = 6). No immunosuppressive agents or steroids were given. Histologic changes were determined and immunohistochemical staining was performed to investigate major histocompatibility complex class I antigens of the transplanted tracheal segment. Results: Two months after tracheal transplantation, 6 surviving ACI rats were killed. Histologically, the epithelium and tracheal cartilage of the transplanted cryopreserved segment displayed normal structure. Immunohistochemical staining showed that the major histocompatibility complex class I antigen of the ACI rat was expressed in the epithelium of the transplanted segment and that the class I antigen of the PVG rat was expressed in the cartilage of the transplanted segment. Conclusions: After transplantation of the cryopreserved trachea, the epithelium of the transplanted cryopreserved segment originated from the recipient epithelium whereas the cartilage retained the structure of the donor trachea. We hypothesize that transplantation of a cryopreserved trachea leads to the growth of the recipient's epithelium over the donor trachea, thereby reducing the antigenicity of the transplant. (J Thorac Cardiovasc Surg 1998;116:624-7)

Immune cells in a mouse airway model of obliterative bronchiolitis.

Obliterative bronchiolitis (OB), a form of chronic lung rejection, affects 50% of all lung-transplant recipients and is a major cause of morbidity and mortality. We used the mouse tracheal allograft model of OB to quantitate inflammatory cells during disease progression to evaluate the pathogenesis of this disorder. Tracheas of BALB/c mice were implanted into C57BL/6, severe combined immunodeficiency (SCID), and BALB/c mice. Cyclosporin was administered at 25 mg/kg/d. Grafts were harvested at 2, 6, 10, and 15 wk, and analyzed immunohistochemically. Tracheal allografts developed epithelial injury and cellular infiltrates at 2 wk, epithelial denudation and complete luminal obliteration at 6 wk, and dense collagenous scarring by 15 wk. SCID allografts and isografts demonstrated intact epithelium throughout, although a mononuclear infiltrate was initially present at 2 wk in the SCID allografts. Immunohistochemical staining, using antibodies to mouse CD4(+) (T-helper lymphocyte), CD8(+) (T-cytotoxic/suppressor lymphocyte), and B lymphocytes, macrophages, and myofibroblasts, revealed large numbers of macrophages and CD4(+) and CD8(+) lymphocytes in allografts at 2 wk, compared with isografts. The allograft CD4(+)/CD8(+) ratio was 0.75 at 2 wk. Allografts demonstrated macrophage, myofibroblast, and CD4(+) predominance at 6 and 10 wk (CD4(+)/CD8(+) = 2/1), but by 15 wk had minimal cellularity and were densely scarred. SCID allografts demonstrated a macrophage-predominant infiltrate at 2 wk, with minimal cellularity at later time points. These results indicate that: (1) OB is predominantly an immunologic airway injury; and (2) CD4(+) and CD8(+) lymphocytes and macrophages play an important role in the evolution of airway inflammation and fibrosis. Additionally, this model suggests that chronic airway fibrosis follows a period of intense airway-directed, cell-mediated rejection.

Experimental study of tracheal allotransplantation with cryopreserved grafts

Objective: Tracheal reconstruction is necessary in patients with extensive tracheal stenosis caused by neoplasm, trauma, and congenital disease. We investigated the possibility of tracheal allotransplantation with cryopreserved grafts in a canine model. Methods: A seven-ring section of thoracic trachea was removed in 19 adult mongrel dogs. In group A ( n = 4), a five-ring tracheal autograft was implanted. In group B ( n = 6), a five-ring allograft was implanted without immunosuppression. In group C ( n = 9), a five-ring cryopreserved tracheal allograft was implanted without immunosuppression. Omentopexy wrapping around the grafts and both anastomotic sites was used in all animals. Results: All grafts survived without any evidence of atrophy or stenosis in group A. All animals in group B died of severe airway obstruction within 1 month, and postmortem examination of these grafts showed epithelial defect and necrotic tracheal cartilage in the scar tissue. In group C, no animals died of asphyxia caused by severe stenosis of the grafts. The graft epithelium was no longer present 20 days after transplantation, and the graft was covered with regenerated epithelium within about 60 days after the operation. Conclusion: These findings show that cryopreserved tracheal allografts can be transplanted by means of omentopexy without immunosuppression and that cryopreservation may reduce tracheal allogenicity. (J Thorac Cardiovasc Surg 1998;116:262-6)

Tracheal allograft transplantation in rats: the role of immunosuppressive agents in development of obliterative airway disease

Tracheal allograft transplantation in rats: the role of immunosuppressive agents in development of obliterative airway disease

Tracheal replacement with cryopreserved tracheal allograft: experiment in dogs

Background. It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether cryopreserved trachea can be used in tracheal replacement, we evaluated the viability of cryopreserved tracheal allografts in a canine model of immunosuppressant-free transplantation. Methods. Cryopreserved tracheal allografts, which had been frozen to −80°C in a programmed freezer and then stored in liquid nitrogen (−196°C) (group 1, n = 6), fresh tracheal autografts (group 2, n = 5), and fresh tracheal allografts (group 3, n = 4) were transplanted into the thoracic segment of the trachea using an omental flap without immunosuppressive agents. Results. All dogs in groups 1 and 2 survived, but in group 3, all 4 died of airway obstruction between 1 month and 2 months after operation. Histologically, the cryopreserved allografts displayed normal epithelium and cartilage, but the fresh allografts showed chronic inflammatory changes, no epithelium, and no cartilage. Conclusions. Cryopreserved tracheal allografts maintain their structural integrity after transplantation. The cryopreservation process seems to reduce the allogenic response of the trachea in canine models. Therefore, we believe the cryopreserved tracheal allograft is an excellent choice for tracheal replacement.

Origin of regenerated epithelium in cryopreserved tracheal allotransplantation

Background. Our previous study showed that a cryopreserved tracheal allograft could be transplanted using omentopexy without immunosuppression. The present study investigated, by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method, whether the regenerated epithelia were of recipient origin or donor origin in a cryopreserved tracheal allotransplantation model. Methods. Twenty-nine mongrel dogs were classified by preoperative peripheral blood PCR-RFLP analysis. The cryopreserved tracheal allografts were implanted into recipient animals that showed a different phenotype from donor animals. A small specimen of epithelia excised from the allograft of animals postmortem was analyzed with the modified PCR-RFLP method. Results. The animals were separated into 16 phenotypes by preoperative PCR-RFLP results, and cryopreserved tracheal allografts transplanted into 8 animals. PCR-RFLP analysis of graft epithelia at 10 days after transplantation showed the donor blood phenotype and analysis of graft epithelia taken from the animals that survived more than 20 days after operation showed the recipient blood and epithelial phenotype. Conclusions. The donor epithelia in the grafts were no longer present within about 20 days after transplantation. The recipient epithelia migrated gradually from the anastomotic site, and the regenerated epithelia that are of recipient origin covered the allograft within about 50 days after transplantation.

Prevention of Obliterative Airway Disease (OAD) without Immunosuppression in Heterotopic Rat Tracheal Allografts: Anastomosis of Recipient Trachea to Donor Trachea Enables Re-epithelialization of Allografts by Recipient Epithelium

Prevention of Obliterative Airway Disease (OAD) without Immunosuppression in Heterotopic Rat Tracheal Allografts: Anastomosis of Recipient Trachea to Donor Trachea Enables Re-epithelialization of Allografts by Recipient Epithelium

Prevention of Obliterative Airway Disease (OAD) without Immunosuppression in Heterotopic Rat Tracheal Allografts: Anastomosis of Recipient Trachea to Donor Trachea Enables Re-epithelialization of Allografts by Recipient Epithelium

Prevention of Obliterative Airway Disease (OAD) without Immunosuppression in Heterotopic Rat Tracheal Allografts: Anastomosis of Recipient Trachea to Donor Trachea Enables Re-epithelialization of Allografts by Recipient Epithelium