Intra-bone bone marrow transplantation from hCD47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival.

BACKGROUND: We have recently demonstrated that human-CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra-bone bone marrow transplantation (IBBMTx) in a pig-to-baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. METHODS: Baboons received GalTKO-hCD47/hCD55Tg (n = 5) or -hCD55Tg (n = 1) or -hCD46/HLA-E Tg (n = 1) pig IBBMTx. Macrochimerism, anti-pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47- porcine lungs 1-3 months later. RESULTS: All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti-pig antibody levels decreased over time and anti-pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1-4 days). CONCLUSION: This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

The Protective Effects of Carbon Monoxide Against Hepatic Warm Ischemia-Reperfusion Injury in MHC-Inbred Miniature Swine.

BACKGROUND: The development of treatment strategies to protect against ischemia-reperfusion injury (IRI) to livers is important not only for liver surgeries but also in regard to increasing the utilization of livers from marginal donors. In this study, we examined whether inhalational carbon monoxide (CO) therapy reduced IRI after a 45-min (min) warm ischemia (WI) in a miniature swine model. MATERIALS AND METHODS: Six CLAWN miniature swine underwent a 45-min hepatic WI induced by clamping the portal vein and proper hepatic artery. Three animals were subjected to control conditions while the remaining three were treated with CO inhalation for a total of 345-min, including 120-min after reperfusion to maintain a concentration of CO-Hb under 15% (CO-treated group). IRI of the livers was evaluated by liver function tests, serum pro-inflammatory cytokines, and liver biopsies. RESULTS: All controls had statistically significant increased levels of liver enzymes compared to the CO-treated group (p < 0.05). In controls, liver biopsies at 2 h after reperfusion showed marked histological changes including diffuse hemorrhage, congestion, necrosis, vacuolization, and neutrophil infiltration with apoptosis. In contrast, the CO-treated group showed less obvious or only minimal histological changes. Furthermore, increases in high-mobility group box 1, TNF-α, and IL-6 in sera that were induced by IRI in controls were markedly inhibited by the CO treatment. CONCLUSION: We demonstrated that low-dose CO inhalation reduces hepatic warm IRI, potentially through downregulation of pro-inflammatory mediators and activation of anti-apoptotic pathways. To our knowledge, this is the first report demonstrating CO inhalation attenuated hepatic IRI following WI in a large animal model.

Catechin and caffeine contents in green tea at different harvest periods and their metabolism in miniature swine.

The catechin content in green tea leaves varies according to cultivation conditions such as intensity of solar radiation, temperature, and precipitation, and thus, there is ambiguity about the best harvest time for obtaining optimal functional effects. In this study, the Yabukita (ordinary) and Benifuki varieties, which contain methylated catechin, were used to determine the difference in green tea catechins according to harvest times and tea manufacturing processes. Caffeine determination was also carried out to provide information about green tea intake for all age-groups of children and pregnant women. Determining the quantity of each catechin was difficult because of degradation, polymerization, and isomerization that had occurred during heat-drying in the refining process. In addition, the absorption of catechin compounds was tested using miniature swine because of their functional and physiological similarity to humans. Benifuki tea leaves contained epigallocatechin-3-(3"-O-methyl) gallate (EGCg3"Me) instead of epigallocatechin-3-(4"-O-methyl) gallate (EGCg4"Me). However, EGCg4"Me was detected during the entire intake period, but EGCg3"Me was not detected in the blood of miniature swine fed Benifuki tea. It is possible that the position of the methyl group was modified by the pig metabolism. Furthermore, caffeine from both Yabukita and Benifuki tea varieties was found to be easily accumulated in miniature swine. These results suggest that nonrefined September-October picking tea (autumn and winter tea) of the Benifuki variety is preferable over the Yabukita variety for consumption by children and pregnant women owing to its lower caffeine content and higher content of methylated catechin.

Preparation of hybrid porcine thymus containing non-human primate thymic epithelial cells in miniature swine.

BACKGROUND: We have achieved greater than a 6-month survival of a life-supporting kidney co-transplanted with a vascularized thymic graft into non-human primates (NHPs). Although we have achieved pig-specific unresponsiveness in vitro, immunosuppression was not able to be fully weaned. Studies in mice and humanized mice suggest that a hybrid pig thymus (Hyb-thy)-containing host thymic epithelial cells (TECs) can optimize intra-thymic selection, achieving xenograft tolerance with improved reconstitution of T-cell function. METHODS: We have tested the feasibility of the preparation of a Hyb-thy that contains NHP TECs in the donor thymic grafts. We first prepared the Hyb-thy in the donor pigs 2-3 weeks before xeno-Tx. We performed six cases of Hyb-thy preparation in six juvenile miniature swine. Two pigs received non-manipulated cynomolgus monkey thymic cells that were isolated from an excised atrophic thymus via injection into their thymic lobes (Group 1). The remaining four received thymic cells that were isolated from non-atrophic thymic glands (Groups 2 and 3). Pigs in Group 2 received unmanipulated thymic cells in one thymic lobe, as well as CD2-positive cell-depleted TEC-enriched cells in the contralateral lobe. Pigs in Group 3 received TEC-enriched cells alone. RESULTS: All thymus-injected pigs received tacrolimus and rapamycin until endpoint (POD16). We detected cynomolgus monkey TEC networks in pig thymus from Groups 1 and 3, while pigs in Group 2 rejected the thymic cells. We demonstrated the preparation of Hyb-thy in pigs using tacrolimus plus rapamycin therapy. CONCLUSIONS: Our results suggest that the enrichment of TEC from the excised NHP thymus facilitated NHP TEC engraftment in pig thymus.

GalT-KO pig lungs are highly susceptible to acute vascular rejection in baboons, which may be mitigated by transgenic expression of hCD47 on porcine blood vessels.

BACKGROUND: Despite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5 days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx). METHODS: Eleven baboons received genetically modified porcine left lungs from either GalT-KO alone (n = 3), GalT-KO/humanCD47(hCD47)/hCD55 (n = 3), GalT-KO/hD47/hCD46 (n = 4), or GalT-KO/hCD39/hCD46/hCD55/TBM/EPCR (n = 1) swine. The first 2 XLTx procedures were performed under a non-survival protocol that allowed a 72-hour follow-up of the recipients with general anesthesia, while the remaining 9 underwent a survival protocol with the intention of weaning from ventilation. RESULTS: Lung graft survivals in the 2 non-survival animals were 48 and >72 hours, while survivals in the other 9 were 25 and 28 hours, at 5, 5, 6, 7, >7, 9, and 10 days. One baboon with graft survival >7 days, whose entire lung graft remained well aerated, was euthanized on POD 7 due to malfunction of femoral catheters. hCD47 expression of donor lungs was detected in both alveoli and vessels only in the 3 grafts surviving >7, 9, and 10 days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss. CONCLUSIONS: To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

Effects of carbon monoxide on early dysfunction and microangiopathy following GalT-KO porcine pulmonary xenotransplantation in cynomolgus monkeys.

BACKGROUND: Despite progress in the current genetic manipulation of donor pigs, most non-human primates were lost within a day of receiving porcine lung transplants. We previously reported that carbon monoxide (CO) treatment improved pulmonary function in an allogeneic lung transplant (LTx) model using miniature swine. In this study, we evaluated whether the perioperative treatment with low-dose inhalation of CO has beneficial effects on porcine lung xenografts in cynomolgus monkeys (cynos). METHODS: Eight cynos received orthotopic left LTx using either α-1,3-galactosyltransferase knockout (GalT-KO; n = 2) or GalT-KO with human decay accelerating factor (hDAF) (GalT-KO/hDAF; n = 6) swine donors. These eight animals were divided into three groups. In Group 1 (n = 2), neither donor nor recipients received CO therapy. In Group 2 (n = 4), donors were treated with inhaled CO for 180-minute. In Group 3 (n = 2), both donors and recipients were treated with CO (donor: 180-minute; recipient: 360-minute). Concentration of inhaled CO was adjusted based on measured levels of carboxyhemoglobin in the blood (15%-20%). RESULTS: Two recipients survived for 3 days; 75 hours (no-CO) and 80 hours (CO in both the donor and the recipient), respectively. Histology showed less inflammatory cell infiltrates, intravascular thrombi, and hemorrhage in the 80-hour survivor with the CO treatment than the 75-hours non-CO treatment. Anti-non-Gal cytotoxicity levels did not affect the early loss of the grafts. Although CO treatment did not prolong overall xeno lung graft survival, the recipient/donor CO treatment helped to maintain platelet counts and inhibit TNF-α and IL-6 secretion at 2 hours after revascularization of grafts. In addition, lung xenografts that were received recipient/donor CO therapy demonstrated fewer macrophage and neutrophil infiltrates. Infiltrating macrophages as well as alveolar epithelial cells in the CO-treated graft expressed heme oxygenase-1. CONCLUSION: Although further investigation is required, CO treatment may provide a beneficial strategy for pulmonary xenografts.

Hydrogen sulfide prevents renal ischemia-reperfusion injury in CLAWN miniature swine.

BACKGROUND: Hydrogen sulfide (H2S) has recently been reported to demonstrate both antiinflammatory and cytoprotective effects; however, its efficacy has not been well documented in large animal models. In this study, we examined whether the administration of H2S offers cytoprotective effects on renal ischemia-reperfusion injury (IRI) in a preclinical miniature swine model. METHODS: Major histocompatibility complex-inbred, CLAWN miniature swine (n = 9) underwent a right nephrectomy, followed by induction of a 120-min period of warm ischemia via placement of clamps on the left renal artery and vein. Group 1 (n = 3) underwent renal ischemia without H2S administration. Groups 2 (n = 3) and 3 (n = 3) received Na2S (prodrug of H2S) 10 min before reperfusion of the ischemic kidneys followed by a 30-min of Na2S postreperfusion intravenously (group 2) or selective administration of Na2S via the left renal artery (group 3). IRI was assessed by kidney biopsies, levels of inflammatory cytokines in sera and kidney tissue. RESULTS: Animals in group 1 had significantly higher serum creatinine levels compared with animals in groups 2 and 3 (P < 0.01). Histology showed severe tubular damage with TUNEL-positive cells in group 1 on postoperative day 2 compared with mild damage in group 2 and minimal damage in group 3. Furthermore, levels of inflammatory cytokines in both serum (interleukin-6 [IL-6], tumor necrosis factor-α, and high-mobility group box 1) and renal tissue (IL-1 and IL-6) in group 3 were markedly lower than in group 2, suggesting beneficial effects of selective Na2S administration. CONCLUSIONS: Na2S administration, especially via an organ selective approach, appears to potentially offer cytoprotective and antiinflammatory effects following renal IRI.

Lung xenotransplantation.

PURPOSE OF REVIEW: This review describes the most recent progress in xeno lung transplantation (XLTx) to date. It describes the potential mechanisms of early xeno lung graft loss, as well as the latest therapeutic strategies to overcome them. RECENT FINDINGS: Using ex-vivo perfusion models of porcine lungs with human blood, the use of genetically modified pig lungs along with novel pharmaceutical approaches has recently been studied. Strategies that have demonstrated improved lung survival include the knockout of known xenoantigens (GalTKO and N-glycolylneuraminic acid-KO), genes that regulate complement activation (hCD46 and hCD55), as well as the inflammation/coagulation cascade (human leukocyte antigen-E, human thrombomodulin, human endothelial protein C receptor, hCD47, hCD39, hCD73 and heme oxygenase-1). Furthermore, pharmacologic interventions including the depletion of pulmonary intravascular macrophages or von Willebrand factor, inhibition of thromboxane synthase and blockade of histamine receptors have also demonstrated protective effects on xeno lung grafts. Using in-vivo pig to nonhuman primate lung transplant models, these approaches have been shown to extend pulmonary xenograft survival to 5 days. SUMMARY: The development of new multitransgenic GalTKO pigs has demonstrated prolongation of porcine xenograft survival; however, advancement in XLTx has remained frustratingly limited. Further intensive and innovative strategies including genetic manipulation of donors, as well as inflammation/coagulation dysregulation, are required to make XLTx a clinical possibility.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

BACKGROUND: Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. MATERIALS AND METHODS: Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. RESULTS: Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. CONCLUSION: This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

Protective effect of neutralization of the extracellular high-mobility group box 1 on renal ischemia-reperfusion injury in miniature swine.

BACKGROUND: Strategies that reduce ischemia-reperfusion injury (IRI) have the potential to expand the numbers of available organs for transplantation. Recent reports in rodent models have demonstrated that high-mobility group box 1 (HMGB1) acts as an alarm in initiating the inflammatory response resulting from ischemic injury. The aim of this study was to evaluate the cytoprotective effects of anti-HMGB1 antibodies on renal IRI in preclinical large animals. METHODS: One hundred twenty minutes of warm and 60 min of cold renal ischemia were induced in 8 CLAWN miniature swine. Three of eight animals received intravenous anti-HMGB1 antibody at 1 mg/kg just before the reperfusion of renal blood flow. Renal function was assessed by serum creatinine and renal biopsy. Serum levels of interleukin (IL)-1ß, IL-6, and HMGB1 were measured. RESULTS: The concentration of HMGB1 increased as early as 30 min after reperfusion and before the elevation of IL-1ß and IL-6. Serum creatinine levels were markedly elevated, peaking at a median of 5 days (peak creatinine levels: 11.6 ± 1.6 mg/dL) and recovering by day 14. Anti-HMGB1 antibody injection dramatically decreased renal damage as well as serum levels of HMGB1 associated with IRI. Renal function returned to near normal by day 9, and peak creatinine levels were markedly lower (7.4 ± 0.2 mg/dL), and biopsies possessed fewer pathologic changes when compared to the control group. CONCLUSION: In this study, we demonstrated the beneficial effects of perioperative administration of anti-HMGB1 antibody in reducing renal IRI in a clinically relevant, large animal model.

Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates.

BACKGROUND: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. METHOD AND RESULTS: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). CONCLUSIONS: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.

Hepatocyte growth factor sustains T regulatory cells and prolongs the survival of kidney allografts in major histocompatibility complex-inbred CLAWN-miniature swine.

BACKGROUND: Although 12 days of high dose of FK506 permits the induction of tolerance of fully major histocompatibility complex (MHC)-mismatched allogeneic kidneys in MGH-miniature swine, we found that the same dose of FK506 is insufficient to induce such tolerance CLAWN-miniature swine. The CLAWN swine model was therefore chosen to study the potential immunoregulatory effects of human-recombinant hepatocyte growth factor (HGF). METHODS: Ten CLAWN miniature swine received fully MHC-mismatched kidneys with 12 days (days 0-11) of FK506. Among these 10 recipients, 4 received 7 or 14 days of human-recombinant HGF starting at day 11. Graft function was assessed by daily serum creatinine and biopsies. Immunologic assays, including CD4/CD25 DP and FoxP3+ cells and development of antidonor antibodies, were performed. RESULTS: Without HGF, all six CLAWN recipients developed severe acute rejection (Cre >9 mg/dL) within 3 weeks of transplantation. In contrast, in the four animals that received HGF for 7 to 14 days, stable renal function was observed for more than 50 days, although all grafts were ultimately rejected by postoperative day 80. Percent FoxP3+ cells in the CD4+CD25+ double positive population (T regulatory cells) in peripheral blood monocyte cells decreased in recipients with FK506 induction monotherapy while no reduction was observed in recipients treated with FK506 and HGF. CONCLUSION: This study demonstrates that in CLAWN swine treated with a dose of FK506 insufficient to induce tolerance across a fully MHC mismatched barrier, a short course of HGF may inhibit acute rejection while maintaining T regulatory cells. To our knowledge, this study provides the first evidence in a large animal transplantation model of HGF's immunoprotective effects.

Autoimmune sensitization to cardiac myosin leads to acute rejection of cardiac allografts in miniature swine.

BACKGROUND: Recent studies in mice and patients suggest that posttransplantation induction of autoimmune responses to tissue-specific antigens contributes to the rejection of major histocompatibility complex mismatched allotransplants. The relevance of this phenomenon to the rejection of major and minor histocompatibility-mismatched allografts performed in large-animal models remains to be established. METHODS: Miniature swine were immunized with cardiac myosin (CM) in Freund's adjuvant and received heterotopic, minor antigen-mismatched heart transplants. T-cell (proliferation and delayed type hypersensitivity [DTH]) and B-cell (antibody) responses specific to CM were measured. The rejection of heart transplants was assessed histologically. RESULTS: Three of four swine that were immunized with CM before receiving a minor antigen-mismatched heart transplant exhibited potent DTH, T-cell proliferation and antibody responses to CM and rejected their grafts acutely. The fourth swine, which failed to mount a significant DTH response to CM and displayed low and transient anti-CM antibody titers, demonstrated long-term allograft survival. CONCLUSIONS: This large-animal study supports the relevance of autoimmunity to CM in the rejection of minor antigen disparate cardiac allotransplants.

Beneficial effects of perioperative low-dose inhaled carbon monoxide on pulmonary allograft survival in MHC-inbred CLAWN miniature swine.

BACKGROUND: We have recently reported that perioperative low-dose carbon monoxide (CO) inhalation decreases lung ischemia-reperfusion injury in miniature swine. The aims of this study were to establish a large animal model of pulmonary allograft rejection using polymerase chain reaction-typed major histocompatibility complex (MHC)-inbred CLAWN miniature swine and to examine the effects of CO on allograft survival. METHODS: Eleven CLAWN miniature swines received fully MHC-mismatched lungs followed by 12 days of tacrolimus (days 0-11; blood level 35-45 ng/mL). Six recipients received tacrolimus alone (control group). Five recipients were additionally treated with inhaled CO (180 min for donors until graft harvest; 390 min for recipients until 2 hr after reperfusion). RESULTS: All recipients treated with tacrolimus alone uniformly rejected their grafts by postoperative day 63 with development of cytotoxic antidonor antibodies. CO treatment was effective in prolonging allograft survival from a mean of 47±7 to 82±13 days (P=0.017), with one CO-treated animal maintaining function until postoperative day 120. Development of antidonor antibodies and donor-specific responsiveness by cell-mediated lympholysis and mixed lymphocyte reaction assays was delayed in animals that received CO therapy. Furthermore, serum concentrations of proinflammatory cytokines (interleukin-1ß and -6) 1 day after transplant were significantly decreased in the CO-treated group. CONCLUSIONS: Fully MHC-mismatched lungs in CLAWN miniature swine were consistently rejected within 63 days, suggesting that this is a robust large animal model ideal for investigating mechanisms and treatment of lung rejection. Perioperative low-dose CO inhalation prolonged graft survival and inhibited antidonor antibody production and was associated with decreased proinflammatory mediators in this model.

Age-related changes in the trachea in healthy adults.

To investigate age-related changes in the shape of trachea, normal male volunteers (n = 83, mean +/- SD: 47.7 +/- 20.2 years old) underwent inspiratory CT scans at full inspiration and lung function tests. Subjects who showed VC < 80% predicted or FEV1 < 80% predicted on lung function tests were excluded. The CT data, which is located at 2.0 cm above the aortic arch, were transferred to a personal computer. The tracheal area (St) and two parameters, Tracheal index (Ti) and Circularity (Ci) indicating the shape of the trachea, were automatically calculated. Ti was defined the ratio of the coronal to the sagittal diameter of the trachea, and the Ci (Ci = 4piS/L2, S: tracheal area, L: tracheal perimeter) was used to indicate the roundness of the trachea. A Ci value of less than 1 indicated the distortion of the roundness. Both St and St/BSA (body surface area) showed a significant correlation with age (r = 0.37, r = 0.52; p = 0.0006, p < 0.0001). Ti was not correlated with age (r = -0.20; p = 0.0697), whereas Ci was significantly correlated with age (r = -0.32; p = 0.00364). There were measurable age related changes of the trachea both in the area and the shape. Aging results in the increased tracheal area and a distortion of the roundness.

Carbon monoxide reduces pulmonary ischemia-reperfusion injury in miniature swine.

OBJECTIVES: Carbon monoxide is produced endogenously as a by-product of heme catalysis and has been shown to reduce ischemia-reperfusion injury in a variety of organs in murine models. The aims of this translational research were to establish an in situ porcine lung model of warm ischemia-reperfusion injury and to evaluate the cytoprotective effects of low-dose inhaled carbon monoxide in this model. METHODS: Warm ischemia was induced for 90 minutes by clamping the left pulmonary artery and veins in 8 Clawn miniature swine (Japan Farm CLAWN Institute, Kagoshima, Japan). The left main bronchus was also dissected and reanastomosed just before reperfusion. Four animals were treated with inhaled carbon monoxide at a concentration of approximately 250 ppm throughout the procedure. Lung function and structure were serially accessed via lung biopsy, chest x-ray films, and blood gas analysis. RESULTS: Carbon monoxide inhalation dramatically decreased the lung injury associated with ischemia and reperfusion. Two hours after reperfusion, the arterial oxygen tension of the carbon monoxide-treated group was 454 +/- 34 mm Hg, almost double the arterial oxygen tension of the control group (227 +/- 57 mm Hg). There were fewer pathologic changes seen on chest x-ray films and in biopsy samples from animals in the carbon monoxide-treated group. Animals in the carbon monoxide-treated group also had fewer inflammatory cell infiltrates and a markedly smaller increase in serum concentrations of the proinflammatory cytokines interleukin 1beta, interleukin 6, and high-mobility group box 1 after ischemia-reperfusion injury. CONCLUSIONS: The perioperative administration of low-dose inhaled carbon monoxide decreases warm ischemia-reperfusion injury in lungs in miniature swine. This protective effect is mediated in part by the downregulation of proinflammatory mediators.

Repetitive gastric aspiration leads to augmented indirect allorecognition after lung transplantation in miniature swine.

INTRODUCTION: Lung transplant recipients with documented gastroesophageal reflux disease (GERD) are at increased risk for graft dysfunction. Here, we present the first large-animal model of gastric aspiration after allogeneic lung transplantation and some preliminary data demonstrating the effect of chronic aspiration on the direct and indirect pathways of allorecognition. METHODS: Left orthotopic lung transplants (n=3) were performed in miniature swine across a major histocompatibility complex class I disparity, followed by 12 days of high-dose cyclosporine A. At the time of transplantation, a transtracheal catheter was placed at the carina, above the bronchial anastomosis. A gastrostomy tube was placed for daily aspiration of gastric contents. Subsequently, graft lungs were instilled with gastric aspirate daily (3 mL/hrX8 hr/day) for 50 days. Recipients were followed up with daily complete blood count, scheduled chest radiographs, and biopsies. In vitro studies, including cell-mediated lympholysis, mixed lymphocyte reactions, and peptide proliferation assays, were performed. Results from these three recipients were compared with those of historical controls (n=6) who were treated identically, except for the tracheal cannulation and simulated gastric aspiration. RESULTS: Two of the experimental animals were euthanized with nonviable lungs soon after the postoperative day 50 biopsy. In both cases the native lung was normal. The third animal survived over 180 days without the evidence of chronic rejection. After immunosuppressive treatment, all animals demonstrated donor-specific hyporesponsiveness by assays of direct alloresponse (cell-mediated lympholysis, mixed lymphocyte reaction). A significant response to synthetic donor-derived class I peptide, however, was seen in all animals. A more pronounced and diffuse response was seen in the animals rejecting their grafts. The historical controls showed medium-term graft survival with evidence of chronic rejection in the majority of animals, as previously reported. CONCLUSION: In a model of GERD after lung transplantation, a spectrum of clinical outcomes was observed. The in vitro data suggest that acid reflux enhances the indirect alloresponse to processed donor class I antigen, giving mechanistic insight into the manner in which GERD may be deleterious to the transplanted lung.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays.

UNLABELLED: Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with (3)H for MLR and (51)Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. OBJECTIVE: The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.

Operational tolerance to class I disparate lungs can be induced despite pretransplant immunization with class I allopeptides.

BACKGROUND: Using a class I-disparate swine lung transplant model, we examined whether an intensive course of tacrolimus could induce operational tolerance and whether preoperative allopeptide immunization would prevent the development of tolerance. METHODS: Left lung grafts were performed using class I-disparate (class II-matched) donors. Recipients were treated with 12 days of postoperative tacrolimus. Three recipients were immunized prior to transplantation with class I allopeptides. Three other recipients were not immunized. RESULTS: The nonimmunized recipients maintained their grafts long term (>497, >451, and >432 days), without developing chronic rejection. The immunized swine also maintained their grafts long term (>417, >402, >401 days), despite developing a variety of in vitro and in vivo responses to the immunizing peptides, as well as having strong mixed lymphocyte reactions to donor cells prior to transplantation. CONCLUSIONS: Using only a brief course of tacrolimus, we have been able to induce a state of operational tolerance in a class I-disparate preclinical lung transplant model. Moreover, preoperative alloimmunization did not block tolerance induction or induce chronic rejection. These data show that it is possible to create a state of operational tolerance to lung allografts even in the presence of donor-sensitized cells.