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Embryonic stem cell (ESC)-based transplantation is considered a promising novel therapy for a variety of diseases. This is bolstered by the suggested immune-privileged properties of ESCs. In this study, we used in vivo bioluminescent imaging (BLI) to non-invasively track the fate of transplanted murine ESCs (mESCs), which are stably transduced with a double fusion reporter gene consisting of firefly luciferase (FLuc) and enhanced green fluorescent protein (eGFP). Following syngeneic intramuscular transplantation of 1 x 10(6) mESCs, the cells survived and differentiated into teratomas. In contrast, allogeneic mESC transplants were infiltrated by a variety of inflammatory cells, leading to rejection within 28 days. Acceleration of rejection was observed when mESCs were allotransplanted following prior sensitization of the host. Finally, we demonstrate that the mESC derivatives were more rapidly rejected compared to undifferentiated mESCs. These data show that mESCs do not retain immune-privileged properties in vivo and are subject to immunological rejection as assessed by novel molecular imaging approaches.

Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

Background: We here describe the pharmacological characteristic, in vivo efficacy, and in vitro mechanisms of a polymer-free leflunomide eluting stent in comparison to its rapamycin-coated equivalent., Methods: Stents were coated with 40 mM solutions of leflunomide (L) or rapamycin (R) or were left uncoated (BM). Neointima formation was assessed 6 weeks after implantation into Sprague Dawley rats by optical coherence tomographies (OCT) and histopathology. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from Sprague Dawley rats to investigate the cell-specific pharmacokinetic effect of leflunomide and rapamycin., Results: HPLC-based drug release kinetics revealed a similar profile with 90% of the drug being released after 12.1+/-0.2 (L) and 13.0+/-0.2 days (R). After 6 weeks, OCTs showed that in-stent luminal obliteration was less for the coated stents (L:12.0+/-9.4%, R:13.3+/-13.1%) when compared to identical bare metal stents (BM:26.4+/-4.7%; p

Recent studies suggest that the mitochondrial aldehyde dehydrogenase (ALDH)2 is involved in vascular bioactivation of nitroglycerin (GTN). However, neither expression of ALDH2 nor its functional role in GTN bioactivation has been reported for the main drug target in humans, namely capacitance vessels. We investigated whether ALDH2 is expressed in human veins and whether inhibition of the enzyme attenuates nitroglycerin effects in these vessels. We determined expression of ALDH2 and dehydrogenase activity in human veins by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescence microscopy. In vitro contraction experiments were performed in the presence or absence of the ALDH inhibitors chloral hydrate, cyanamide, and ethoxycyclopropanol. Concentration response curves were determined for the alpha-agonist phenylephrine, nitroglycerin, and the direct NO donor diethylamine NONOate (DEA-NONOate). ALDH2 expression was largely confined to smooth muscle cells as determined by confocal immunofluorescence microscopy. Contractile responses to phenylephrine were unaffected by all ALDH inhibitors tested. In clear contrast, the ALDH inhibitors significantly reduced the potency of nitroglycerin by approximately 1 order of magnitude (P < or = 0.01). Neither of the inhibitors affected the potency of the direct NO donor DEA-NONOate, which ruled out nonspecific effects on the NO signaling cascade. In human capacitance vessels, ALDH2 is a key enzyme in the biotransformation of the frequently used antianginal drug nitroglycerin.

Background: Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348., Methods: (1) Detailed pharmacokinetic data were obtained in rats; (2) multiple in vitro enzyme inhibition assays were performed to characterize the drug; (3) prevention of acute rejection was investigated in animals treated with different doses of R348 or rapamycin for 5 days; and (4) cardiac allograft survival after a 10-day treatment period was studied for various regimens of R348, tacrolimus, or rapamycin; combination indices were calculated to evaluate drug interactions., Results: (1) Plasma levels of R348's active metabolite R333 sustained high for 8 hr or more, depending on the dose. (2) In vitro enzyme assays showed potent inhibition of JAK3- and Syk-dependent pathways. (3) R348 40 mg/kg preserved graft function, significantly reduced graft infiltration, and decreased histologic ISHLT rejection scores on postoperative day 5. Results were similar to those of rapamycin 3 mg/kg. Likewise, both drugs significantly reduced the cellular Th1 and Th2 immune responses, as determined by enzyme-linked immunosorbent assays. Intragraft inflammatory cytokine upregulation was similarly suppressed by R348 and rapamycin. R348 10 mg/kg was subtherapeutic. (4) Allograft survival was similar for R348 20 and 40 mg/kg, which was comparable with therapeutically dosed tacrolimus or rapamycin. In combination regimens, R348 demonstrated highly beneficial synergistic interactions with tacrolimus., Conclusions: R348 is a promising novel JAK3/Syk-inhibitor with favorable pharmacokinetics and biological activity. It effectively diminishes acute cardiac allograft rejection and is suitable for combination regimens with tacrolimus.

Background: This study aimed at investigating the efficacy of the novel immunosuppressant FK778 to prevent the development and progression of chronic allograft vasculopathy (CAV)., Methods: Orthotopic aortic transplantations were performed in the PVG-to-ACI rat model and followed over the course of 120 days. Immunosuppression with FK778 (20 mg/kg) or sirolimus (2 mg/kg) was either started early or delayed when CAV was already present. Trough levels were monitored. Aortic luminal obliteration was quantified using computer morphometry and intragraft cytokine profiles were analyzed with Western Blotting. Donor-reactive antibodies were quantified by flow cytometry., Results: Untreated animals developed CAV with luminal obliteration of 25.2+/-13.6% and 41.4+/-23.3% after 80 and 120 days, respectively. Continuous immunosuppression with FK778 or sirolimus effectively prevented the development of vasculopathy. When the start of the immunosuppressive regimen was delayed until postoperative day 80, FK778 and sirolimus inhibited a progression of established CAV but did not reverse the luminal obliteration. Intragraft tumor growth factor-beta activity increased over the course of time in untreated recipients but was significantly suppressed after continuous immunosuppression with either agent. Expression of platelet-derived growth factor, intercellular adhesion molecule-1, and vascular adhesion molecule-1 also was moderately suppressed. A stable elevation of donor-reactive IgG-antibody levels was found over 120 days in the absence of treatment. With FK778 or sirolimus, antibody levels were effectively decreased. FK778 was very well tolerated and only sirolimus showed side effects with elevation of BUN, cholesterol, triglycerides, and ALT after 120 days., Conclusions: FK778 prevents the development of CAV and inhibits a progression of established disease. It shows a similar efficacy but a safer drug profile when compared to sirolimus.

We investigated the effect of the bovine hemoglobin based oxygen carrier HBOC-200 (Oxyglobin) in rat aortic endothelial cells (RAEC) on the activation of heme oxygenase 1 (HO-1). RAEC were incubated in the presence of 75 microM (G1) or 225 microM (G2) HBOC-200. The positive control (G3) was performed by incubation with 50 microM Hemin. For negative control (G4) cells were incubated with medium alone. G2 and G3 reached a significant increase of bilirubin concentration compared to G4. A positive HO-1 signal in the Western Blot was seen in G3 12 and 24 h after incubation. The Western Blot of G1, G2 and G4 showed no HO-1 signal. These data suggest that HBOC-200 in the applied dosage cannot induce HO-1 expression in RAEC, and may be degraded by isoenzymes at a lower level.

Atrial septal defects are among the most common congenital anomalies requiring surgical repair. Thrombus formation after patch-based repair is a recognized complication, usually manifested by an embolic event. However, thromboembolic complications after primary repair of atrial septal defects are exceedingly rare. We present a 38-year-old woman found to have a right atrial mass diagnosed as a myxoma by echocardiography and magnetic resonance imaging 3 years after primary atrial septal defect repair. However, final pathology revealed an organized thrombus. A review of the literature and clinical management of postoperative atrial thrombi are discussed.

This study aimed to investigate the pharmacokinetics after tacrolimus aerosol inhalation and to assess its efficacy to suppress acute and chronic airway allograft rejection. Orthotopic tracheal transplantations were performed and tacrolimus (4 mg/kg) was administered orally (PO) or via aerosol (AER). Tracheal tissue level AUCs(0-12) were similar in both treatment groups, but blood AUCs(0-12) were approximately 5.5-fold lower with AER (p < 0.001). Interestingly, only PO animals showed elevated BUN, cholesterol and triglycerides on POD 60 (p < 0.05). Histology of grafts harvested after 6 and 60 days revealed that both treatment groups were similarly effective in suppressing graft mononuclear infiltration (p < 0.001). Cellular immune activation (assessed by IFN-gamma- and IL-4-ELISPOTS), however, was far more effectively suppressed by tacrolimus PO (p < 0.001). In both treatment groups, the vigorous alloreactive IgM-antibody surge was effectively inhibited (p < 0.001). Due to the insufficient systemic cellular immunosuppression, discontinuation of tacrolimus AER resulted in a far stronger (3.5-fold) graft infiltration on POD 8 compared to PO (p < 0.001). Tacrolimus aerosol reduces systemic side effects and effectively protects the airway graft from early cellular rejection and chronic obliterative airway disease.

Background: Different animal models have been developed to study the pathogenesis and treatment of obliterative airway disease (OAD). Here we describe the techniques of heterotopic and orthotopic tracheal transplantations in the rat, comparing the kinetics of systemic host immune response and of histopathologic OAD development., Methods: Heterotopic and orthotopic tracheal transplantations were performed in both allogeneic (Brown Norway-to-Lewis) and syngeneic (Lewis-to-Lewis) models. Grafts were harvested after 7, 30, and 60 days post-transplant for histologic evaluation and analysis of host cellular and humoral response., Results: Syngeneic tracheal grafts did not develop luminal obliteration and were morphologically indistinguishable from native tracheas. In heterotopic allografts, airway epithelium was rapidly destroyed and OAD progressed with complete luminal occlusion by 30 days. Orthotopic allografts showed enhanced early infiltration (1298+/-45 vs. 674+/-75 cells/high power field, p<0.001) with concomitant greater day 7 luminal narrowing (45+/-6% vs. 14+/-3%, p<0.001). In this model, donor-type BN epithelium (62+/-17%, 21+/-19%, and 1+/-1% on days 7, 30, and 60) was gradually replaced by recipient-type epithelial cells (2+/-4%, 70+/-22%, and 98+/-2%). OAD developed with circular orientation of cells and connective tissue fibers to 45+/-6% obliteration by day 60. Cellular host response, as determined by IFN-gamma-ELISPOT assay (548+/-132 vs. 402+/-197 spots, p=0.046) and anti-donor alloreactive IgM antibody production (2827+/-148 vs. 1565+/-393 mean channel fluorescence, p<0.001) were significantly stronger in rats bearing orthotopic vs. heterotopic allografts., Conclusions: The orthotopic tracheal transplantation model may be more representative of OAD found in human lung transplant recipients and we therefore encourage the wider use of this model.

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development., ((c) 2007 Wiley-Liss, Inc.)

Objective: The aim of this study was to assess the efficacy of FK778 to prevent acute and chronic allograft rejection compared with other immunosuppressive agents., Materials and Methods: Heterotopic Brown-Norway (BN)-to-Lewis rat cardiac transplantations and heterotopic BN-to-Lewis tracheal transplantations were performed to study acute heart rejection and the development of chronic obliterative airway disease (OAD), respectively. Recipients were treated with FK778, tacrolimus, MMF, or sirolimus for 10 days (acute rejection study) or 28 days (chronic OAD study) at varying doses., Results: In untreated recipients, cardiac allograft survival was 6.2 +/- 0.4 days. FK778 (20 mg/kg), tacrolimus (2 or 8 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly prolonged graft survival to 17.0 +/- 2.8, 18.5 +/- 2.7, 25.0 +/- 2.5, 20.7 +/- 3.8, 14.5 +/- 2.2, and 23.2 +/- 1.5 days, respectively (P < .05). Tracheal grafts in untreated recipients showed intense infiltration and complete luminal obliteration by day 28. FK778 (20 mg/kg), tacrolimus (1 or 4 mg/kg), MMF (10 or 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly inhibited tracheal luminal obliteration (19.5% +/- 16.4%, 44.2% +/- 33.6%, 12.3% +/- 3.3%, 61.7% +/- 18.6%, 18.3% +/- 11.3%, 55.0% +/- 30.9%, and 8.5% +/- 3.5% (P < .05). All 4 high-dose groups showed similar efficacy., Conclusions: When used in therapeutic doses, tacrolimus and sirolimus were more effective than FK778 to prolong cardiac allograft survival. However, with its antiproliferative effects on smooth muscle cells, its good tolerability, and its blockade of cytomegalovirus replication, FK778 proved effective to prevent chronic OAD development. Thus, FK778 may acquire an important role in maintenance therapy for the prevention of long-term fibroproliferative complications.

Background: Mesenchymal stem cells (MSCs) show differentiation capacity along mesenchymal lineages and have the potential to aid tissue regeneration. MSC transplantation strategies are therefore currently being assessed following injury to various organs. However, potential MSC migration to these organs after intravenous (IV) MSC injection continues to be impeded by cell trapping within the lung., Methods: Mouse MSCs were isolated, purified, transfected with firefly luciferase, and labeled with CSFE. Their size was assessed in vitro. To estimate the diameter of mouse pulmonary capillaries, fluorescence-labeled microspheres of different sizes were injected with or without sodium nitroprusside (SN) pretreatment. The lungs were harvested after 30 seconds and mean numbers of trapped microspheres per high-power field (HPF) were calculated. After IV injection of MSC suspensions (with or without SN), their dynamic distribution was monitored by in vivo luciferine imaging as well as by histopathology., Results: The diameter of suspended MSCs in vitro was 15 to 19 microm. Whereas nearly no 4-microm microspheres could be detected in lung sections, the numbers of trapped 10- and 15-microm microspheres could be significantly decreased by prior SN injection from 19.3 +/- 11.1 to 6.0 +/- 1.6 cells/HPF (P = .004) and from 34.9 +/- 11.9 to 25.6 +/- 8.1 cells/HPF (P = .028), respectively. Within seconds after MSC IV injection, the vast majority of cells was found in the lungs. However, cell trapping in the pulmonary microvasculature was significantly reduced by pre-treatment with SN., Conclusions: We demonstrate that cell trapping in lungs can be reduced with IV SN pretreatment, increasing MSC passage through the lung capillaries, and potentially facilitating cell access to injured organs.

Mesenchymal stem cells (MSCs) are widely used for experimental regenerative strategies. Due to their differentiation capacity into mesenchymal lineages, they are a potential cellular source for tissue regeneration. Because there is no specific antigen that can be used to define MSCs directly, there is no consensus about how to isolate them. Here we describe a simple protocol to isolate, purify, and culture expand murine bone marrow MSCs using magnetic cell sorting and plastic adherence. We further show that cytokine supplementation enhances MSC proliferation without jeopardizing their pluripotency.

The rat heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease (OAD) and to assess immunosuppressive strategies for chronic rejection. Despite its widespread application, the heterotopic transplantation model does have a number of limitations like the lack of air flow and mucociliary clearance. The present article provides a detailed description of the surgical technique for orthotopic tracheal transplantations, which may share more similarities with lung transplants in humans. The technique is easy to learn, the procedure is well tolerated by the animals, and the grafts develop OAD lesions similar to those of human obliterative bronchiolitis., ((c) 2007 Wiley-Liss, Inc.)

Background: This study examines the efficacy of FK778 regimens for prevention of different phases of xenograft rejection., Methods: Antibody and complement tissue depositions were measured by immunofluorescence in a discordant ex vivo rat-to-human heart perfusion model of hyperacute rejection with immunosuppressant-enriched human blood. The concordant hamster-to-rat aortic xenotransplantation model was used to assess host cellular (lymphocyte activation, mixed lymphocyte reaction [MLR]) and humoral responsiveness (xenoantibody production) as well as histologic xenograft rejection. Recipients were treated for 14 days with FK778, tacrolimus, sirolimus or combination regimens at varying doses., Results: Antibody binding during hyperacute rejection was unaffected by the immunosuppressive treatment, but complement deposition was reduced in the following order: tacrolimus > FK778 approximately sirolimus. FK778 most effectively reduced complement factor 5 in vitro. In untreated rats with hamster aortic xenografts, a large infiltrative response was observed within the grafts with extensive myocyte necrosis. Tacrolimus > FK778 approximately sirolimus dose-dependently diminished xenograft infiltration and in the same order reduced vessel-wall myocyte necrosis. Tacrolimus approximately FK778 > sirolimus reduced in vivo lymphocyte CD25 expression and tacrolimus > FK778 approximately sirolimus diminished MLR. Xenoreactive IgM and IgG antibody production levels were vigorously upregulated a few days after transplantation, but were significantly reduced by tacrolimus > FK778 approximately sirolimus. Combination regimens revealed no significant benefit when compared with the corresponding monotherapy groups., Conclusions: FK778 mildly interfered with hyperacute rejection and markedly suppressed acute humoral and cellular aortic xenograft rejection. However, T-cell-dependent host responses were most potently suppressed by tacrolimus, and the overall efficacy of FK778 was similar to that of sirolimus.

Objective: To investigate the efficacy of the malononitrilamide FK778 to prevent vascular smooth muscle cell (SMC) migration/proliferation, and vascular fibrosis, the key events in restenosis development using in vivo and in vitro studies., Background: Since the high rate of restenosis after percutaneous transluminal coronary angioplasty limited its long-term success, the implementation of locally delivered antiproliferative/immunosuppressive agents became advantageous., Methods: Rats underwent balloon denudation of the abdominal aorta and received sirolimus, tacrolimus, or FK778 for 28 days in varying doses. Aortas were harvested for histologic evaluation, profibrotic gene expression, and organ chamber studies. Antifibrotic, antiproliferative and antimigratory effects of the immunosuppressants were further evaluated in vitro., Results: Histology of untreated animals revealed marked intimal hyperplasia with moderate luminal obliteration. Neointima formation was dose-dependently attenuated by all three agents with FK778 and sirolimus being most efficacious. Organ chamber relaxation studies showed a leftward shift of the nitroglycerin and the acetylcholine dose-responses in all treatment groups, indicating diminished endothelial dysfunction. In vivo, only FK778 treatment revealed a significant downregulation of the TGF-beta/vasorin system which could be explained by upregulation of the TGF-beta-inhibitory mediator SMAD7. In vitro, FK778 showed most potent antiproliferative and antimigratory effects on SMC compared with sirolimus and tacrolimus. Only the antiproliferative effect of FK778 was due to pyrimidine synthesis blockade and could be reversed by uridine supplementation., Conclusions: The malononitrilamide FK778 proved highly efficacious against restenosis development by targeting two major components of intimal hyperplasia: SMC proliferation/migration and vascular fibrosis. Thus, the introduction of malononitrilamide-loaded stents may be a promising effort for future strategies., (2007 S. Karger AG, Basel)

Background: Bronchiolitis obliterans is the most significant complication adversely affecting the survival of lung allograft recipients. Injury and loss of epithelium are associated with obliteration of the airway lumen. The aim of this study was to examine the effects of various immunosuppressants on airway epithelium., Methods: Tracheae from Brown Norway donors were heterotopically transplanted into the greater omentum of Lewis (allografts) or Brown Norway (isografts) animals. Recipients were treated for 28 days with FK778 (20 mg/kg), tacrolimus (4 mg/kg), or sirolimus (2 mg/kg). Tracheal segments were evaluated for the degree of luminal occlusion as well as the type and percent of luminal epithelial cell coverage., Results: All agents inhibited peritracheal infiltration and luminal obliteration. Tacrolimus- more than sirolimus-treated recipients showed partial preservation of the luminal epithelial coverage, whereas animals that received FK778 showed no respiratory epithelium. The epithelial loss was accompanied by the appearance of fibrous tissue, which replaced the mucosa., Conclusions: Tacrolimus as well as sirolimus effectively prevented the development of obliterative airway disease whereas tacrolimus and, to a lesser degree, sirolimus preserved epithelial cells as a source of protective cytokines. With FK778 significant airway obliteration was suppressed despite complete epithelial loss. Thus, FK778-treated animals displayed an epithelial-independent inhibitory effect on myofibroblast proliferation.

This study examined the efficacies of sirolimus and the novel immunosuppressive agent FK778 to prevent obliterative airway disease (OAD). Tracheae from Brown-Norway donors were heterotopically transplanted in the greater omentum of Lewis rats. Recipients were treated for 28 days with sirolimus (0.5 or 2 mg/kg), FK778 (5 or 20 mg/kg), or combination regimens (0.5 + 5 mg/kg, 2 + 20 mg/kg). Tracheal segments were evaluated for degree of luminal obliteration, percentage of luminal epithelial cell coverage, and peritracheal infiltration. In vitro smooth muscle cell (SMC) proliferation and migration assays were performed to assess direct nonimmune-related effects of the drugs. Sirolimus 2 mg/kg and FK778 20 mg/kg effectively reduced graft infiltration and prevented airway obliteration, whereas FK778 5 mg/kg was insufficient. Sirolimus 0.5 mg/kg at least showed moderate inhibitory effects on luminal obliteration and graft infiltration. Combination regimens revealed no significant beneficial effects. Both sirolimus and FK778 barely showed preserved epithelial coverage. Within the range of relevant concentrations, FK778 showed more potent anti-proliferative and anti-migratory effects on SMC in vitro than sirolimus. Both agents proved effective to prevent OAD development without preserving relevant amounts of epithelium. The anti-proliferative potency on SMCs seems to be an especially important mechanism for FK778. De novo combination regimens revealed no beneficial interaction and thus remain doubtful.

Purpose: The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection., Methods: Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-alpha was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade., Results: TNF-alpha stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation., Conclusions: FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.

Background: The effectiveness of the novel immunosuppressive agent FK778 and of tacrolimus to prevent the development of obliterative airway disease (OAD) was investigated in an animal model., Methods: Tracheae from Brown-Norway donors were heterotopically transplanted in the greater omentum of Lewis rats. Recipients were treated for 28 days with FK778 (5 or 20 mg/kg), tacrolimus (1 or 4 mg/kg) or combination regimens at varying doses (5 + 1 mg/kg, 10 + 2 mg/kg or 20 + 4 mg/kg). Grafts were harvested and processed for histologic and immunohistochemical evaluation. Lymphocyte surface antigen expression was quantified and in vitro smooth muscle cell (SMC) proliferation assays were performed., Results: In untreated recipients, very large amounts of infiltrating CD4+, CD8+ and ED1+ mononuclear cells were observed in the peritracheal region with epithelial loss and complete luminal obliteration. Granulation tissue consisted of alpha-actin-positive cells and collagen-rich fibrosis. FK778 and tacrolimus as well as combination regimens of both agents dose-dependently inhibited peritracheal infiltration and luminal obliteration. Only tacrolimus-treated recipients showed preserved luminal epithelial coverage with airway goblet cells, whereas, in animals that received FK778, no epithelium was found. Both agents equally suppressed in vivo lymphocyte CD25 expression. Only FK778-treated animals were completely free of adverse drug side effects. FK778 but not tacrolimus showed potent anti-proliferative effects on SMC in vitro., Conclusions: Although both agents proved effective to prevent OAD development, histology revealed major differences. The anti-proliferative potency of FK778 on SMC may be an important mechanism of action. Combination regimens showed favorable drug interaction and allowed dose reduction of both agents to achieve maximal immunosuppressive efficacy.

Background: Since the two estrogen receptor isoforms ERalpha and ERbeta have been discovered it is unclear by which receptor immunomodulating or feminizing effects are mediated. In this study, the effects of the two selective ERalpha- and ERbeta-agonists ethinylestradiol and biochaninA, respectively, on acute cardiac allograft rejection, uterus growth, vascular adhesion molecule and MHC-II expression were investigated and verified using in vitro cell culture., Methods: Heterotopic Lewis to ovarectomized F344 cardiac transplantations were performed. The study groups received supplemental biochaninA or ethinylestradiol, the control group received no treatment. Grafts and uteri were harvested on the fifth postoperative day and blood was taken for hormone plasma level quantifications. Purified Lewis aortic endothelial cell cultures were pre-treated with biochaninA or ethinylestradiol and stimulated with TNF-alpha or IFN-gamma for quantification of ICAM-1/VCAM-1 and MHC-II expression. Endothelium-lymphocyte adhesion assays were performed using purified F344 lymphocytes., Results: Both biochaninA and ethinylestradiol treatment significantly reduced graft mononuclear infiltration of CD8(+) and ED1(+) cells and markedly reduced ISHLT grading compared to untreated controls. Either agent significantly inhibited lymphocyte adhesion, endothelial VCAM-1 upregulation during graft rejection and during TNF-alpha-stimulation in vitro, whereas no effect was observed for ICAM-1 upregulation. BiochaninA but not ethinylestradiol significantly reduced endothelial MHC-II upregulation in vivo and in vitro. Only ethinylestradiol treatment strongly affected uterus growth in ovarectomized recipients., Conclusions: Only the treatment with the phytoestrogen biochaninA reduced endothelial MHC-II expression in vivo and in vitro and weakened allograft rejection without affecting the reproductive system. Supplemental phytoestrogens may therefore provide further benefits in the clinical setting.

The adhesion molecules, P-selectin, ICAM-1, and VCAM-1 are important mediators of T-cell adhesion and T-cell co-stimulation. We investigated the effect of the malononitrilamide FK778 on cardiac allograft survival, acute allograft rejection, and adhesion molecule up-regulation in a heterotopic, cardiac transplantation model. Rats received low- or high-dose FK778 or no treatment. Grafts were harvested on the fifth postoperative day for histologic examinations. To assess allograft survival, recipients were treated for a maximum of 10 days and grafts were harvested after cessation of the contractile activity. FK778 low dose showed a mild but significant decrease in mononuclear infiltration but failed to markedly reduce histologic rejection, adhesion molecule up-regulation, or to prolong allograft survival. However, high-dose FK778 treatment significantly reduced early up-regulation of P-selectin, ICAM-1, and VCAM-1, abolished infiltration, reduced histologic rejection and resulted in prolonged cardiac allograft survival. Therefore, FK778 is a novel, highly desirable immunosuppressive drug for transplantation medicine.

Background: The malononitrilamide FK778 is a novel derivate of leflunomide and interacts with T- and B-cell function by inhibiting de novo pyrimidine synthesis. We investigated the effects of FK778 upon acute cardiac allograft rejection and upon adhesion molecule upregulation in experimental transplantation and by using in vitro cell culture., Methods: Heterotopic, abdominal cardiac transplantations were performed in the Brown Norway (BN) to Lewis (Lew) rat model. The study groups received daily low- or high-dose FK778 immunosuppression. FK778 plasma levels were quantified by HPLC. Grafts were harvested on the fifth postoperative day for histologic and immunohistologic examinations using computerized morphometry. Purified BN aortic endothelial cell cultures were pretreated with low- or high-dose FK778 according to FK778 plasma levels and were stimulated with tumor necrosis factor (TNF)-alpha. Adhesion molecule expression was quantified by immunofluorescence, FACS analysis, and Western blotting. Lymphocyte-endothelium adhesion assays were performed using purified Lew lymphocytes and radiolabeled TNF-alpha was used for receptor binding assays., Results: FK778 treatment dose-dependently reduced graft mononuclear infiltration of CD4(+), CD8(+), and ED1(+) cells, but only high-dose FK778 treatment significantly reduced early upregulation of ICAM-1 and VCAM-1 in vivo. FK778 also dose-dependently reduced TNF-alpha-stimulated endothelial adhesion molecule upregulation in vitro, whereas the effect on VCAM-1 was more dominant. We did not find evidence that FK778 interferes with surface receptor binding of TNF-alpha. Lymphocyte adhesion to endothelial cell monolayers was significantly attenuated by FK778., Conclusion: Besides its inhibitory effect on pyrimidine synthesis, FK778 directly reduces endothelial adhesion molecule upregulation and attenuates lymphocyte-endothelium interaction, which is a critical step in graft rejection.

Objective: The synthetic malononitrilamide FK778 inhibits T- and B-cell responsiveness, phagocyte effector function, exerts inhibitory activity against cytomegalovirus, and is thus one of the most promising new immunosuppressive drugs. The aim of this study was to evaluate the combination of FK778 and tacrolimus in a high-responder rat cardiac transplantation model., Methods: The Brown Norway-Lewis rat strain combination was used to investigate graft survival after 10 days of posttransplant oral therapy with FK778 (5 or 20 mg/kg), tacrolimus (2 or 8 mg/kg), or combination regimens at varying doses (5+2 mg/kg, 10+1 mg/kg, or 20+8 mg/kg). Grafts were harvested after cessation of cardiac contractions. Combination indices (CI) were calculated for drug combinations., Results: In untreated recipients, allograft survival was 6.2+/-0.4 days. FK778 at 20 mg/kg and tacrolimus at 2 or 8 mg/kg significantly prolonged graft survival to a mean survival time (MST) of 17.0+/-2.8, 18.5+/-2.7, and 25.0+/-2.5 days, respectively. The two low-dose drug combinations achieved a graft survival of 23.2+/-2.9 and 25.2+/-3.1 days, which was significantly longer compared with FK778 at 5 mg/kg, FK778 at 20 mg/kg, and tacrolimus at 2 mg/kg (P

FK778 blocks the dihydro-orotate dehydrogenase, necessary for pyrimidine synthesis, and mycophenolate mofetil (MMF) inhibits the inosine monophosphate dehydrogenase, a crucial enzyme for purine biosynthesis. Beneficial immunosuppressive effects have been suggested for the combination of both drugs. The Brown Norway-Lewis rat heterotopic heart transplantation model was used. FK778 (5 and 20 mg/kg/day), MMF (10 and 40 mg/kg/day), or a combination of both drugs for 10 days was used for prevention of acute graft rejection. Grafts of untreated animals were rejected after 6.2 +/- 0.4 days. Low-dose FK778 and low-dose MMF administration did not result in a significantly prolonged graft survival (6.7 +/- 0.8 and 8.7 +/- 1.4 days; P=not significant). Grafts of rats treated with high-dose FK778 or high-dose MMF survived significantly longer (17.0 +/- 2.8 and 20.7 +/- 3.8 days; P<0.01). Concomitant use of low-dose FK778 with low-dose MMF produced synergistic interactions (mean survival time 12.3 +/- 2.9 days; P<0.01; combination index=0.85). High-dose drug combination (mean survival time 24.0 +/- 1.4 days) showed antagonistic drug interaction (combination index=1.55) with increased toxic side effects.

We report the case of a 31-year-old woman with a 4-year history of recurrent palpitations, presenting with an increased frequency of paroxysms caused by ventricular tachycardias during pregnancy. A cardiac tumor of unknown origin infiltrating the right ventricle was diagnosed. Three weeks after prophylactic abrasion the tumor was totally excised with the use of cardiopulmonary bypass including restoration of the right ventricular wall and the tricuspid valve. Histology confirmed diagnosis of a benign cardiac lipoma. The postoperative course was uneventful and the patient was discharged 7 days after surgery. There was no episode of ventricular tachycardias during the 6-month follow-up.

In cardiac xenotransplantation, transfections with recombinant adeno-associated viruses could be a promising way of modulating the cardiomyocyte genome. Proteins like TRAIL, hDAF, Il-10, Il-4, and beta-galactosidase may be expressed on cardiomyocyte surfaces in order to prevent cellular rejection processes. However, after intracoronary perfusion with transfecting viruses, it takes up to 4 weeks before expression of the transgene can be detected. Mouse models have been established to create reliable transfection protocols. Currently, the major problem involves performing the intraaortic injection in mice without causing a lethal pneumothorax. Here we describe a new technique for chest opening to safely reach the mouse ascending aorta without opening the pleural space. This U-sternotomy reduces the risk of animal death and therefore the amount of quite expensive virus solutions needed., (Copyright 2003 Wiley-Liss, Inc.)

Objective: Skeletal myoblasts have been proven to survive transplantation into myocardial scar tissue. The objective of this study was to evaluate whether these cells can also be transferred into vital myocardium and maintain their ability for cell division after transplantation. In addition, an intravital fluorescence dye for marking these cells was evaluated., Material and Methods: Skeletal myoblasts were harvested from male Lewis rats (n = 6) and then expanded in culture. Before implantation, these cells were trypsinized and labeled using an intravital fluorescence dye (PKH-26). Syngenic myoblast transfer to recipient female Lewis rats (n = 36) was used to simulate autologous transplantation. Under general anesthesia, the rats received injections of 106 myoblasts via a subxyphoidal approach into the apex of the heart. The animals were then divided into 3 groups (n = 10 each). The animals were sacrificed at several time points, and the hearts were harvested for histologic examination: group A, 7 days postoperatively; group B, 14 days postoperatively; and group C, 28 days postoperatively. An additional group, group D (n = 6), served as a control group; these animals were injected with only cell medium. Corresponding to the study groups, 2 animals of this control group were sacrificed at each time point, and the hearts were explanted. At histological examination, 8- m sections were investigated to identify surviving stained cells. For further evaluation, the sections were stained using monoclonal antibodies against n-cam, desmin, and a- actin., Results: No fluorescing cells were found in any hearts of rats in the control group. Surviving fluorescing myoblasts were found in 9 of 10 hearts of groups A and C and 8 of 10 hearts of group B. Labeled myoblasts were located in the intercellular spaces between the myocardial fibers. Fibrotic or inflammatory reactions could not be identified around the injection site in any hearts of the study groups. Immunohistochemical staining results showed that the labeled cells expressed n-cam, desmin, and a-actin. The myoblasts had regained their physiologic structures and had started to form myofibers. In groups B and C, more n-cam-positive cells than labeled cells were found, indicating further cell division., Conclusions: Intravital fluorescence staining with PKH- 26 dye proved to be an easy and reliable method for identifying cells after cellular transplantation. Myoblasts survived an intracardiac transfer, regaining their physiologic structures and maintaining their ability for further cell division.

Event-related potentials (ERPs) following infrequent and frequent stimuli were studied as subjects moved from wakefulness to sleep. Subjects were instructed to respond to the infrequent "target" stimuli (attend condition) or to ignore the stimuli (ignore condition). Parietal P300, prominent following target ERPs in wakefulness under the attend condition, disappeared in association with reduced behavioral responsiveness and emergence of a central negativity (N350). The N350 and preceding and following positivities (P220 and P450) became the dominant feature of both target and nontarget ERPs under both attend and ignore conditions. The P220-N350-P450 complex was larger and peak latencies were shorter under the attend condition. Peak amplitudes tended to be larger following targets, especially under the attend condition. The findings suggest that, although the processes underlying P300 are less likely to be engaged, processing of stimulus deviance and task relevance continues in sleepiness and sleep, and is reflected by variance in N350 and related activity.