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Supplemental Digital Content is available in the text., Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Resumen Introduccion y objetivos Los valores plasmaticos de galectina-3 (Gal-3) estan elevados y se correlacionan con la mortalidad total y cardiovascular en pacientes con insuficiencia cardiaca, pero su correlacion con el pronostico tras el trasplante cardiaco (TxC) es desconocida. El objetivo fue describir la tendencia evolutiva y el valor pronostico de este biomarcador tras el TxC. Metodos Mediante enzimoinmunoensayo, se midieron las concentraciones plasmaticas de Gal-3 en muestras de suero de 122 receptores de TxC, antes y 1, 3, 6 y 12 meses despues de este. Mediante regresion de Cox se analizo el valor pronostico del valor plasmatico de Gal-3 a los 12 meses del TxC. El objetivo primario del estudio fue la variable combinada muerte o disfuncion del injerto. Resultados Las concentraciones de Gal-3 disminuyeron progresivamente durante el primer ano tras el TxC (medianas: pretrasplante, 19,1 ng/ml; 1 ano postrasplante, 14,6 ng/ml; p Conclusiones Las concentraciones plasmaticas de Gal-3 disminuyeron progresivamente durante el primer ano tras el TxC. Un valor plasmatico elevado de Gal-3 1 ano tras el TxC se correlaciono con un pronostico adverso.

[Abstract] Introduction and objectives: Circulating galectin-3 (Gal-3) is elevated and significantly correlates with all-cause and cardiovascular mortality in patients with heart failure. However, the relationship between serum Gal-3 and heart transplant (HT) outcomes is unclear. The aim of this study was to describe the longitudinal trend and prognostic value of Gal-3 levels after HT. Methods: Banked serum samples were available from 122 HT recipients, collected before transplant and at 1, 3, 6, and 12 months posttransplant. Gal-3 levels in these serum samples were measured by enzyme immune assay. Multivariable Cox regression was performed to determine the prognostic value of 12-month posttransplant Gal-3 serum levels. The primary endpoint was the composite variable all-cause death or graft failure over long-term posttransplant follow-up. Results: Circulating Gal-3 concentration steadily decreased during the first year after HT (median values: pretransplant, 19.1 ng/mL; 1-year posttransplant, 14.6 ng/mL; P

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

[Abstract] Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling. This work was supported by the British Heart Foundation, the European Research Council (ERC Advanced award) and the Medical Research Council. P.E. is supported by The Barts Charity Cardiovascular Programme Award G00913. We also thank K. Hartmann for her technical assistance and Biobank of ‘A Coruña’ (XXIAC-Instituto de Investigación Biomédica de A Coruña) for providing healthy heart tissue samples. T.E. acknowledges support from NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and KCL; the Centre of Excellence in Medical Engineering funded by the Welcome Trust and EPSRC (WT 088641/Z/09/Z) and the KCL Comprehensive Cancer Imaging Centre funded by the Cancer Research UK (CRUK) and EPSRC in association with MRC and DoH. We acknowledge the metabolic flux analysis facility of the Barts School of Medicine and Dentistry created with the support of the Barts and the London Charity, grant MGU0401 Barts Charity; MGU0401

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton's jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4-5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.

The endogenous mechanisms contributing to tissue survival following myocardial infarction are not fully understood. We investigated the alterations in the mitochondrial proteome after ischemia-reperfusion (I/R) and its possible implications on cell survival. Mitochondrial proteomic analysis of cardiac tissue from an in vivo porcine I/R model found that surviving tissue in the peri-infarct border zone showed increased expression of several proteins. Notably, these included subunits of the mitochondrial pyruvate carrier (MPC), namely MPC1 and MPC2. Western blot, immunohistochemistry, and mRNA analysis corroborated the elevated expression of MPC in the surviving tissue. Furthermore, MPC1 and MPC2 protein levels were found to be markedly elevated in the myocardium of ischemic cardiomyopathy patients. These findings led to the hypothesis that increased MPC expression is cardioprotective due to enhancement of mitochondrial pyruvate uptake in the energy-starved heart following I/R. To test this, isolated mouse hearts perfused with a modified Krebs buffer (containing glucose, pyruvate, and octanoate as metabolic substrates) were subjected to I/R with or without the MPC transport inhibitor UK5099. UK5099 increased myocardial infarction and attenuated post-ischemic recovery of left ventricular end-diastolic pressure. However, aerobically perfused control hearts that were exposed to UK5099 did not modulate contractile function, although pyruvate uptake was blocked as evidenced by increased cytosolic lactate and pyruvate levels. Our findings indicate that increased expression of MPC leads to enhanced uptake and utilization of pyruvate during I/R. We propose this as a putative endogenous mechanism that promotes myocardial survival to limit infarct size.

[Abstract] The purpose of the current study was to establish a valid protocol for nerve cryopreservation, and to evaluate if the addition of albumin supposed any advantage in the procedure. We compared a traditional cryopreservation method that uses dimethyl sulfoxide (DMSO) as cryoprotectant, to an alternative method that uses DMSO and albumin. Six Wistar Lewis rats were used to obtain twelve 20 mm fragments of sciatic nerve. In the first group, six fragments were cryopreserved in 199 media with 10% DMSO, with a temperature decreasing rate of 1 °C per minute. In the second group, six fragments were cryopreserved adding 4% human albumin. The unfreezing process consisted of sequential washings with saline in the first group, and saline and 20% albumin in the second group at 37 °C until the crioprotectant was removed. Structural evaluation was performed through histological analysis and electronic microscopy. The viability was assessed with the calcein-AM (CAM) and 4′,6-diamino-2-fenilindol (DAPI) staining. Histological results showed a correct preservation of peripheral nerve architecture and no significant differences were found between the two groups. However, Schwann cells viability showed in the CAM-DAPI staining was significantly superior in the albumin group. The viability of Schwann cells was significantly increased when albumin was added to the nerve cryopreservation protocol. However, no significant structural differences were found between groups. Further studies need to be performed to assess the cryopreserved nerve functionality using this new method.

[Abstract] Background: Insulin-like growth factor 1 (IGF-1) was found to stimulate Schwann cell mitosis. Exogenous IGF-1 may improve nerve regeneration after cryopreservation. Objective: To evaulate the effect of intraneural administration of IGF-1 in cryopreserved nerve isografts. Methods: Eighteen millimeter grafts were used for bridging an 18-mm defect in the rat sciatic nerve. A total of 57 rats were randomly divided into three groups: (1) autograft (Group 1); (2) cryopreserved isograft (Group 2); (3) cryopreserved isograft with intraneural IGF-1 administration (Group 3). 12 weeks after surgery, functional recovery (Sciatic functional index [SFI], Swing speed [SS], nerve conduction velocity [NCV], amplitude of compound motor action potentials [CMAP], and gastrocnemius muscle index [GMI]) and nerve regeneration (myelin sheath area, total fiber counts, fiber density, and fiber width) were all evaluated. Results: The intraneural injection of IGF-1 significantly improved SFI and SS at weeks 10 and 12. There were no statistical differences between Groups 1 and 3 in any of the SFI or SS evaluations. CMAP and NCV in Group 1 were significantly higher than in Groups 2 and 3, and Group 3 had significantly higher CMAP and NCV compared to Group 2. No significant differences were found in fiber width. The number of nerve fibers, percentage of myelinated fibers, fiber density, and GMI was significantly higher in Group 1 compared to Group 2, but no significant differences were found between Groups 1 and 3. Conclusion: The results show that intraneural injection of IGF-1 in an 18 mm cryopreserved isograft improve axonal regeneration and functional recovery.

Human amniotic membrane (HAM) has useful properties as a dermal matrix substitute. The objective of our work was to obtain, using different enzymatic or chemical treatments to eliminate cells, a scaffold of acellular HAM for later use as a support for the development of a skin equivalent. The HAM was separated from the chorion, incubated and cryopreserved. The membrane underwent different enzymatic and chemical treatments to eliminate the cells. Fibroblasts and keratinocytes were separately obtained from skin biopsies of patients following a sequential double digestion with first collagenase and then trypsin–EDTA (T/E). A skin equivalent was then constructed by seeding keratinocytes on the epithelial side and fibroblasts on the chorionic side of the decellularizated HAM. Histological, immunohistochemical, inmunofluorescent and molecular biology studies were performed. Treatment with 1 % T/E at 37 °C for 30 min totally removed epithelial and mesenchymal cells. The HAM thus treated proved to be a good matrix to support adherence of cells and allowed the achievement of an integral and intact scaffold for development of a skin equivalent, which could be useful as a skin substitute for clinical use.

[Background]: Heart failure (HF) is a complex syndrome associated with a maladaptive innate immune system response that leads to deleterious cardiac remodeling. However, the underlying mechanisms of this syndrome are poorly understood. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a newly recognized innate immune sensor involved in cardiovascular diseases. [Objectives]: This study evaluated the role of NOD1 in HF progression. [Methods]: NOD1 was examined in human failing myocardium and in a post-myocardial infarction (PMI) HF model evaluated in wild-type (wt-PMI) and Nod1 mice (Nod1-PMI). [Results]: The NOD1 pathway was up-regulated in human and murine failing myocardia. Compared with wt-PMI, hearts from Nod1-PMI mice had better cardiac function and attenuated structural remodeling. Ameliorated cardiac function in Nod1-PMI mice was associated with prevention of Ca dynamic impairment linked to HF, including smaller and longer intracellular Ca concentration transients and a lesser sarcoplasmic reticulum Ca load due to a down-regulation of the sarcoplasmic reticulum Ca-adenosine triphosphatase pump and by augmented levels of the Na/Ca exchanger. Increased diastolic Ca release in wt-PMI cardiomyocytes was related to hyperphosphorylation of ryanodine receptors, which was blunted in Nod1-PMI cardiomyocytes. Pharmacological blockade of NOD1 also prevented Ca mishandling in wt-PMI mice. Nod1-PMI mice showed significantly fewer ventricular arrhythmias and lower mortality after isoproterenol administration. These effects were associated with lower aberrant systolic Ca release and with a prevention of the hyperphosphorylation of ryanodine receptors under isoproterenol administration in Nod1-PMI mice. [Conclusions]: NOD1 modulated intracellular Ca mishandling in HF, emerging as a new target for HF therapy., This work was supported by grants CP11/00080 and PI14/01078 from Instituto de Salud Carlos III (ISCIII); SAF2014-52492-R, SAF2014-57190-R, and RTC2015-3741 from Ministerio de Economía y Competitividad; Fondos Fondo Europeo de Desarrollo Regional; and Red de Investigación Cardiovascular (RIC) RD12/0042/0019. RIC is a network funded by the Carlos III Health Institute. Dr. Fernández Velasco is Miguel Servet researcher of ISCIII.

Resumen La realidad de la investigacion biomedica en Espana, exige tener un conocimiento actualizado de la realidad investigadora y de su marco etico/legal. Los estudios de investigacion con muestras biologicas humanas deben realizarse con un numero de muestras lo suficientemente amplio para reflejar la diversidad de la poblacion humana. Asimismo, deben cumplir los requisitos estandarizados de calidad optima para garantizar los resultados de la investigacion a desarrollar. Ademas, la investigacion con seres humanos, y la obtencion y/o derivacion de muestras biologicas humanas e informacion clinica a estudios de investigacion, esta sujeta a una serie de requisitos y restricciones legales. Los biobancos y las redes de biobancos se constituyen como la estructura optima que favorece el almacenamiento de grandes volumenes de muestras biologicas humanas gestionadas en base a criterios que garanticen su optima calidad, armonizacion y seguridad, respetando en todo momento los requisitos eticos y legales que garantizan los derechos de los ciudadanos.

Background: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Methods: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. Results: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. Conclusions: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

Background— After myocardial ischemia, extracellular matrix (ECM) deposition occurs at the site of the focal injury and at the border region. Methods and Results— We have applied a novel proteomic method for the analysis of ECM in cardiovascular tissues to a porcine model of ischemia/reperfusion injury. ECM proteins were sequentially extracted and identified by liquid chromatography tandem mass spectrometry. For the first time, ECM proteins such as cartilage intermediate layer protein 1, matrilin-4, extracellular adipocyte enhancer binding protein 1, collagen α-1(XIV), and several members of the small leucine-rich proteoglycan family, including asporin and prolargin, were shown to contribute to cardiac remodeling. A comparison in 2 distinct cardiac regions (the focal injury in the left ventricle and the border region close to the occluded coronary artery) revealed a discordant regulation of protein and mRNA levels; although gene expression for selected ECM proteins was similar in both regions, the corresponding protein levels were much higher in the focal lesion. Further analysis based on >100 ECM proteins delineated a signature of early- and late-stage cardiac remodeling with transforming growth factor-β1 signaling at the center of the interaction network. Finally, novel cardiac ECM proteins identified by proteomics were validated in human left ventricular tissue acquired from ischemic cardiomyopathy patients at cardiac transplantation. Conclusion— Our findings reveal a biosignature of early- and late-stage ECM remodeling after myocardial ischemia/reperfusion injury, which may have clinical utility as a prognostic marker and modifiable target for drug discovery.

Mesenchymal stem cells (MSCs) may be among the first stem cell types to be utilized in the clinic for cell therapy, because of their ease of isolation and extensive differentiation potential. Using a porcine model, we have established several cell lines from MSCs to facilitate in vitro and in vivo studies of their potential use for cellular therapy. Bone marrow-derived primary MSCs were immortalized using the pRNS-1 plasmid. We obtained four stable immortalized cell lines that exhibited higher proliferative capacities than the parental cells. All four cell lines displayed a common phenotype similar to that of primary mesenchymal cells, characterized by constitutively high expressions of CD90, CD29, CD44, SLA I and CD46, while CD172a, CD106 and CD56 were less expressed. Remarkably, treatment with 5-azacytidine-stimulated porcine MSCs lines to differentiate into cells that were positive for cardiac phenotypic markers, such as α-actin, connexin-43, sarcomeric actin, serca-2 and, to a lesser extent, desmin and troponin-T. These porcine MSC lines will be valuable biological tools for developing strategies for ex vivo expansion and differentiation of MSCs into a specific lineage. Copyright © 2011 John Wiley & Sons, Ltd.

Severe allograft dysfunction after heart transplant (HT), without ischemia or evidence of cellular rejection upon endomyocardial biopsy (EMB), is a rare but potentially fatal condition that suggests humoral rejection (HR). Its incidence, and the methods of choice for its diagnosis and management, remain uncertain. We retrospectively studied 445 HT patients (April 1991-December 2003) to determine incidence of HR diagnosed by clinical and conventional histopathological criteria. We used immunofluorescence (IF) techniques to test archived frozen EMB issue for IgM, IgG, C1q, C3, fibrin and C4d. Twelve patients (2.7%) fulfilled the criteria for HR after a mean time post-HT of 21.3 +/- 24.7 months (range: 2-72 months). Patients were treated with high doses of steroids and plasmapheresis, with successful recovery in 11 cases. IF studies using classical markers were mainly negative for the six patients with enough EMB tissue for testing. All six patients showed positivity for C4d during the HR episode but not before or after. Humoral rejection was observed in less than 3% of HT patients. Plasmapheresis treatment was highly effective. Classical IF tests were not useful for diagnosis, but C4d appears to be useful both for confirmation of diagnosis and for monitoring response to treatment.

BACKGROUND Nonhuman primates are potential permissive animals for studying the risk of in vivo infection with porcine endogenous retrovirus (PERV). Anti-alphaGal natural antibodies are considered one of the barriers for preventing PERV infection, and it has been postulated that reduction of these antibodies could increase the risk of this infection. The aim of this study was to investigate the role of GAS 914, which depletes anti-alphaGal antibodies, in the potential in vivo transfer of PERV after pig-to-baboon organ xenotransplantation. METHODS Twenty-seven baboons underwent xenotransplantation with hDAF or hMCP/hDAF transgenic pig organs, including heterotopic heart (n = 14) and kidney (n = 13) transplants. All of them received GAS 914 along with different immunosuppression protocols. PERV sequences were investigated by reverse-transcriptase polymerase chain reaction and by polymerase chain reaction assays in samples obtained at autopsy. The presence of PERV-specific antibodies and/or pig xenomicrochimerism was also evaluated. RESULTS PERV RNA was not detected in any baboon plasma sample. In addition, all plasma samples were negative for PERV antibodies. However, PERV DNA sequences were detected in peripheral blood mononuclear cells from 6 of 14 (43%) animals investigated. Porcine mitochondrial DNA was also found in all of these positive samples and in six of the eight (75%) samples with negative PERV DNA, indicating that the detection of PERV sequences was attributable to xenochimerism. PERV-positive cells as a result of xenochimerism were also found in eight of nine (89%) spleen and lymph node tissue samples tested. CONCLUSIONS Sustained depletion of anti-alphaGal antibodies does not augment the risk of PERV infection in pig-to-baboon organ transplantation.

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-αGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-αGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.

Background A flow cytometry complement-mediated cytotoxicity assay (FCCA) using fluorescein diacetate (FDA) and propidium iodide (PI) to measure antibody-dependent toxicity is useful to determine the success of xenotransplant organs. We evaluated the validity of different mathematical models as a measure of cytotoxicity in FCCA. Methods Sera from untreated baboons (n = 7) and from immunosuppressed animals (n = 5) undergoing different xenotransplantation protocols with pig organs were tested by endogenous FCCA and a similar assay also using exogenous complement, and the results were compared with those of a complement-dependent hemolytic assay to detect anti-pig antibodies (APHA). The influence of PI/FDA staining and the use of several mathematical models were analyzed. Results For both groups of animals, we observed high correlations between the endogenous and exogenous FCCA pathways and between calculations based on PI and FDA staining. Of the four mathematical models tested—the Von Krogh equation, two exponential models, and area under the curve—the Von Krogh equation was the most appropriate in terms of goodness of fit and concordance with APHA. Conclusions FDA/PI FCCA is useful to measure endogenous and exogenous complement-mediated cytotoxicities, and it has advantages related to identification of potential new xenoantibodies. Although all four mathematical models produced acceptable solutions, the Von Krogh equation was the best option. © 2004 Wiley-Liss, Inc.

BACKGROUND The impact of anti-Galalpha1-3Gal (alphaGal) antibodies on the acute humoral xenograft rejection (AHXR) of pig organs transplanted in baboons is unclear. METHODS Twenty-three baboons underwent heterotopic pig heart transplantation (Tx). Groups A (n = 5) and B (n = 6) received non-transgenic and human decay accelerating factor (hDAF) pig hearts, respectively, without any treatment. Groups C (n = 5) and D (n = 7) were transplanted with non-transgenic and hDAF organs, respectively, and the exclusive treatment was repeated extracorporeal immunoadsorptions (EIA) before and after Tx with an alphaGal column containing disaccharide (DI), trisaccharide (TRI) 2 and pentasaccharide (PENTA) oligosaccharides. RESULTS In group A, 3 of 5 xenografts underwent hyperacute rejection (HAR). No xenograft from groups B, C and D experienced HAR, most of them failing from AHXR. Immediately after Tx and up to day 2, the level of immunoglobulin (Ig)M and IgG anti-alphaGal DI, TRI2 and TRI6, and anti-pig hemolytic antibody (APHA) antibodies decreased in all the groups by 80 to 96% compared with the concentration present before Tx. From day 3 to AHXR, a sustained increase of anti-alphaGal IgM DI, TRI2 and TRI6, and APHA occurred in all groups. EIA depleted anti-alphaGal IgM and APHA before Tx, but it did not modify the increase of these antibodies after Tx. Baboon serum samples before Tx, pre-incubated in vitro with 1 mg/ml of DI, TRI2 and TRI6, had an average of 93% reduction of anti-alphaGal IgM antibodies specific against each one of these alphaGal oligosaccharides. In contrast, at AHXR, the average reduction after in vitro pre-incubation with either 1 or 5 mg/ml of DI, TRI2 and TRI6 was 40%. CONCLUSIONS The EIA reduces anti-alphaGal and APHA antibodies, preventing the HAR of non-transgenic pig hearts transplanted in baboons, as does hDAF expression. However, EIA does not modify the level of anti-alphaGal IgM and APHA antibodies after Tx nor the AHXR of either non-transgenic or hDAF pig organs. The increase in anti-alphaGal IgM after Tx was similar for the different antibodies of the anti-alphaGal polymorphism, and was only partially neutralized in vitro with the specific alphaGal oligosaccharide.

In this report, we describe the characterization of a novel activation antigen on porcine lymphocytes recognized by mAb 5A6/8. This antigen was detected on B and T cells 24 h after treatment with various stimuli. It was also found on alveolar macrophages, and at low levels on untreated monocytes. MAb 5A6/8 precipitated two bands of 45 and 50 kDa under non-reducing conditions, and of 22 and 28 kDa under reducing conditions. The cellular distribution, expression kinetics and/or molecular size of the 5A6/8 antigen differ from those of other known lymphocyte activation antigens. MAb 5A6/8 was able to inhibit lymphocyte proliferative responses driven by different stimuli, suggesting a role for this molecule in the events that lead to lymphocyte activation. © INRA, EDP Sciences, 2004.

We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Low-complexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC-MALDI-TOF/TOF analysis, purified β-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated -20 °C freezer in the Biobank facility. Samples were kept dried at -20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC-MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins - serum albumin and serotransferrin - were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC-MS/MS, β-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage - up to 3 months - of dried peptide extracts.

The reality of biomedical research in Spain requires having an updated knowledge of the research reality and its ethical/legal framework. Research studies with human biological samples should be made with a sufficiently large number of samples to reflect the diversity of the human population, which meets the standard requirements to ensure optimum quality of the research results for further development. Furthermore, research with humans and obtaining and/or deriving human biological samples and clinical research studies information are subject to a number of legal requirements and restrictions. Biobanks and biobank networks are established as the optimal structures that favor the storage of large volumes of human biological samples based on criteria to ensure their optimum quality, harmonization and security, respecting at all times, the ethical and legal requirements guaranteeing the rights of citizens.

We describe in this report the production and characterization of monoclonal antibodies (mAb) to the swine homologues of CD11a and CD18 antigens, and their use for phenotypic and functional analysis of porcine leukocytes. Monoclonal antibodies BL1H8 and BL2F1 precipitated two bands of approximately 170 and 95 kDa, whereas mAb BA3H2 brought down three bands of 170, 155 and 95 kDa, from alveolar macrophage lysates. Clearance of macrophage lysates with mAbs BL1H8 and BL2F1 resulted in complete removal of the 170-kDa band. The cell distribution of the molecules recognized by these mAbs was similar to that of human LFA-1. It was found on all leukocytes, although its expression varied among the different leukocyte subpopulations, with monocytes, granulocytes and a subset of CD8+ cells expressing the highest levels. Cross-blocking studies showed that these antibodies recognize different epitopes on porcine LFA-1. Both anti-LFA-1 mAbs strongly inhibited the mitogenic response of PBMC to ConA, whereas the anti-CD18 mAb had no effect. These anti-LFA-1 mAbs also inhibited the mixed lymphocyte reaction (MLR) and the NK cell-mediated lysis of K-562 cells.

The effect of porcine reproductive and respiratory syndrome (PRRS) virus infection on the synthesis and secretion of TNF-α and other pro-inflammatory cytokines by porcine alveolar macrophages (PAM) was investigated as well as the effect that TNF-α has on the replication of this virus. A clear reduction of phorbol myristate acetate (PMA)-induced expression of TNF-α mRNA was observed in cells incubated with PRRS virus. Moreover, the presence of PRRS virus also induced a decrease in IL-1 α and MIP-1 β mRNAs expression with respect to PMA-stimulated uninfected cells. According to these results, exposure to the PRRS virus led to a reduction of the TNF-α protein in supernatants of PMA-stimulated PAM. On the other hand, addition of recombinant porcine TNF-α to cultures clearly reduced virus replication; however the addition of TNF-α to cultures containing IFN-α did not result in a further reduction of the produced by IFN-α alone. This indicates the lack of synergy in the effect of these cytokines on viral replication. (C) 2000 Published by Elsevier Science B.V.

An in vivo porcine model of myocardial infarction was developed with the aim of comparing the effectiveness for cardiac repair of intracoronary, transthoracic, or transendocardial delivery strategies for bone marrow mesenchymal stem cells (BMMSC) using an analysis of expression levels of transcripts related to various cellular processes at 8 heart regions using quantitative reverse transcriptase polymerase chain reaction. We observed significant rises in cardiomyogenic markers Mef2C, Gata4 and Nkx2.5, and contractibility marker Serca2A at infarcted regions for cell-treated pigs. We also observed differences in Sdf1 expression related to the organ stress response between delivery strategies. Unexpectedly, increased expression of Col1A1 was detected in 2 cell-treated groups at various heart regions. Our results suggest improvements in both contractility and cardiomyogenic capability of damaged tissue after BMMSC injection, but also warned us about the relevance of the chosen delivery strategy and potential undesired effects like increasing fibrosis after treatment.

Myocardial infarction is one of the main causes of mortality in developed countries. Injection of bone marrow mesenchymal stem cells (BMMSC) with the ability to regenerate lost cardiomyocytes is a promising therapy for heart failure. To evaluate this strategy, an in vivo porcine model of infarction was used. Gene expression profiles of 3 groups of pigs (n = 5 each) were analyzed and compared by real-time reverse transcription-polymerase chain reaction (RT-PCR). One of the groups underwent anterior descending coronary occlusion followed by BMMSC injection; a placebo group was injected with culture medium without cells after infarction; and a third group was formed by healthy pigs. Four weeks later, cells or medium was administered by intracoronary injection and, a month later, animals were sacrificed and samples collected. Genes related to cardiomyogenesis (Mef2C, Gata4, Nkx2.5), mobilization and homing of resident or circulating stem cells (Sdf1, Cxcr4, c-Kit), contractibility (Serca2a), and fibrosis (CollA1) were analyzed. Gene expression profiles changed in various heart areas in the 3 groups. Expression of genes related to cardiomyogenesis decreased in infarcted zones compared with homologous regions of healthy hearts. Sdf1 expression increased in the apex of infarcted hearts. Serca2a expression was reduced in the ventricles and atria of infarcted hearts. Also, increases in Cxcr4 and CollA1 expression were observed in infarcted hearts of cell-treated pigs compared with the placebo group. In conclusion, infarction induced changes in genes involved in various biological processes. Intracoronary injection of heterologous BMMSC resulted in localized changes in the expression of Cxcr4 and Col1A1.

This report describes the obtention and characterization of two monoclonal antibodies (mAbs), 6E3 7 [mAb 6E3 7 was submitted to the Second International Swine CD Workshop, where it has been assigned to CD45R] and 3C3 9 , which recognize the isoform of highest molecular weight of porcine CD45. This conclusion is based on their cell reactivity and tissue distribution, identical to that reported for the human high molecular weight isoform of CD45, and on data from immunoprecipitation and immunoblotting analyses which show that these mAbs react with the largest polypeptide of those precipitated by mAb 2A5, that recognizes an epitope shared by all CD45 isoforms. These mAbs react with 60% of peripheral blood mononuclear cells (PBMC) but not with alveolar macrophages, granulocytes, platelets or erythrocytes. Antigen expression on PBMC is heterogeneous and is reduced after in vitro activation with mitogens. B cells and CD8+ T cells express more antigen than CD4+ T cells. Using immunoperoxidase techniques, the antigen was detected on B cell areas of lymph nodes and Peyer's patches, and on a subpopulation of medullary thymocytes. These mAbs will be useful reagents for functional and phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) and is a common complication of various cardiovascular diseases. However, little is known about proteins in the cardiac extracellular space. Proteomics analysis of cardiac ECM can be challenging due to the presence of more abundant intracellular proteins, the low degree of solubility of integral ECM proteins, and the presence of abundant posttranslational modifications. Here we describe an extraction methodology based on tissue decellularization, which allows the biochemical subfractionation of extracellular proteins in cardiac tissue. These relatively low-complexity protein fractions are suitable for analysis by gel-LC-MS/MS and other proteomics techniques.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) and is a common complication of various cardiovascular diseases. However, little is known about proteins in the cardiac extracellular space. Proteomics analysis of cardiac ECM can be challenging due to the presence of more abundant intracellular proteins, the low degree of solubility of integral ECM proteins, and the presence of abundant posttranslational modifications. Here we describe an extraction methodology based on tissue decellularization, which allows the biochemical subfractionation of extracellular proteins in cardiac tissue. These relatively low-complexity protein fractions are suitable for analysis by gel-LC-MS/MS and other proteomics techniques.

[Abstract] Idiopathic dilated cardiomyopathy (IDC) is a structural heart disease with strong genetic background. The different single nucleotide polymorphisms (SNPs) that constitute mitochondrial haplogroups could play an important role in IDC progression. The aim of this study was to test frequencies of mitochondrial haplogroups in healthy controls (n = 422) and IDC patients (n = 304) of a Caucasian Spanish population. To achieve this, ten major European haplogroups were identified. Frequencies and Odds Ratios for the association between IDC and haplogroups were calculated in both groups. We found that compared to healthy controls, the prevalence of haplogroup H was significantly higher in IDC patients (40.0% vs 50.7%, p-value = 0.040). Instituto de Salud Carlos III; PS09/00840

Based on the results from different studies, it is recommended to use multiple assays to fully monitor a peripheral antibody response in recipients of pig xenografts. In particular, the complement-dependent hemolytic assay (CH50) determines total endogenous complement activity; the cytolytic activity of anti-pig antibodies (APA) is also measured by hemolytic assay; an ELISA is used for quantification of anti-galactose α1,3galactose (Gal) IgG and IgM antibodies and flow cytometry for determining the antiendothelial cell (anti-EC) antibodies. The APA is recommended because this assay measures cytolytic antibodies unlike the other assays which are binding assays. The anti-Gal ELISA is especially useful as it specifically quantifies antibodies against the major xenoantigen in the pig-to-primate transplant combination. Thus, it provides different information than the APA assay and is particularly useful to follow up treatment modalities aimed at blocking/inhibiting the anti-Gal antibodies. Finally, the anti-EC assay measures IgG and/or IgM antibodies reactive to Gal and non-Gal antigens expressed on endothelial cells. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome rejection. Here, we describe in detail the procedures of these techniques.

[Abstract] Background. Since mitochondria are the principal source of reactive oxygen species (ROS), these organelles may play an important role in ischemic cardiomyopathy (IC) development. The mitochondrial genome may influence this disease. The aim of the present study was to test the relationship between IC development and the impact of single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) defining the mitochondrial haplogroups in a population study. Methodology and principal findings. Ten major European haplogroups were identified by using the single base extension technique and by polymerase chain reaction-restriction fragment length polymorphism. Frequencies and Odds Ratios for the association between IC patients (n = 358) and healthy controls (n = 423) were calculated. No convincing associations between classical risk factors for ischemic cardiomyopathy development and haplogroups were found. However, compared to healthy controls, the prevalence of haplogroup H was significantly higher in IC patients (40.0% vs 50.0%, p-value = 0.039) while the frequency of haplogroup J was significantly lower (11.1% vs 5.6%, p-value = 0.048). The analysis of the SNPs characterizing the European mtDNA haplogroups showed that the m.7028C allele (40.0% vs 50.0%, p-value = 0.005) and m.14766C allele (43.0% vs 54.2%, p-value = 0.002) were overrepresented in IC patients, meanwhile the m.10398G allele (19.8% vs 13.1%, p-value = 0.015) and m.4216C allele (22.2% vs 16.5%, p-value = 0.044) were found as protective factors against IC. Conclusions and significance. Our results showed that the haplogroups H and J were found as a risk and protective factors for ischemic cardiomyopathy development, respectively. Instituto de Salud Carlos III; PS09/00840

Based on the results from different studies, it is recommended to use multiple assays to fully monitor a peripheral antibody response in recipients of pig xenografts. In particular, the complement-dependent hemolytic assay (CH50) determines total endogenous complement activity; the cytolytic activity of anti-pig antibodies (APA) is also measured by hemolytic assay; an ELISA is used for quantification of anti-galactose α1,3galactose (Gal) IgG and IgM antibodies and flow cytometry for determining the antiendothelial cell (anti-EC) antibodies. The APA is recommended because this assay measures cytolytic antibodies unlike the other assays which are binding assays. The anti-Gal ELISA is especially useful as it specifically quantifies antibodies against the major xenoantigen in the pig-to-primate transplant combination. Thus, it provides different information than the APA assay and is particularly useful to follow up treatment modalities aimed at blocking/inhibiting the anti-Gal antibodies. Finally, the anti-EC assay measures IgG and/or IgM antibodies reactive to Gal and non-Gal antigens expressed on endothelial cells. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome rejection. Here, we describe in detail the procedures of these techniques.

We evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model.Myocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10(6) 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo group.Infarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.

Stem cell therapy constitutes an exciting, powerful therapy to repair the heart. Nevertheless, there are numerous doubts about the best route of stem cell administration to achieve implantation into the injured myocardium. Development of a preclinical, large animal model may be useful to obtain a better approach to clinical situations. The aim of this work was to study the effectiveness of various routes of heterologous bone marrow mesenchymal stem cell (MSCs) administration in a porcine model of myocardial infarction. MSC treated with 5-azacytidine were stained with a fluorescent compound (DiO) before their administration to previously infarcted pigs via 3 routes: intracoronary (IC), intramyocardial (IM), or endocardial (EC; n = 5 each group). Healthy, noninfarcted animals were used as a control group. At 30 days after delivery, hearts were divided into 12 parts: infarcted zone (1-6), right-left atria, interatrial and interventricular septa, and right-left ventricles. In each zone we looked for and quantified, injected fluorescence-stained cells. In the animals in which presence of DiO-stained cells was detected, cells were located preferentially in the infarcted zone and not in the atria, ventricles, or septa. Comparing various administration routes, the mean number of engrafted cells within the infarct zone was significantly greater after IC infusion than either IM or EC injection. Fluorescent cells were not observed in healthy zones of the myocardium or in healthy animals.

The contribution of the hypervariable region spanning amino acid residues 62 to 80 to the serologic determinants of HLA-A2 and HLA-B7 has been examined by site-directed mutagenesis. Three HLA-A2 mutants, having changes as in HLA-B7 at positions 62, 76, and at the complete 65-to-80 segment, respectively, were obtained and expressed on class I HLA-deficient human cells upon transfection. The reactivity of 19 monoclonal antibodies (mAbs) against both broad public and allospecific determinants on HLA-A2 and HLA-B7 was analyzed. The results indicate that: (1) the change at residue 62 abrogated recognition of the corresponding HLA-A2 mutant by mAb MA2.1 (anti-A2 + B17); (2) the change at residue 76 did not effect any of the determinants analyzed, although its side chain is easily accessible at the surface of the molecule; (3) the replacement of the whole 65-to-80 segment in HLA-A2 by that from HLA-B7 abrogated recognition by MA2.1 and by 108-2C5, a mAb recognizing a public determinant from the HLA-A locus. Such replacement led to gaining the determinants recognized by mAbs GS145.2 (anti-B7 + B27) and SFR8-B6 (anti-Bw6); and (4) the HLA-A2-reactive mAbs whose reactivity was known to be abrogated by changes in alpha 2 were unaffected by the changes introduced in alpha 1, underlining the frequent segregation of serologic determinants on class I antigens to single domains.

Toll-like receptors (TLR) are a group of pattern recognition molecules that play a crucial role in innate immunity. TLR2 recognises a variety of microbial components leading to the development of inflammatory and immune responses. To characterise the expression and functional properties of porcine TLR2 (pTLR2), we have raised a panel of monoclonal antibodies (mAb) against this molecule. Mouse 3T3 cell transfectants expressing pTLR2 were used for immunisation of mice. The specificity of these antibodies was confirmed by their reactivity with CHO cells transfected with pTLR2 but not with pTLR4 or with non-transfected cells. Using one of these mAbs, named 1H11, pTLR2 was found on cells of the innate immune system, including monocytes, macrophages, and granulocytes, but not on peripheral blood lymphocytes. Staining of tissue sections showed that pTLR2 is also expressed on epithelial cells lining the tracheobronchial and intestinal tracts, bile ducts in the liver and renal tubules, and on the basal layer of the epidermis. This distribution is consistent with a surveillance function at entry sites, allowing for early detection of microbial invasion. toll-like receptor / monoclonal antibody / monocyte / epithelial cell / pig

This study investigated the potential transmission of porcine endogenous retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.

c-kit (CD117) plays an important role in the early stages of haematopoiesis. Previous studies of porcine haematopoietic stem cells have relied for their identification on the use of the c-kit ligand stem cell factor. Here, we describe a new mAb, 2B8/BM, that recognizes a 155-kDa protein expressed on a small subset (2-8%) of bone marrow haematopoietic cells. 2B8/BM+ cells have a blast appearance, and are mostly negative for lineage-specific markers or express low levels of CD172a or SLA-II. In in vitro colony-forming unit assays these cells were able to give rise to erythroid and myeloid colonies. Altogether these data suggested that the 2B8/BM antigen might be the porcine orthologue of the human c-kit. This specificity was confirmed by the binding of mAb 2B8/BM to CHO cells transfected with a plasmid encoding the porcine c-kit ectodomain. This antibody can facilitate the isolation and enrichment of porcine stem cells to be used in procedures aimed to induce xenograft tolerance or to test their potential to repair damaged tissues and organs. © 2007 Elsevier B.V. All rights reserved.