Intramuscular Gene Transfer of Interleukin-10 Reduces Neutrophil Recruitment and Ameliorates Lung Graft Ischemia-Reperfusion Injury

Interleukin-10 (IL-10) has potent anti-inflammatory properties but its direct effects on neutrophil trafficking in lung transplant ischemia-reperfusion (I/R) injury are unknown. This study was performed to determine if recipient intramuscular IL-10 gene transfer reduces neutrophil infiltration in lung isografts and ameliorates I/R injury. Twenty-four hours before transplantation, recipient rodents received intramuscular injection with 1 x 10(10) plaque-forming units (pfu) adenovirus encoding human IL-10 (hIL-10), 1 x 10(10) pfu adenovirus control encoding p-galactosidase, or saline. Gene expression in muscle and plasma was confirmed. Lung grafts were harvested, stored at 4 degrees C for 18h, and assessed 24 h after transplantation. Peak muscle and plasma expression of hIL-10 was achieved 24h after gene transfer and returned to baseline by 7 days (p < 0.05 vs. controls). Gene transfer of hIL-10 reduced neutrophil sequestration and emigration in lung grafts as measured by morphometry and myeloperoxidase activity (p < 0.03 vs. controls). Furthermore, hIL-10 improved graft oxygenation and reduced lung edema (p <0.01 vs. controls). Intramuscular gene transfer of hIL-10 releases hIL-10 protein into plasma and reduces neutrophil sequestration and emigration in lung isografts. This is associated with a reduction in I/R injury with improved isograft oxygenation and reduced tissue edema. Intramuscular gene transfer may be a useful strategy to reduce clinical l/R injury.     

Protection against ischemia/reperfusion injury by the cavitary two-layer method in canine small intestinal transplantation with reduction of reactive oxygen species

BACKGROUND: Ischemia and reperfusion (I/R) injury is a major determinant of early graft dysfunction and long-term graft survival in small intestinal transplantation. The cavitary two-layer method (TLM) has been reported to be superior to the University of Wisconsin cold storage method (UWM) in long-term preservation of canine small intestine. This study was designed to evaluate the protective effect of the cavitary TLM against I/R injury in canine small intestinal transplantation. METHODS: Intestinal grafts harvested from beagles were allotransplanted after 24-hour preservation by UWM (group 1) or the cavitary TLM (group 2). The graft in the controls (group 3) was immediately allotransplanted without preservation. I/R injury was assessed by functional success rates, biochemical assay, graft adenosine triphosphate (ATP) and lipid peroxidation (LPO) concentrations, and histopathologic examination including TUNEL staining for apoptosis. RESULTS: In group 1, ATP recovery was delayed after reperfusion, and most recipients died with hemorrhage of the grafts and lungs. In group 2, graft ATP concentrations recovered rapidly, and I/R injury was prevented with reduced LPO production, resulting in good outcome. CONCLUSIONS: The cavitary TLM protected intestinal grafts against I/R injury evidenced by maintenance of graft ATP levels and reduction of LPO production compared with UWM in canine small intestinal transplantation.     

Ex Vivo Application of Carbon Monoxide in University of Wisconsin Solution to Prevent Intestinal Cold Ischemia/Reperfusion Injury

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.     

Early Renal Ischemia-Reperfusion Injury in Humans Is Dominated by IL-6 Release from the Allograft

The pathophysiology of ischemia/reperfusion (I/R) injury is complex, and current knowledge of I/R injury in humans is incomplete. In the present study, human living-donor kidney transplantation was used as a highly reproducible model to systematically study various processes potentially involved in early I/R injury. Unique, direct measurements of arteriovenous concentration differences over the kidney revealed massive release of interleukin (IL)-6 in the first 30 minutes of graft reperfusion and a modest release of IL-8. Among the assessed markers of oxidative and nitrosative stress, only 15( S )-8- iso -PGF_2α was released. When assessing cell activation, release of prothrombin factor 1 + 2 indicated thrombocyte activation, whereas there was no release of markers for endothelial activation or neutrophil activation. Common complement activation complex sC5b-9 was not released into the bloodstream, but was released into urine rapidly after reperfusion. To investigate whether IL-6 plays a modulating role in I/R injury, a mouse experiment of renal I/R injury was performed. Neutralizing anti-IL-6 antibody treatment considerably worsened kidney function. In conclusion, this study shows that renal I/R in humans is dominated by local IL-6 release. Neutralization of IL-6 in mice resulted in a significant aggravation of renal I/R injury.     

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation with and without donor-specific cell augmentation

BACKGROUND: Immunomodulatory strategies such as donor-specific bone marrow or blood transfusions have been used to promote engraftment after intestinal transplants. We previously showed that delivery of donor antigen via the portal vein can effectively reduce the rate of intestinal graft rejection. The purpose of our current study was to investigate the impact of donor-specific cell augmentation (blood versus bone marrow) via the portal vein on cytokine expression in intestinal grafts versus native livers. MATERIAL AND METHODS: We performed heterotopic small intestinal transplants between male Brown-Norway (donor) and female Lewis (recipient) rats. We studied 10 groups according to the type of donor-specific cell augmentation and the use and dose of immunosuppressive therapy. For cell augmentation, donor-specific blood or bone marrow was transfused via the donor portal vein immediately before graft implantation. For immunosuppression, tacrolimus was used post-transplant at a high or low dose. Control rats received neither immunosuppression nor cell augmentation. Tissue samples for histological assessment were obtained at designated time points. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta). Chimerism levels were determined using Q-PCR analysis. RESULTS: Without concurrent immunosuppression, neither portal donor-specific blood nor bone marrow transfusion reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood (versus bone marrow) transfusion. Irrespective of the type of donor-specific cell augmentation, severe rejection caused strong cytokine expression in the grafts of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha; in the native livers, mainly of TNF-alpha (with IFN-gamma showing hardly any increase). In general, rejection caused stronger cytokine expression in the grafts than in the native livers. Mild rejection correlated well with strong intragraft expression of IL-6, TNF-alpha, and TNF-beta (early rejection markers); severe rejection with IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha (late rejection markers).In addition to cell augmentation per se, the type of cell augmentation also had an impact on cytokine expression in both grafts and native livers. Cell-augmented (versus tacrolimus-treated) rats showed hardly any differences in intragraft cytokine expression, but the expression of almost all cytokines was significantly stronger in the native livers. With immunosuppression, bone marrow infusion increased intragraft cytokine expression of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF alpha, as well as liver cytokine expression of IL-1 beta, compared to blood transfusion. This finding reflected the more advanced rejection stages in the bone marrow infused group; different types of donor-specific cell augmentation had similar effects on liver cytokine expression. In the absence of myoablative therapy, chimerism levels were low, in both cell-augmented and non-cell-augmented groups. CONCLUSIONS: Rejection and donor-specific cell augmentation independently causes differences in intragraft versus native liver cytokine expression after intestinal transplants. Portal donor-specific blood transfusion, as compared with donor-specific bone marrow infusion, lowered the incidence of rejection and diminished intragraft cytokine up-regulation.     

Preservation injury and acute rejection of rat intestinal grafts: Protection afforded by pyruvate

Pyruvate has been shown to prevent intestinal mucosal injury after ischemia-reperfusion. The aim of the present study was to determine whether pyruvate can (1) prevent postreperfusion mucosal injury occurring after intestinal preservation and subsequent transplantation and (2) exert a protective effect on the intestinal graft mucosa during acute rejection. Preservation mucosal injury was evaluated, after 2 hours of reperfusion, by comparing grafts transplanted in a rat syngeneic combination (ACI to ACI) after 2 hours of cold preservation using pyruvate (n = 6) or placebo (n = 6). Mucosal parameters obtained during acute rejection (allogeneic combination: ACI to Lewis) were compared between placebo-treated (n =6) and pyruvate-treated (n = 6) animals. Tissue injury was evaluated by histopathologic examination, oxygen free radical production by luminol-enhanced chemiluminescence, and degree of neutrophil infiltration by myeloperoxidase staining. After reperfusion of the preserved grafts and during acute rejection, mucosal oxygen free radical levels and the number of infiltrating neutrophils were significantly (P <0.05) increased in the untreated grafts, whereas there was a statistically significant inhibition of these parameters in those treated with pyruvate. Mucosal injury, seen after reperfusion of the preserved grafts, was prevented by pyruvate. The histopathologic abnormalities observed in the untreated grafts during rejection were also significantly reduced by pyruvate. Treatment with pyruvate before cold preservation of intestinal grafts, in this rat model, reduced reperfusion mucosal injury, neutrophil infiltration, and oxygen free radical production. Oxygen free radicals were produced in the mucosa of the graft during acute rejection and their production was reduced by pyruvate, which exerted a protective effect on the rejecting allograft mucosa.     

Intragraft gene expression profile during acute cellular rejection in clinical small bowel transplantation: a case report

BACKGROUND: Acute cellular rejection (ACR) is a common complication of small bowel transplantation (SBTx) and the major cause of graft loss. However, little is known regarding the genetic graft response to ACR in clinical transplants. In this study, we have determined a genetic expression profile of intestinal graft response to ACR after living related (LR) SBTx. RESULTS: By identifying the expression profiles of reported markers of rejection we were able to identify 57 genes that had significantly increased (more than twofold) expression in response to ACR. Known markers of rejection identified: MMP-9, MMP-2, VIP, IFNgamma, IL-2R, MADCAM-1, HSP-60, and HSP-70 all had greater than twofold increased expression after ACR diagnosed (week 3 to week 6). The newly identified genes were: IFI27, EPST11, APAF1, LAP3, STK6, and MDK. CONCLUSION: Newly identified up-regulated genes in response to ACR in small bowel graft are involved in the immune response, cell adhesion, neurogenesis, cell division and proliferation, DNA replication/repair, protein ubiquitin/proteolysis, and apoptosis. TNFalpha up-regulated early at week 2 biopsy may be an early genetic marker of ACR in SBTx.     

Protection Against Ischemia/Reperfusion Injury in Cardiac and Renal Transplantation with Carbon Monoxide, Biliverdin and Both

Both carbon monoxide (CO) and biliverdin, products of heme degradation by heme oxygenase, have been shown to attenuate ischemia/reperfusion (I/R) injury. We hypothesized in this study that dual-treatment with CO and biliverdin would induce enhanced protective effects against cold I/R injury. Heterotopic heart and orthotopic kidney transplantation were performed in syngeneic Lewis rats after 24-h cold preservation in UW solution. While monotherapy with CO (20 ppm) or biliverdin (50 mg/kg, ip) did not alter the survival of heart grafts, dual-treatment increased survival to 80% from 0% in untreated recipients, with a significant decrease of myocardial injury and improved cardiac function. Similarly, dual-treatment significantly improved glomerular filtration rates of renal grafts and prolonged recipient survival compared to untreated controls. I/R injury-induced up-regulation of pro-inflammatory mediators (e.g. TNF-alpha, iNOS) and extravasation of inflammatory infiltrates were significantly less with dual-treatment than untreated controls. In addition, dual-treatment was effective in decreasing lipid peroxidation and improving graft blood flow through the distinctive action of biliverdin and CO, respectively. The study shows that the addition of byproducts of heme degradation with different mechanisms of action provides enhanced protection against transplant-associated cold I/R injury of heart and kidney grafts.     

Protective effect of carbon monoxide inhalation for cold-preserved small intestinal grafts

BACKGROUND: Heme oxygenase (HO)-1 system has been shown to provide protection against oxidative stress through the degradation of heme to biliverdin, free iron, and carbon monoxide (CO). This study investigated cytoprotective efficacy of CO at a low concentration on cold ischemia/reperfusion (I/R) injury of transplanted intestine. METHODS: Lewis rat recipients of syngenic orthotopic small intestinal transplantation with 6 hours UW cold preservation were either kept in room air (air-treated control) or exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. RESULTS: In air-treated grafts, mRNA levels for interleukin-6, intracellular adhesion molecule-1, cyclooxygenase-2, and inducible nitric oxide synthase promptly increased. Sequential histopathologic analysis of untreated grafts revealed initial rapid epithelial loss, subsequent recruitment of inflammatory infiltrates, and local hemorrhage in the lamina propria, which extended downward to the epithelial crypt and muscle layer with time. CO effectively blocked proinflammatory cascade during I/R injury, inhibited upregulation of inflammatory molecules and ameliorated intestinal tissue injuries. Beneficial effects of CO were associated with improved graft blood flow without inhibiting endogenous HO-1 activity. Recipient animal survival was significantly improved with CO to 100% versus 58% in air-treated controls. CONCLUSIONS: These results indicate a significant role for CO in protecting the intestine from cold I/R injury associating with small intestinal transplantation.     

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

INTRODUCTION: Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. METHODS: Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1). the type of donor-specific cell augmentation and (2). the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. RESULTS: Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1alpha, IL-1beta, IFN-gamma, and TNF-alpha; liver expression mainly involved TNF-alpha. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1alpha, IL-1beta, IFN-gamma, as well as TNF-alpha, and liver expression of IL-1beta. CONCLUSIONS: (1). Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2). Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3). In our study, TNF-alpha appeared to be the cytokine most strongly associated with rejection.     

基质金属蛋白酶-9 与小体积移植肝早期损伤的相关性研究

目的 观察不同体积肝移植术后早期移植肝内基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的表达及活化特点,探讨MMP-2和MMP-9在小体积移植肝早期损伤中的作用机制. 方法 随机将108只SD大鼠分成3组,每组36只.分别为:全肝(100%肝体积)移植组、半肝(50%肝体积)移植组和小体积肝(25%肝体积)移植组.分别检测移植肝再灌注后0.5、6、12、24、48、72 h的肝功能及移植肝组织中丙二醛(MDA)和髓过氧化物酶(MPO)浓度,观察移植肝组织病理学特征,并运用双抗夹心酶联免疫吸附试验(ELISA)、实时定量聚合酶链反应(PCR)、明胶酶谱和免疫组织化学方法检查移植肝中MMP-2和MMP-9的表达情况. 结果 与全肝和半肝移植组比较,小体积肝移植组再灌注后6～24 h MMP-9表达明显升高;而且MMP-9活化和表达增高伴随着移植肝功能损害和严重的缺血再灌注损伤.MMP-9早期表达都集中在移植肝门静脉周围,与门静脉高灌注密切相关. 结论 MMP-9表达升高是小体积移植肝早期重要的致损伤因素;肝移植术后门静脉高灌注可能是触发MMP-9活化和表达的重要原因.     

血红素氧合酶-1减轻大鼠移植肝缺血再灌注损伤的作用及机制

目的 探讨血红素氧合酶-1(HO-1)减轻大鼠移植肝缺血再灌注损伤的作用及其机制.方法 选择近交系雄性SD大鼠作为肝移植的供、受者;采用单纯随机方法将48只SD大鼠随机分为对照组、抑制组和诱导组(每组供、受者各8只).对照组:供肝不用任何药物处理;抑制组:在获取供肝前24 h,经供者腹腔注射HO-1抑制剂锌原卟啉20 mg/kg进行预处理;诱导组:在获取供肝前24 h,经供者腹腔注射HO-1诱导剂钴原卟啉5 mg/kg进行预处理.获取供肝后,在4℃UW液中冷保存24 h.肝移植前检测供肝HO-1的表达水平;肝移植后6 h采血并获取移植肝标本,分离培养枯否细胞;检测受者的肝功能;检测枯否细胞培养上清中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量;观察移植肝组织病理学表现以及枯否细胞CD14 mRNA的表达水平和蛋白含量测定.结果 移植前诱导组供肝HO-1的表达水平明显增高.移植后诱导组血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量明显降低;移植肝组织病理学损伤减轻;枯否细胞培养上清中TNF-α和IL-6含量减少;而且枯否细胞上的CD14 mRNA表达水平和蛋白含量也明显低于抑制组.结论 诱导供肝HO-1表达上调可能抑制了枯否细胞的激活,从而减轻大鼠移植肝缺血再灌注损伤.     

瘦素对肠缺血/再灌注损伤大鼠肝脏功能的影响及其机制

目的:探讨瘦素(leptin)对大鼠肠缺血/再灌注(I/R)损伤时肝脏的保护作用及其机制。方法:99只Wistar大鼠随机分成11组:假手术组,肠缺血45min再灌注15min、30min、1h、3h、6h的肠I/R损伤组和相应时间点的leptin治疗组,每组9只大鼠。肠I/R损伤组和leptin治疗组建立大鼠肠I/R损伤模型;leptin治疗组于再灌注的同时腹腔注射leptin0.2mg/kg。假手术组术后1h处死,其余各组大鼠于相应时间点处死,检测血清丙氨酸转氨酶(ALT),肝组织中白细胞介素-6(IL-6)、IL-10和超氧化物歧化酶(SOD)水平的动态变化。结果:肠I/R组血清ALT随着时间变化逐渐升高,肝组织中的IL-6水平于再灌注3h显著升高,而IL-10水平基本处于平稳的波动状态,SOD活性于再灌注早期呈略微下降趋势,1h后逐步回升至假手术组水平;leptin治疗后血清ALT水平基本稳定在较低水平,再灌注3h后,肝组织IL-6水平较损伤组明显降低,同时SOD活性显著提高,IL-10水平于再灌注15min时突然增高,随即恢复到假手术组水平。结论:leptin对大鼠肠I/R造成的肝脏功能损伤具有改善作用,可能与其调节肝组织中的细胞因子释放、提高肝脏的抗氧化损伤能力有关。     

CD62 blockade with P-Selectin glycoprotein ligand-immunoglobulin fusion protein reduces ischemia-reperfusion injury after rat intestinal transplantation

BACKGROUND: Intestinal transplantation (ITx) is severely limited by ischemia-reperfusion (I/R) injury. This study investigates I/R injury and ameliorates its consequences by using a recombinant protein targeted against selectins (recombinant P-selectin glycoprotein ligand-immunoglobulin [rPSGL-Ig]). METHODS: An isogeneic model of ITx was undertaken with control animals (no therapy) and treatment animals (rPSGL-Ig). Survival was assessed. Separate groups underwent an analysis examining tissue at multiple time points after I/R injury including histopathology; myeloperoxidase staining; immunostaining for CD3 and ED2; polymerase chain reaction analysis of interleukin (IL)-8/cytokine-inducible neutrophil chemoattractant, IL1beta, IL-6, interferon-gamma, IL-2, IL-4, and IL10; and western blots for hemoxygenase-1, BCL-2, and BCL-xl. Standard statistical analysis was undertaken. RESULTS: Treatment with rPSGL-Ig resulted in significantly improved survival after ITx. Analysis demonstrated diminished injury on histopathology and reduced tissue infiltration of neutrophils and lymphocytes. Significant differences in the cytokine profile after ITx were seen between the two groups including the production of inflammatory cytokines at 24 hr and the Th1 and Th2 cytokines at 2 and 4 hr. Last, treatment resulted in increased production of hemoxygenase, BCL-2, and BCL-xl. CONCLUSION: The results of this investigation of I/R injury after ITx revealed that rPSGL-Ig treatment led to marked improvement in outcome. The mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration leading to a decreased inflammatory response possibly driven by Th2 cytokines. The results not only lend insight into the mechanisms behind I/R injury after ITx but also demonstrate a potential therapeutic modality to ameliorate its consequences.     

Role of tumour necrosis factor in lung injury caused by intestinal ischaemia-reperfusion

BACKGROUND: Despite the well known inflammatory effects of tumour necrosis factor alpha (TNF), the mechanism of TNF-mediated lung injury following ischaemia-reperfusion (I/R) is still unclear. In this study, the role of TNF in the development of acute lung injury following intestinal I/R was investigated. METHODS: Male Wistar rats underwent either sham operation (n = 10), 1 h of superior mesenteric artery occlusion and 2 h of reperfusion (I/R, n = 10), or pretreatment with anti-TNF polyclonal antibody 2 mg/kg and I/R (n = 6). Lung injury was evaluated by Evans blue dye concentration, immunohistochemical staining and morphometric analysis. Intestinal injury was assessed by Evans blue dye concentration and histological examination. RESULTS: Intestinal I/R resulted in lung injury characterized by an increase in Evans blue dye concentration, neutrophil sequestration, and obvious staining for expression of pulmonary CD11b and CD18. Pretreatment of animals with anti-TNF antibody led to a reduction in the sequestration of neutrophils, and a decrease in expression of pulmonary intracellular adhesion molecule 1 and CD18. Anti-TNF antibody pretreatment also reduced the intestinal microvascular injury but not histological grade after intestinal I/R. CONCLUSION: Treatment with an anti-TNF antibody resulted in a significant attenuation of lung injury following intestinal I/R. The data indicate that TNF is an important trigger for upregulation of pulmonary endothelial and neutrophil adhesion molecules after intestinal I/R.     

Interleukin-4 secretion by the allograft fails to affect the allograft-specific interleukin-4 response in vitro.

BACKGROUND: The role of the cytokine, interleukin (IL)-4, in allograft rejection and protection is not clear. We have previously shown that IL-4 transgenically expressed in a pancreas allograft does not protect the allograft from rejection. Here, we analyze the effect of the transgenically expressed IL-4 on the cytokine profile of the allograft-specific immune response. METHODS: C57BL/6SCID mice were infused with small numbers of spleen cells from C57BL/6 donors. The former received pancreas grafts from 1- to 2-day-old BALB/c donors which did or did not transgenically express IL-4 in the graft. Three weeks after the cell infusion, the spleens were removed and the splenocytes were restimulated in vitro with BALB/c APC, and third party BALB.K APC. IL-2 and IL-4 levels in the culture supernatants were measured. RESULTS: The presence of a pancreatic allograft induced an increase in the levels of both IL-2 and IL-4 in culture supernatants from splenocytes of mice receiving grafts compared with mice not receiving grafts. The presence of IL-4 transgenically expressed in the pancreas allograft had no effect on the in vitro cytokine profile. CONCLUSIONS: from these results we conclude that the failure of transgenically expressed IL-4 to protect the allograft was not associated with up-regulation of a graft antigen-specific IL-4 response.     

Riboflavin-mediated reduction of oxidant injury, rejection, and vasculopathy after cardiac allotransplantation

BACKGROUND: Riboflavin is a well-known nutritional supplement that has been shown to exhibit antioxidant properties and protect tissue from oxidative damage. We hypothesized that riboflavin given during cardiac ischemia-reperfusion (I/R) might reduce subsequent acute rejection, after allotransplantation, and coronary allograft vasculopathy (CAV). METHODS: A murine heterotopic cardiac transplantation model was used to test whether riboflavin improves I/R injury and acute/chronic rejection. RESULTS: Riboflavin significantly reduced oxidant production and inflammatory mediator production induced by I/R injury, as evidenced by decreased levels of malondialdehyde, myeloperoxidase activity, and tumor necrosis factor alpha. Administration of riboflavin also improved graft survival and suppressed T-cell infiltration and donor-reactive alloantibody formation during the early period after allotransplantation. A murine long-term cardiac allograft model using immunosuppression (preoperative anti-murine CD4 and anti-CD8) was employed to investigate the effect of riboflavin against CAV at 60 days. Riboflavin-treated grafts exhibited a significant decrease in the severity of coronary artery luminal occlusion as compared with saline-treated grafts (17.4+/-1.8% vs. 43.5+/-5.6%, P=0.0012). However, there was no significant effect of riboflavin to reduce donor-reactive alloantibodies in this chronic model. CONCLUSIONS: These data indicate that riboflavin improves early I/R injury and reduces the development of CAV, most likely due to alloantigen-independent effects such as reduced early graft oxidant stress. Riboflavin administered in the setting of cardiac allograft transplantation appears to be a powerful means to reduce early graft lipid peroxidation, leukocytic infiltration, and cytokine production as well as to suppress the late development of cardiac allograft vasculopathy.