Upregulation of endothelin converting enzyme-1 in host liver during chronic cardiac allograft rejection

Endothelin regulates cytokine expression in vitro and in vivo. This study investigated the effects of chronic allograft rejection on hepatic endothelin-converting enzyme-1 (ECE-1) gene expression and endothelin-1 (ET-1) plasma clearance. Using the Lewis-F344 minor histocompatibility mismatch model of heterotopic cardiac transplantation, hepatic ECE-1 gene expression was measured by real-time polymerase chain reaction and host plasma clearance of ET-1 was measured 8 weeks after transplantation in the absence of immunosuppression. In animals undergoing allograft rejection, hepatic ECE-1 gene expression increased 2-fold (P < 0.05), whereas no effect of rejection on ET-1 clearance from plasma was observed. In summary, upregulation of ECE-1 gene expression occurs in the liver of the host during chronic allograft rejection. Because the liver represents both a key organ for cytokine production and for endothelin metabolism, increased hepatic ECE-1-mediated ET-1 synthesis may contribute to host responses and cytokine production during allograft rejection.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Reflection, refraction, and rejection : copper smelting heritage and the execution of environmental policy

This dissertation examines the global technological and environmental history of copper smelting and the conflict that developed between historic preservation and environmental remediation at major copper smelting sites in the United States after their productive periods ended. Part I of the dissertation is a synthetic overview of the history of copper smelting and its environmental impact. After reviewing the basic metallurgy of copper ores, the dissertation contains successive chapters on the history of copper smelting to 1640, culminating in the so-called German, or Continental, processing system; on the emergence of the rival Welsh system during the British industrial revolution; and on the growth of American dominance in copper production the late 19th and early 20th centuries. The latter chapter focuses, in particular, on three of the most important early American copper districts: Michigan’s Keweenaw Peninsula, Tennessee’s Copper Basin, and Butte-Anaconda, Montana. As these three districts went into decline and ultimately out of production, they left a rich industrial heritage and significant waste and pollution problems generated by increasingly more sophisticated technologies capable of commercially processing steadily growing volumes of decreasingly rich ores. Part II of the dissertation looks at the conflict between historic preservation and environmental remediation that emerged locally and nationally in copper districts as they went into decline and eventually ceased production. Locally, former copper mining communities often split between those who wished to commemorate a region’s past importance and develop heritage tourism, and local developers who wished to clear up and clean out old industrial sites for other purposes. Nationally, Congress passed laws in the 1960s and 1970s mandating the preservation of historical resources (National Historic Preservation Act) and laws mandating the cleanup of contaminated landscapes (CERCLA, or Superfund), objectives sometimes in conflict – especially in the case of copper smelting sites. The dissertation devotes individual chapters to the conflicts that developed between environmental remediation, particularly involving the Environmental Protection Agency and the heritage movement in the Tennessee, Montana, and Michigan copper districts. A concluding chapter provides a broad model to illustrate the relationship between industrial decline, federal environmental remediation activities, and the growth of heritage consciousness in former copper mining and smelting areas, analyzes why the outcome varied in the three areas, and suggests methods for dealing with heritage-remediation issues to minimize conflict and maximize heritage preservation.

Analysis of independent microarray datasets of renal biopsies identifies a robust transcript signature of acute allograft rejection

Transcriptomics could contribute significantly to the early and specific diagnosis of rejection episodes by defining 'molecular Banff' signatures. Recently, the description of pathogenesis-based transcript sets offered a new opportunity for objective and quantitative diagnosis. Generating high-quality transcript panels is thus critical to define high-performance diagnostic classifier. In this study, a comparative analysis was performed across four different microarray datasets of heterogeneous sample collections from two published clinical datasets and two own datasets including biopsies for clinical indication, and samples from nonhuman primates. We characterized a common transcriptional profile of 70 genes, defined as acute rejection transcript set (ARTS). ARTS expression is significantly up-regulated in all AR samples as compared with stable allografts or healthy kidneys, and strongly correlates with the severity of Banff AR types. Similarly, ARTS were tested as a classifier in a large collection of 143 independent biopsies recently published by the University of Alberta. Results demonstrate that the 'in silico' approach applied in this study is able to identify a robust and reliable molecular signature for AR, supporting a specific and sensitive molecular diagnostic approach for renal transplant monitoring.

Interference during the implicit learning of two different motor sequences

It has been demonstrated that learning a second motor task after having learned a first task may interfere with the long-term consolidation of the first task. However, little is known about immediate changes in the representation of the motor memory in the early acquisition phase within the first minutes of the learning process. Therefore, we investigated such early interference effects with an implicit serial reaction time task in 55 healthy subjects. Each subject performed either a sequence learning task involving two different sequences, or a random control task. The results showed that learning the first sequence led to only a slight, short-lived interference effect in the early acquisition phase of the second sequence. Overall, learning of neither sequence was impaired. Furthermore, the two processes, sequence-unrelated task learning (i.e. general motor training) and the sequence learning itself did not appear to interfere with each other. In conclusion, although the long-term consolidation of a motor memory has been shown to be sensitive to other interfering memories, the present study suggests that the brain is initially able to acquire more than one new motor sequence within a short space of time without significant interference.

Chronic rejection in lung allografts: immunohistological analysis of fibrogenesis

In ongoing chronic rejection after lung transplantation, alveolar interstitial fibrosis develops. However, little is known about the mechanisms involved. In order to investigate these mechanisms, expression of extracellular matrix molecules (ECM) (undulin, decorin, tenascin, laminin, and fibronectin) and cytokines [transforming growth factor (TGF)-beta 1, TGF-beta 3, platelet-derived growth factor (PDGF), and PDGF receptor] were semiquantitatively evaluated in chronically rejected lung allografts, using standard immunohistochemical techniques. Additionally, the presence of macrophages was analysed. The present study demonstrates an increased infiltration of macrophages with a concomitant upregulation of cytokines (TGF-beta 1, TGF-beta 3, and PDGF) and an increased deposition of ECM in chronic lung rejection. These cytokines have an important role in the stimulation of fibroblasts which are a major source of ECM. Upregulated expression of ECM in the alveolar interstitial space leads to alveolar malfunction by thickening of the wall and, thus, is one of the causative factors of respiratory dysfunction in chronic lung graft rejection.

Treatment of recurrent rejection in heart transplantation: cytolytic therapy or bolus steroids?

OBJECTIVES: The treatment of recurrent rejection in heart transplant recipients has been a controversial issue for many years. The intent of this retrospective study was to perform a risk-benefit analysis between treatment strategies with bolus steroids only versus anti-thymocyte globulins (RATG; 1.5 mg/kg q 4 days). METHODS: Between 1986 and 1993, 69 of 425 patients (17 male, 52 female; mean age 44 +/- 11 years) who had more than one rejection/patient per month (rej/pt per mo) in the first 3 postoperative months were defined as recurrent rejectors. RESULTS: Repetitive methylprednisolone bolus therapy (70 mg/kg q 3 days) was given in 27 patients (group M; 1.4 +/- 0.2 rej/pt per mo) and RATG therapy for one of the rejection episodes of the 42 remaining patients (group A; 1.5 +/- 0.2 rej/pt per mo). The quality of triple drug immunosuppression in the two study groups was comparable. The rejection-free interval (RFI) following RATG treatment in group A was 21.6 +/- 10 days and 22 +/- 11 in group M. In group M, 3 of 27 patients (11%) had a rejection treatment-related infection (2 bacterial; 1 viral) versus 6 of the 42 patients of group A (14.2%; bacterial 1, viral 5). During postoperative months 3-24, 0.15 +/- 0.12 rej/pat per mo were observed in group M and 0.21 +/- 0.13 rej/pat per mo in group A (n.s.). In this 21-month period cytolytic therapy for rejection was initiated in 8 of the remaining 21 patients of group M (38%) and 15 of the remaining 37 patients of group A (40.5%). The absolute survival and the individual causes of death were not affected by the type of initial treatment of recurrent rejection. The actuarial freedom of graft atherosclerosis is comparable in the two groups with 78% in group A versus 79% in group M free of graft atherosclerosis at 3 years postoperatively. CONCLUSIONS: A comparison of cytolytic therapy versus repeated applications of bolus steroids for treatment of recurrent rejection reveals no significant difference in the long-term patient outcome with respect to the incidence of future rejection episodes and survival.

Tricuspid valve regurgitation attributable to endomyocardial biopsies and rejection in heart transplantation

In the present report the prevalence, severity, and risk factors of tricuspid valve regurgitation (TR) in 251 heart transplant recipients have been analyzed retrospectively. Tricuspid valve function was studied by color-flow Doppler echocardiogram and annual heart catheterization. The presence or severity of TR was graded on a scale from 0 (no TR) to 4 (severe). Additional postoperative data included rate of rejection, number of endomyocardial biopsies, incidence of transplant vasculopathy, and preoperative and postoperative hemodynamics. The incidence of grade 3 TR increases from 5% at 1 year to 50% at 4 years after transplantation. Multivariate analysis showed rate of rejection and donor heart weight to be significant risk factors. The ischemic intervals as well as the preoperative and postoperative pulmonary hemodynamics did not affect the severity or prevalence of TR. These results indicate that various factors appear to have an impact on the development of TR and that the prevalence might be lowered by a reduction of the number of biopsies performed and when possible, oversizing of donor hearts.

The impact of early postoperative cyclosporine serum levels on the incidence of cardiac allograft rejection

The introduction of cyclosporine A (CyA) into the immunosuppressive therapy has significantly improved the results of heart transplantation (HTX). Its nephrotoxicity and hepatotoxicity, however, often limit the perioperative and postoperative use of this drug. The purpose of this retrospective study was to evaluate the effect of early postoperative CyA blood levels on the incidence of early as well as late cardiac rejection and patients' survival. Between October 1985 and June 1991, HTX was performed in 311 patients. Standard immunosuppression consisted of azathioprine (1-2 mg/kg), prednisolone (0.5 to 0.1 mg/kg) and CyA. Rabbit-antithymocyte-globulin (RATG - 1.5 mg/kg) was administered for the first 4 days postoperatively. Moderate rejection was treated with 3 x 500 mg methylprednisolone, severe rejection with RATG (1.5 mg/kg three times a day). Patients were excluded from this study because of a positive cross-matching, early death unrelated to rejection or alternate forms of immunosuppression (n = 111). Follow-up was complete in 200 patients (mean age 44 +/- 11; 18 female, 182 male; 204,233 patient days) with a total of 5380 biopsies. The cohort was divided into group I (no CyA for day 0 to 2; n = 108) and group II (CyA during day 0 to 2; n = 92) according to the onset of CyA therapy. In 101 patients (group A) the mean CyA blood level was less than 150 ng/ml from day 0 to 14 and in 99 patients more than 150 ng/ml (group B).(ABSTRACT TRUNCATED AT 250 WORDS)

Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study. In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood. The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts. Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.

ABO blood group incompatible haematopoietic stem cell transplantation and xenograft rejection

The current organ shortage in transplantation medicine stimulates the exploration of new strategies to expand the donor pool including the utilisation of living donors, ABO-incompatible grafts, and xenotransplantation. Preformed natural antibodies (Ab) such as anti-Gal or anti-A/B Ab mediate hyperacute graft rejection and thus represent a major hurdle to the employment of such strategies. In contrast to solid organ transplantation (SOT), ABO blood group incompatibilities are of minor importance in haematopoietic stem cell transplantation (HSCT). Thus, ABO incompatible HSCT may serve as an in vivo model to study carbohydrate antigen (Ag)-mismatched transplantations such as ABO-incompatible SOT or the effect of preformed Ab against Gal in xenotransplantation. This mini-review summarises our clinical and experimental studies performed with the support of the Swiss National Science Foundation program on Implants and Transplants (NFP-46). Part 1 describes data on the clinical outcome of ABO-incompatible HSCT, in particular the incidence of several immunohaematological complications, acute graft-versus-host-disease (GvHD), and the overall survival. Part 2 summarises the measurements of anti-A/B Ab in healthy blood donors and ABO-incompatible HSCT using a novel flow cytometry based method and the potential mechanisms responsible for the loss of anti-A/B Ab observed following minor ABO-incompatible HSCT, ie the occurrence of humoral tolerance. Part 3 analyses the potential of eliminating Gal expression as well as specific complement inhibitors such as dextran sulfate and synthetic tyrosine analogues to protect porcine endothelial cells from xenoreactive Ab-mediated damage in vitro and in a hamster-to-rat heart transplantation model. In conclusion, due to similarities of the immunological hurdles of ABO incompatible transplantations and xenotransplantation, the knowledge obtained from both fields might lead to new strategies to overcome humoral rejection in transplantation.

Endothelial cell protection by dextran sulfate: a novel strategy to prevent acute vascular rejection in xenotransplantation

We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cell-mediated cytotoxicity towards porcine cells in vitro. We therefore hypothesized that DXS, combined with cyclosporine A (CyA), could prevent acute vascular rejection (AVR) in the hamster-to-rat cardiac xenotransplantation model. Untreated, CyA-only, and DXS-only treated rats rejected their grafts within 4-5 days. Of the hearts grafted into rats receiving DXS in combination with CyA, 28% survived more than 30 days. Deposition of anti-hamster antibodies and complement was detected in long-term surviving grafts. Combined with the expression of hemoxygenase 1 (HO-1) on graft EC, these results indicate that accommodation had occurred. Complement activity was normal in rat sera after DXS injection, and while systemic inhibition of the coagulation cascade was observed 1 h after DXS injection, it was absent after 24 h. Moreover, using a fluorescein-labeled DXS (DXS-Fluo) injected 1 day after surgery, we observed a specific binding of DXS-Fluo to the xenograft endothelium. In conclusion, we show here that DXS + CyA induces long-term xenograft survival and we provide evidence that DXS might act as a local EC protectant also in vivo.

Xenograft rejection: IgG1, complement and NK cells team up to activate and destroy the endothelium

Acute vascular rejection represents a formidable barrier to clinical xenotransplantation and it is known that this type of rejection can also be initiated by xenoreactive antibodies that have limited complement-activating ability. Using a sophisticated mouse model, a recent study has provided in vivo evidence for the existence of an IgG(1)-mediated vascular rejection, which uniquely depends on both the activation of complement and interactions with FcgammaRIII on natural killer (NK) cells.

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. METHODS: To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). RESULTS: siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. CONCLUSION: In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.