Long-term survival of lung allografts is limited by chronic rejection (CR). Oxidative stress (OxS) plays a central role in the development of CR. We investigated the influence of pirfenidone (alone ...or in combination with everolimus) on OxS and CR. A rat model of left lung allo-transplantation (F344-to-WKY) was used to evaluate the effects of pirfenidone alone 0,85% in chow from postoperative day (POD) -3 to 20/60 and in combination with everolimus 2,5 mg/kg bw daily from POD 7 to 20/60. Allografts of non-treated animals, everolimus treated animals and right, non-transplanted lungs were used as references. Immunohistology of myeloperoxidase (MPO), haemoxygenase-1 (HO-1), iron and platelet-derived-growth-factor-receptor-alpha (PDGFR-a) were performed. On POD 20, all groups showed severe acute rejection (ISHLT A3-4/B1R-B2R). Groups treated with pirfenidone showed a lower interstitial inflammatory infiltration and a lower participation of highly fibrotic degenerated vessels (ISHLT-D2R). In the long term follow up (POD 60), pirfenidone alone significantly reduced chronic airway rejection (ISHLT-C; p≤0.05), interstitial fibrosis (IF; p≤0.05), content of collagen (p≤0.05), expression of PDGFR-a (p≤0.05) and the deposition of iron (p≤0.05). All groups treated with pirfenidone showed a high expression of the cytoprotective enzyme HO-1 (p≤0.05). The additional application of everolimus resulted in a significant decrease of chronic airway rejection (ISHLT-C; p≤0.05), vasculopathy (ISHLT; p≤0.05) and IF (p≤0.05). In conclusion, early application of pirfenidone inhibited the progression of CR by its anti-fibrotic and anti-oxidative properties. The additional application of an m-TOR-inhibitor increased the anti-fibrotic effects of pirfenidone which resulted in a reduction of CR after experimental LTx.
Bronchiolitis obliterans (BO) is a progressive and fatal disease after lung transplantation (LTX). Dysregulated growth factor-induced proliferation of myofibroblasts seems to be responsible for the ...development of BO. The aim was to confirm the efficacy of both inhibitors of receptor tyrosine kinases (RTKI) and of mammalian target of rapamycin (mTORI) after rat LTX. We used a rat model of left lung allo-transplantation (F344-to-WKY) to evaluate the effect of imatinib (RTKI; 20 mg/kg/day; postoperative day (POD) 0-100) alone or in combination with everolimus (mTORI; 2.5 mg/kg/day; POD 14-100). Non-treated animals were the reference. In non-treated rats, acute rejection (AR) peaked between POD 20 and 30 (19/19) and ended in chronic rejection (CR) on POD 60/100 (12/12). Imatinib alone did not prevent AR (6/6), but attenuated the degree of degenerated bronchioles on POD 30 (non-treated, 57%; imatinib, 4%), and increased the allografts free of CR on POD 60/100 (3/12). A combination of imatinib and everolimus significantly reduced AR, attenuated fibrotic degenerated bronchioles (5%) and vessels (non-treated, 24%; combination therapy, 11%) on POD 30, and reduced fibrotic degenerated vessels (non-treated, 97%; combination therapy, 43%) and bronchioles (non-treated, 88%; combination therapy, 34%) on POD 60/100. Fifty percent of the animals were completely free of BO and vasculopathy. In conclusion, co-application of RTKI and mTORI attenuated the development of BO and vasculopathy. Thus, imatinib might be an interesting therapeutic approach after LTX.
Abstract Background Chronic rejection (CR) in terms of bronchiolitis obliterans (BO) and vascular sclerosis (VS) still represents the major obstacle for pulmonary graft survival in the medium and ...long term course after lung transplantation (LTX). Aside from nonspecific stimuli, early acute rejection (AR) seems to be causative especially in cases of a late diagnosis or inadequate treatment. This study investigated the effects of FTY720, a new immunosuppressant that promotes lymphocyte sequestration into lymph nodes and Peyer's patches, on the development of CR after experimental LTX. Methods A total of 50 rats underwent allogenic (F344-to-WKY) and syngenic (WKY-to-WKY) left LTX. Group 1 animals had no treatment. Group 2 animals were administered FTY720 (3 mg/kg body weight per day) at the maximum time of AR (day 14) and continued up to day 100 after LTX. Group 3 animals were treated with the same dosage of FTY720 from day 0 to 100. The grades of AR and CR were classified according to the criteria of the International Society for Heart and Lung Transplantation. Results Within 14 days after allogenic LTX, all nontreated rats developed early AR followed by severe CR with VS and BO. Similar data were observed for FTY720 treatment of existing AR (group 2). Only early administration of FTY720 (at the time of LTX) significantly reduced the proportion of animals with severe acute vascular rejection ( P < .001). However, all of these allografts showed high-grade acute airway inflammation. After long-term application, the chronic inflammatory response was absent; none of the allografts developed BO and VS. Conclusion Only application of FTY720 immediately after LTX prevented lymphocyte recirculation and lung injury.
ABSTRACT
Chronic allograft rejection and bronchiolitis obliterans (BO) limited successful long-term outcome after lung transplantation (LTX). Reliable animal models are needed to study the ...pathogenesis of BO and to develop effective therapeutic strategies. The relevance of an available experimental LTX model without immunosuppression-the Fischer (F344)-Wistar Kyoto (WKY) rat strain combination-was analyzed. The kinetics of acute and chronic rejection and respective graft histopathology were described in the F344-to-WKY rat strain combination after allogeneic LTX. A modified classification of chronic lung allograft rejection was introduced to describe obliterative changes in the airways after rat LTX. Animals from Harlan Winkelmann (HW) and Charles River (CR) were examined. Within 14 days after LTX, allografts showed moderate to severe acute vascular and bronchiolar inflammation. In contrast to rats from CR, animals from HW showed a delayed acute rejection process. Mid-term phase after LTX (days 20-40) presented a "borderline form" consisting of both acute and first signs of chronic airway rejection. On postoperative day (POD) 60, first signs of airway remodelling and distinct BO were diagnosed in 80% of animals from HW. At the same time, the allografts with BO-like lesions increased up to 100% in rats from CR. The F344-to-WKY rat LTX model allows detailed assessment of all features of acute and chronic pulmonary rejection representing a clinically relevant model. However, due to breeding differences resulting in various sublines of the same rat strain, the source and husbandary history of the animals is important for analysis of immuno-mediated processes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Immunosuppressive therapy required to treat rejection after lung transplantation (LTx) contributes significantly to the pathogenesis of cytomegalovirus (CMV) infection and disease. In a weak ...allogeneic left LTx model in the rat (Fisher 344 F344 to Wistar Kyoto WKY rats) we analyzed the influence of acute CMV infection on postoperative day (POD) 3, with application of standard triple‐drug immunosuppression (TD‐IS) (cyclosporin A, azathioprine, prednisolone) on late outcome after LTx. Native right lungs and syngeneic grafts (WKY to WKY) served as controls. Rats were sacrificed on POD 15, 30, 60, and 100. TD‐IS completely prevented acute and chronic rejection in non‐infected rats. Allografts of CMV‐infected rats treated with TD‐IS showed only mild perivascular infiltrations in 6/10 rats (POD 15 and 30), which persisted up to POD 100 in 4/10 rats. In the long‐term course, mild isolated interstitial and alveolar changes were found in 40% of these animals. In conclusion, rat CMV infection partially neutralized the immunosuppressive effect of TD‐IS. However, an amplification of CMV infection under TD‐IS can be controlled and does not result in fatal outcome.
Minimized extracorporeal circulation (MECC) is a promising alternative to standard extracorporeal circulation (ECC) and its use is increasing in routine coronary bypass surgery. We analyzed the ...clinical outcome of patients with reduced left ventricular function who underwent coronary artery bypass surgery with MECC or with standard ECC.
From January 2003 to September 2008, 238 patients with a left ventricular function < 30 % underwent bypass surgery with ECC or MECC. The primary end point of our retrospective observational study was 30-day mortality. Secondary endpoints were the transfusion requirements, as well as intensive care and the in-hospital course.
Demographic data, comorbidities and left ventricular function were similar in the study groups. MECC patients had a tendency towards a lower 30-day mortality rate, a better postoperative renal function and reduced ventilation times. Extracorporeal circulation time and postoperative high-dose inotropic support were significantly lower in the MECC group, while the stays in the intensive care unit and in hospital were comparable between the two groups. In our study, age in the ECC group, and previous infarction and New York Heart Association grade IV in the MECC group were preoperative risk factors associated with a higher mortality.
Coronary bypass surgery using MECC is feasible and safe for patients with severely impaired left ventricular function. It is a promising alternative to ECC with a low mortality rate and a more favorable postoperative course.
Organ rejection and inflammation are accompanied by endothelial cell activation. An in vitro model with patient-specific endothelial cells was used to study the impact of mTOR inhibitors on cell ...growth and release of proinflammatory cytokines.
Confluent monolayers of human saphenous vein endothelial cells were pretreated with everolimus or sirolimus followed by induction with tumour necrosis factor-α (TNF-α).
Incubation with sirolimus or everolimus resulted in a dose-dependent deceleration of cell growth. Compared to control, cell count at high concentrations ceased to increase and remained at 60%. This mitotic arrest was accompanied by a dose-dependent inhibition of the TNF-α–induced in situ synthesis and release of interleukin-6 per cell by 60%.
Under conditions mimicking cytokine-induced cell activation a predominant inhibitory effect of everolimus compared to sirolimus on endothelial cell proliferation was observed paralleled by an inhibition of proinflammatory cytokines. This might attenuate the acute proinflammatory status after transplantation.
1 Institute of Pathology and 2 Clinic of Cardiothoracic Surgery, University of Regensburg, Regensburg, Germany; and 3 BioMedES, Aberdeen, United Kingdom
Submitted 25 May 2005
; accepted in final form ...30 November 2005
The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 µm were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for >8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs.
endothelium; angiogenesis; human umbilical vein endothelial cell; multicellular spheroid; coculture; tubular structures
Address for reprint requests and other correspondence: L. A. Kunz-Schughart, Institute of Pathology, Univ. of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany (e-mail: leoni.kunz-schughart{at}klinik.uni-regensburg.de )
Abstract Sometimes intravenous administration of cyclosporine (CsA) is essential before oral administration is possible. There are only a few reports available on the interindividual variability of ...CsA metabolism and different metabolite pattern depending on intravenous versus oral administration of CsA in heart transplant (HTx) patients. For effective inhibition of calcineurin we used a short infusion reaching peak concentrations after 2 hours. In a prospective cross-over study we compared the pharmacokinetics of CsA and its metabolites after oral (2.0 mg/kg body weight) versus intravenous (0.7 mg/kg body weight; 2-hour infusion) CsA administration (single test dose) in 7 pre-HTx patients. The pharmacokinetic parameters of CsA and its metabolites were analyzed using high-pressure liquid chromatography. The pharmacokinetic parameter area under the concentration time curve (AUC(0-∞) ) of CsA after intravenous administration was significantly lower (2903 ng*h*mL−1 ) than that after oral administration (4344 ng*h*mL−1 ; P = .01). Peak concentrations, time to peak concentration, and terminal elimination half life were not significantly different. Short-time infusion of CsA resulted in a significant decrease in the AUC of the metabolites AM1 (3-fold), AM9 (10-fold), and AM1c (3-fold). A 2-hour infusion of CsA is just as effective as oral administration and the reduced amount of metabolites is advantageous for the patient.
Oxygenator thrombosis is a serious complication in extracorporeal membrane oxygenation (ECMO) and may necessitate a system exchange. Coagulation and fibrinolysis parameters, flow dynamics, and gas ...transfer performance are currently used to evaluate the degree of oxygenator thrombosis, but there is no technical approach for direct visualization and quantification of thrombotic deposits within the membrane oxygenator (MO). We used multidetector computed tomography (MDCT) with three-dimensional postprocessing to assess the incidence of oxygenator thrombosis, to quantify thrombus extent, and to localize clot distribution. Twenty heparin-coated MOs after successful weaning were analyzed. Mean ECMO support time was 7 ± 4 days, mean activated partial thromboplastin time (aPTT) during ECMO was 59 ± 20 seconds. Thrombotic deposits were detected in all MOs. The mean clot volume was 51.7 ± 22.3 cm. All thrombotic deposits were located in the venous, i.e., inlet part of the device, without apparent evidence of embolization in patients. There was no correlation between clot volume and ECMO support time or aPTT. Clot formation within the MO is a common finding in ECMO despite adequate systemic anticoagulation. The clinical significance of thrombus formation and its influence on gas exchange capacity and hemostatic complications have to be addressed in further studies.
