Nitrite reduces acute lung injury and improves survival in a rat lung transplantation model.

Ischemia/reperfusion injury (IRI) is the most common cause of early mortality following lung transplantation (LTx). We hypothesized that nitrite, an endogenous source of nitric oxide (NO), may protect lung grafts from IRI. Rat lung grafts were stored in preservation solution at 4°C for 6 hours. Both grafts and recipients were treated with nitrite. Nitrite treatment was associated with significantly higher levels of tissue oxygenation, lower levels of cytokines and neutrophil/macrophage infiltration, lower myeloperoxidase activity, reduced oxidative injury and increased cGMP levels in grafts than in the controls. Treatment with either a nitric oxide scavenger or a soluble guanylyl cyclase (sGC) inhibitor diminished the beneficial effects of nitrite and decreased cGMP concentrations. These results suggest that nitric oxide, generated from nitrite, is the molecule responsible for the effects of nitrite via the nitric oxide/sGC/cGMP pathway. Allopurinol, a xanthine oxidoreductase (XOR) inhibitor, abrogated the protective effects of nitrite, suggesting that XOR is a key enzyme in the conversion of nitrite to nitric oxide. In vitro experiments demonstrated that nitrite prevented apoptosis in pulmonary endothelial cells. Nitrite also exhibits longer survival rate in recipients than control. In conclusion, nitrite inhibits lung IRI following cold preservation and had higher survival rate in LTx model.

Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury.

The susceptibility of xenografts to hyperacute rejection is postulated to reflect in part failure of complement regulatory proteins (CRPs) to control activation of heterologous complement on graft endothelium. To test this concept, transgenic swine expressing the human CRP decay accelerating factor and CD59 were developed using a novel expression system involving transfer of the proteins from erythrocytes to endothelial cells. Hearts from transgenic swine transplanted into baboons had markedly less vascular injury and functioned for prolonged periods compared to hearts from nontransgenic swine. These results indicate that expression of human CRPs in xenogeneic organs may contribute to successful xenografting and suggest that intercellular protein transfer might be a useful approach for expression of heterologous proteins in endothelial cells.

Lung transplantation in a recipient with novel 2009 H1N1 influenza: lessons learned.

Immunosuppressed patients are at an increased risk for severe disease from influenza A (H1N1). We report a case of a patient who died of septic complications from H1N1 acquired at the time of single lung transplant.

Alemtuzumab induction prior to cardiac transplantation with lower intensity maintenance immunosuppression: one-year outcomes.

Induction therapy with alemtuzumab (C-1H) prior to cardiac transplantation (CTX) may allow for lower intensity maintenance immunosuppression. This is a retrospective study of patients who underwent CTX at a single institution from January 2001 until April 2009 and received no induction versus induction with C-1H on a background of tacrolimus and mycophenolate. Those with C-1H received dose-reduced calcineurin inhibitor and no steroids. A total of 220 patients were included, 110 received C-1H and 110 received no induction. Recipient baseline characteristics, donor age and gender were not different between the two groups. Mean tacrolimus levels (ng/mL) for C-1H versus no induction: months 1-3 (8.5 vs. 12.9), month 4-6 (10.2 vs. 13.0), month 7-9 (10.2 vs. 11.9) and month 10-12 (9.9 vs. 11.3) were all significantly lower for the C-1H group, p < 0.001. There were no differences between the C-1H and no induction groups at 12 months for overall survival 85.1% versus 93.6% p = 0.09, but freedom from significant rejection was significantly higher for the C-1H group, 84.5% versus 51.6%, p < 0.0001. In conclusion, induction therapy after CTX with C-1H results in a similar 12 month survival, but a greater freedom from rejection despite lower calcineurin levels and without the use of steroids.

Lung transplantation in the United States, 1998-2007.

This article highlights trends and changes in lung and heart-lung transplantation in the United States from 1998 to 2007. The most significant change over the last decade was implementation of the Lung Allocation Score (LAS) allocation system in May 2005. Subsequently, the number of active wait-listed lung candidates declined 54% from pre-LAS (2004) levels to the end of 2007; there was also a reduction in median waiting time, from 792 days in 2004 to 141 days in 2007. The number of lung transplants performed yearly increased through the decade to a peak of 1 465 in 2007; the greatest single year increase occurred in 2005. Despite candidates with increasingly higher LAS scores being transplanted in the LAS era, recipient death rates have remained relatively stable since 2003 and better than in previous years. Idiopathic pulmonary fibrosis became the most common diagnosis group to receive a lung transplant in 2007 while emphysema was the most common diagnosis in previous years. The number of retransplants and transplants in those aged > or =65 performed yearly have increased significantly since 1998, up 295% and 643%, respectively. A decreasing percentage of lung transplant recipients are children (3.5% in 2007, n = 51). With LAS refinement ongoing, monitoring of future impact is warranted.

In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium.

Many proteins are associated with the outer layer of the cell membrane through a posttranslationally added glycosyl phosphatidylinositol (GPI) anchor. The functional significance of this type of protein linkage is unclear, although it results in increased lateral mobility, sorting to the apical surface of the cell, reinsertion into cell membranes, and possibly cell signaling. Here evidence is presented that GPI-linked proteins can undergo intermembrane transfer in vivo. GPI-linked proteins expressed on the surface of transgenic mouse red blood cells were transferred in a functional form to endothelial cells in vivo. This feature of GPI linkage may be potentially useful for the delivery of therapeutic proteins to vascular endothelium.

Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury.

Ischemia/reperfusion (I/R) injury during small intestinal transplantation (SITx) frequently causes complications including dysmotility, inflammation and organ failure. Recent evidence indicates hydrogen inhalation eliminates toxic hydroxyl radicals. Syngeneic, orthotopic SITx was performed in Lewis rats with 3 h of cold ischemic time. Both donor and recipient received perioperative air or 2% hydrogen inhalation. SITx caused a delay in gastrointestinal transit and decreased jejunal circular muscle contractile activity 24 h after surgery. Hydrogen treatment resulted in significantly improved gastrointestinal transit, as well as jejunal smooth muscle contractility in response to bethanechol. The transplant induced upregulation in the inflammatory mediators CCL2, IL-1 beta, IL-6 and TNF-alpha were mitigated by hydrogen. Hydrogen significantly diminished lipid peroxidation compared to elevated tissue malondialdehyde levels in air-treated grafts demonstrating an antioxidant effect. Histopathological mucosal erosion and increased gut permeability indicated a breakdown in posttransplant mucosal barrier function which was significantly attenuated by hydrogen treatment. In recipient lung, hydrogen treatment also resulted in a significant abatement in inflammatory mRNA induction and reduced neutrophil recruitment. Hydrogen inhalation significantly ameliorates intestinal transplant injury and prevents remote organ inflammation via its antioxidant effects. Administration of perioperative hydrogen gas may be a potent and clinically applicable therapeutic strategy for intestinal I/R injury.

Unexpected Neoplasms in Lungs Explanted From Lung Transplant Recipients: A Single-Center Experience and Review of Literature.

INTRODUCTION: Lung transplantation is a common treatment for various indications, but undiagnosed neoplasms are found in 0.5% to 2.4% of explanted lungs. We report the largest single-institution series of patients with unexpected neoplasms in explanted lungs and compare rates of undiagnosed malignancies before and after the 2005 Lung Allocation Score (LAS) update. METHODS: We reviewed the medical records of patients who underwent lung transplantation at the Cleveland Clinic from 1990 to 2014. In cases of neoplasm discovered on explant, tumor type, pathological stage, recurrence, and date of death were recorded. RESULTS: From January 1, 1990 to June 30, 2014, 1303 patients underwent lung transplantation at the Cleveland Clinic. The overall mean smoking history was 35 pack-years, and 25 undiagnosed lung malignancies were found upon explant in 24 transplant recipients (1.84%). In the post-LAS era (ie, 2005 onward), 20/812 lung transplant recipients had 21 incidental neoplasms in their explanted lungs (2.5%). Seventeen of these 25 tumors occurred in patients with interstitial lung disease; 8 occurred in patients with centrilobular emphysema. Eight tumors recurred (6 in patients with interstitial lung disease and 2 in patients with emphysema). The most common histological tumor types were adenocarcinomas (n = 14) and squamous cell carcinomas (n = 7). CONCLUSIONS: Unexpected neoplasms were found in 1.84% of lung transplant recipients' explanted lungs, with a slightly higher incidence (2.46%) in the post-LAS era. Neoplasms were more common in patients with interstitial lung diseases than in patients with centrilobular emphysema. Explanted lungs should be pathologically examined for evidence of tumor foci because this can impact post-transplantation management.

C4d deposition in lung allografts is associated with circulating anti-HLA alloantibody.

UNLABELLED: The complement activation demonstrated by vascular C4d deposition is used to diagnose antibody-mediated rejection (AMR) in renal allografts, but remains controversial in lung transplantation (LTX). METHODS: C4d deposition was assessed by immunohistochemistry in 192 lung transplant biopsies from 32 patients. ELISA analysis was performed on 415 serum samples in those 32 temporally and rejection-grade matched LTX patients; 16 patients developed HLA-Ab, while the other 16 patients remained negative. The specificity of C4d staining was further compared in 18 additional LTX patients without HLA-Ab or acute cellular rejection (ACR), but in the presence of CMV-pneumonitis or reperfusion injury. RESULTS: Specific subendothelial C4d deposition was seen in 5 of 16 (31%) patients with HLA-Ab and was absent in 16 patients without HLA-Ab (p<0.05). All patients with specific C4d deposition exhibited donor-specific HLA-Ab. There were 13 patients with bronchiolitis obliterans syndrome in the group of 16 HLA-Ab positive patients, versus 2/16 in ELISA-negative patients (p<0.005). One of 7 patients with CMV pneumonitis and 2 of 11 patients with reperfusion injury also showed C4d positivity (not statistically significant). CONCLUSIONS: In this study, specific subendothelial C4d deposition was a marker for the involvement of HLA-Ab in lung allograft rejection. The patchy nature, low sensitivity, and specificity of C4d staining might limit clinical use in protocol biopsies. However, in patients with decreasing pulmonary function, refractory ACR and/or HLA-Ab, specific C4d deposition may serve as a marker of coexistent AMR.

Transgenic expression of human complement regulatory proteins in mice results in diminished complement deposition during organ xenoperfusion.

Complement activation is an essential step in the hyperacute rejection of a vascularized xenograft. Endothelial cell-associated complement regulatory proteins limit complement activation in most settings, but are not able to limit the extensive complement activation that occurs in xenografts, at least in part due to their species specificity. To overcome this problem we and others have sought to express human complement regulatory proteins in the organs of potential donor animals. As an initial step toward evaluating this concept we tested organs from transgenic mice expressing human CD59 and/or decay-accelerating factor (DAF) in two in vitro perfusion systems for the ability to control activation of heterologous complement. In the first system, mouse hearts were perfused on a Langendorff circuit with 50% human plasma. Immunopathologic analysis of heart biopsies revealed deposition of human IgG, IgM, and C4 in both control and transgenic organs. The hearts from mice transgenic for human CD59 had substantially less and in some cases no membrane attack complex (MAC) and hearts from CD59/DAF transgenic mice had substantially less or no C5b and MAC. In the second system, mouse hearts were perfused with baboon blood through arterial lines inserted into baboons. Immunopathologic analysis of serial biopsies revealed the deposition of IgG, IgM, and C4 in control and transgenic hearts. Compared with controls, less MAC was deposited in many CD59-expressing hearts and less C5b and MAC in DAF-expressing hearts. These results demonstrate that human complement regulatory proteins expressed in a xenogeneic organ are able to contribute to the control of complement activation in that organ and support the concept that expression of these human molecules would help protect a xenogeneic organ transplanted into a human.

Protection of xenogeneic cardiac endothelium from human complement by expression of CD59 or DAF in transgenic mice.

We investigated the ability of membrane-bound human complement regulatory proteins to control complement-driven humoral immune reactions on murine microvasculature. The human complement regulatory proteins CD59 and DAF were expressed using heterologous promoters in a variety of tissues in transgenic mice. Animals expressing these gene products are healthy and exhibit significant levels of endothelial cell expression of CD59 and DAF in cardiac muscle. Transgenic hearts perfused with human plasma exhibited profound reductions in the level of complement deposition compared with nontransgenic controls. We have also produced transgenic pigs that express these two human genes. Our results indicate that expression of complement regulatory proteins can control activation of complement and suggest that these proteins may have therapeutic applications in some inflammatory diseases and in the development of xenogeneic organs for human transplantation.

Variation in the level of xenoantigen expression in porcine organs.

Hyperacute rejection of vascularized porcine to primate xenografts is initiated by the binding of xenoreactive natural antibodies to donor endothelium. We tested the hypothesis that the level of xenoantigen expression varies in the population of potential porcine donors and may determine the amount of binding of xenoreactive natural antibodies to a porcine organ perfused by xenogeneic blood. Two hundred ninety pigs were studied using an inhibition ELISA that quantitated the xenoantigen level on porcine platelets. Based on this assay, the levels of xenoantigen expression in the population adhered to a normal distribution. Kidneys from pigs found to express high antigen levels and kidneys from pigs found to express low antigen levels were perfused with baboon blood using an extracorporeal circuit. In multiple experiments, a significant difference was observed in the amount of xenoreactive natural antibody adsorbed by high antigen versus low antigen organs. Normalizing for the weight of the perfused organs and for levels of natural antibody in individual baboons, high antigen organs adsorbed 3.6 +/- 1.3 U of xenoreactive natural antibody/g and low antigen organs adsorbed -0.8 +/- 1.0 U of xenoreactive natural antibody/g (P < 0.002). Immunopathology of tissues from the perfused organs demonstrated more deposition of IgM and C4 in high than in low xenoantigen organs. The quantitative relationship between binding of xenoreactive natural antibodies to platelets and to whole organs suggests that platelets are a valid representation of endothelial cell antigen expression in vivo. Despite the probable importance of Gal alpha(1-3)Gal as an epitope recognized by xenoreactive natural antibodies, differences in the binding to platelets or to organs of the GS-I-B4 lectin that recognizes that sugar had no correlation with the differences in binding of IgM to these tissues. Variation in expression of xenoantigen may be exploited to selectively breed donors for xenotransplantation that are less susceptible to attack by xenoreactive natural antibodies.

Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage.

We characterize a line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor. These genes, under the control of heterologous promoters, are expressed in a variety of organs, including the vasculature of the heart, kidney, and liver. We demonstrate that moderate levels of these gene products are sufficient to protect peripheral blood cells from human or baboon complement. Using pig to baboon heterotopic heart transplants, we show that expression of these proteins is sufficient to block the complement-mediated damage that is the hallmark of such xenografts, when nontransgenic organs are used. These results indicate that there is significant species specificity of intrinsic complement regulatory protein function. This specificity is evident in transgenic organs in which low levels of human CD59 and human decay-accelerating factor expression significantly effect the humoral immune response that causes xenograft rejection. This result suggests that transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunological barriers that currently block the use of xenogeneic organs for human transplantation.

Characterization of transgenic pigs expressing functionally active human CD59 on cardiac endothelium.

The critical shortage of human donor organs has generated interest in the potential for porcine to human xenotransplantation. The initial immunological barrier to xenotransplantation is hyperacute rejection, which is mediated by xenoreactive antibodies and complement, and results in rapid and irreversible tissue destruction. While endogenous complement regulatory proteins (CRPs) protect cells from injury caused by autologous complement, they are relatively species specific and most likely ineffectual in this setting. This has led to the hypothesis that expression of human CRPs in transgenic pigs may affect susceptibility to complement-mediated tissue injury in a porcine-to-human xenograft. Using specific lines of transgenic pigs that express low levels of human CD59, a CRP that acts at the terminal stage of the complement cascade, we present evidence that shows that the human CD59 protein inhibits membrane attack complex assembly and reduces tissue damage when the heart is transplanted to a baboon. Examination by immunohistochemistry of transgenic porcine hearts after transplantation revealed markedly reduced deposition of C5b and MAC, but a similar level of C3 deposition as compared with transplanted control hearts. This finding supports the concept that the species specific function of CRPs contributes to the humoral barrier to xenotransplantation and, given the low level of human CD59 protein expression in the porcine heart, argues that the human protein contributes a unique rather than an additive function in regulation of complement in a xenogeneic setting.

Humoral responses to pig-to-baboon cardiac transplantation: implications for the pathogenesis and treatment of acute vascular rejection and for accommodation.

Organs transplanted between phylogenetically-disparate species, such as from the pig into the primate, are subject to hyperacute and acute vascular rejection. Hyperacute rejection of a porcine organ by a primate is thought to be initiated by the binding of xenoreactive natural antibodies to Galalpha1-3Gal expressed on the endothelial lining of blood vessels in the xenograft. The factor(s) which initiates acute vascular rejection is uncertain; however, there is some evidence implicating xenoreactive antibodies. The nature of the humoral response which might contribute to acute vascular rejection of a porcine organ was investigated in baboons which received a porcine cardiac xenograft plus immunosuppression with methylprednisolone, azathioprine, and cyclosporine. Following rejection and surgical removal of the xenografts, the serum concentration of xenoreactive antibodies increased in untreated animals but in immunosuppressed animals was similar to the concentration in preimmune serum. The antibodies in the sensitized recipients were specific for Galalpha1-3Gal (70-95%) and other determinants (5-30%). However, cross-blocking studies showed that, following xenotransplantation, the immunosuppressed baboons had no detectable IgM or IgG directed against "new" endothelial antigens. These results indicate that antibodies made by immunosuppressed individuals in response to xenotransplantation are much like xenoreactive natural antibodies and suggest that acute vascular rejection might in some cases be addressed by therapeutic strategies aimed at those antibodies.

A clinical trial combining donor bone marrow infusion and heart transplantation: intermediate-term results.

BACKGROUND: Donor chimerism (the presence of donor cells of bone marrow origin) is present for years after transplantation in recipients of solid organs. In lung recipients, chimerism is associated with a lower incidence of chronic rejection. To augment donor chimerism with the aim to enhance graft acceptance and to reduce immunosuppression, we initiated a trial combining infusion of donor bone marrow with heart transplantation. Reported herein are the intermediate-term results of this ongoing trial. METHODS: Between September 1993 and August 1998, 28 patients received concurrent heart transplantation and infusion of donor bone marrow at 3.0 x 10(8) cells/kg (study group). Twenty-four contemporaneous heart recipients who did not receive bone marrow served as controls. All patients received an immunosuppressive regimen consisting of tacrolimus and steroids. RESULTS: Patient survival was similar between the study and control groups (86% and 87% at 3 years, respectively). However, the proportion of patients free from grade 3A rejection was higher in the study group (64% at 6 months) than in the control group (40%; P =.03). The prevalence of coronary artery disease was similar between the two groups (freedom from disease at 3 years was 78% in study patients and 69% in controls). Similar proportions of study (18%) and control (15%) patients exhibited in vitro evidence of donor-specific hyporesponsiveness. CONCLUSIONS: The infusion of donor bone marrow reduces the rate of acute rejection in heart recipients. Donor bone marrow may play an important role in strategies aiming to enhance the graft acceptance.

In the lung aerosol cyclosporine provides a regional concentration advantage over intramuscular cyclosporine.

BACKGROUND: Acute rejection remains an almost universal complication among lung transplant recipients. Refractory rejection as well as chronic systemic immunosuppression is associated with significant morbidity and mortality. Recent studies suggest that aerosol cyclosporine may address these issues by effectively preventing acute cellular rejection while maintaining low systemic drug concentrations. This study was designed to evaluate the concentrations of cyclosporine in blood and lung tissue after aerosol and intramuscular administration. METHODS: Lewis rats were divided into 4 experimental groups: Groups A (n = 33) and B (n = 30) received aerosol cyclosporine 3 and 5 mg/kg, respectively; Groups C (n = 33) and D (n = 30) received systemic cyclosporine 5 and 15 mg/kg, respectively. We used high-performance liquid chromatography to quantitate blood and lung tissue cyclosporine levels at timed intervals. We used the trapezoidal rule to approximate area under the concentration vs time curve (AUC). RESULTS: Aerosol delivery of cyclosporine resulted in higher and more rapid peak drug levels in lung tissue samples than did systemic delivery. At an equivalent 5 mg/kg dose, the cyclosporine AUC was 3 times higher with aerosol delivery than with intramuscular delivery in lung tissue (477,965 vs 157,706 ng x hour/g, respectively). The lung tissue: blood AUC ratio was highest in the aerosol groups (27.3:1 and 17.4:1) compared with the intramuscular groups (8.1:1 and 9.4:1). CONCLUSION: Local aerosol inhalation delivery of cyclosporine provides a regional advantage over systemic intramuscular therapy by providing higher peak concentrations and greater lung tissue exposure.

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement.

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.

Resource utilization and outcome of liver transplantation for alcoholic cirrhosis. A case-control study.

Liver transplantation for alcoholic cirrhosis remains controversial at some transplantation centers. We compared resource utilization and outcome in alcoholic and nonalcoholic cirrhotic patients undergoing liver transplantation. Data were collected from 56 patients who underwent transplantation for alcohol-related cirrhosis from August 1985 to February 1991 and compared with data from a control group matched for age, sex, Child-Pugh class, and date of transplantation. No significant differences were noted in the resource utilization variables examined or in outcome (as assessed by indicators of early graft function, frequency of sepsis, incidence of rejection, renal function, and retransplantation rate). One-year survival was not significantly different (75% for the alcoholic cirrhotic group vs 76% for the nonalcoholic cirrhotic group). We conclude that liver transplantation for end-stage alcohol-related cirrhosis provides excellent results and that resource utilization appears to be equivalent to that for patients undergoing transplantation for non-alcohol-related cirrhosis.

Extracorporeal life support after heart or lung transplantation.

Extracorporeal life support (ECLS) has been used in 10 patients after heart (5 patients), lung (3 patients), and heart-lung (2 patients) transplantation. The age range was 7 months to 55 years. Cardiopulmonary failure leading to institution of ECLS was due to acute postoperative organ malfunction in 4 patients (2 survived), subacute organ malfunction in 3 patients (none survived), and late rejection or infection in 3 patients (2 survived). Neurologic complications occurred in 3 patients (1 survived) and bleeding, in 5 patients (2 survived). Six patients (60%) were successfully weaned from ECLS, and 4 (40%) survived to leave the hospital. Survival was associated with younger age, shorter duration of ECLS, and longer interval from operation to initiation of ECLS but not to reason for initiating ECLS. Extracorporeal life support is feasible for sustaining both adults and children after heart, lung, or heart-lung transplantation. Best results were obtained in patients with conditions that, in retrospect, were treatable and reversible within days. More experience is needed to predict preoperatively which patients will benefit most from ECLS.