Protective function of pirfenidone and everolimus on the development of chronic allograft rejection after experimental lung transplantation.

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.

Synergism of imatinib mesylate and everolimus in attenuation of bronchiolitis obliterans after rat 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.

Early administration of FTY720 prevents chronic airway as well as vascular destruction in experimental rat lung transplantation.

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.

Relevance of maintenance triple-drug immunosuppression to bridle the amplification of rat cytomegalovirus infection after experimental lung transplantation.

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.

Lung transplantation in the fischer 344-wistar kyoto strain combination is a relevant experimental model to study the development of bronchiolitis obliterans in the rat.

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.

Minimized extracorporeal circulation in coronary artery bypass surgery is equivalent to standard extracorporeal circulation in patients with reduced left ventricular function.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A prospective cross-over study comparing the pharmacokinetics of cyclosporine A and its metabolites after oral versus short-time intravenous cyclosporine A administration in pre-heart transplant patients.

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-infinity)) 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.

[The STEMMAT-project as part of health initiative BayernAktiv: adult stem cells from umbilical cord and cord blood as alternative to embryonic stem cell research]. / Das STEMMAT-Projekt als Teil der Gesundheitsinitiative BayernAktiv: Adulte Stammzellen aus Nabelschnur und -blut als Alternative zur embryonalen Stammzellforschung.

Adult stem cells from umbilical cord and cord blood are an interesting alternative to embryonic stem cells because such research is commonly recognized as ethical undisputed and many aspects are still insufficiently investigated. In the context of the STEMMAT research project (STEM = Stem Cell and MAT = Material) different aspects of stem cells from umbilical cord and cord blood are investigated, to improve basic science understanding and potentially leading someday to a clinical application.

Predominant inhibition of interleukin-6 synthesis in patient-specific endothelial cells by mTOR inhibitors below a concentration range where cell proliferation is affected and mitotic arrest takes place.

UNLABELLED: 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. MATERIAL AND METHODS: Confluent monolayers of human saphenous vein endothelial cells were pretreated with everolimus or sirolimus followed by induction with tumour necrosis factor-alpha (TNF-alpha). RESULTS: 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-alpha-induced in situ synthesis and release of interleukin-6 per cell by 60%. CONCLUSIONS: 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.

The spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of Erythrina caffra trypsin inhibitor and the recombinant substitution variant recSerETI.

A recombinant substitution mutant (recSerETI) of the Erythrina caffra trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable. Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Five repetitive cycles of guanidinium chloride (GdmCl)-induced denaturation-renaturation yield the native mutant protein with its inhibitory activity fully restored. The only difference between the wildtype and the mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein. The pH range of stability is shifted from pH 1-9.5, for wtETI, to pH 2-9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis. The mid-point of the thermal unfolding transition of the mutant protein is at approximately 65 degrees C, as compared to approximately 75 degrees C for the wildtype protein.

Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization.

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.

Erythrina caffra trypsin inhibitor retains its native structure and function after reducing its disulfide bonds.

Erythrina trypsin inhibitor (ETI) from the seeds of Erythrina caffra is a high-affinity inhibitor of trypsin, chymotrypsin and tissue plasminogen activator. Its 172 amino acid polypeptide chain is stabilized in its compact, native state by two disulfide bonds. In spite of their conservation in all trypsin inhibitors of the soybean trypsin inhibitor (STI-Kunitz) family, their state of oxidation is essential only for protein stability but not for inhibitory function. Reduction/reoxidation of ETI in the presence of glutathione reshuffling buffer (GSH/GSSG; pH 8.3) not only allows the inhibitor to be restored in its native structure, but also does not interfere with its binding affinity; carboxymethylation or carboxamidomethylation of the free thiol groups does not affect K1 significantly (for trypsin (KI)ETIox = 2.3 nM, (KI)ETICM = 1.9 nM; for chymotrypsin (KI)ETIox = 30 microM, (KI)ETICM = 25 microM). The two cystine cross-bridges in the native ETI lead to enhanced stability toward pH and chaotropic agents. As taken from intrinsic protein fluorescence at acid pH and varying ionic strength (pH < 4, I = 0.01 to 0.15 M), the oxidized inhibitor retains its spectral properties, whereas reduced and carboxymethylated or carboxamidomethylated ETI undergo at least partial denaturation. At alkaline pH, the oxidized protein is stable up to pH 9.5, whereas the reduced protein undergoes structural alterations at pH > 7, reaching a final plateau at pH 10.0 to 10.5. In the case of urea (U) or guanidinium chloride (GdmCl) denaturation at pH 7.0, structural transitions of the oxidized inhibitor show "hysteresis" with half-concentrations (cU)1/2 approximately 10 M and (cGdmCl)1/2 approximately 4.5 M for denaturation, and (cU)1/2 = 4.7 M and (cGdmCl)1/2 = 1.5 M for renaturation. In contrast, the reduced (and chemically modified) inhibitors exhibit true equilibrium transitions at (cU)1/2 = 0.9 M and (cGdmCl)1/2 = 0.5 M, respectively. Reduction/reoxidation in the absence and in the presence of denaturants (GdmCl) can also be applied to ETI covalently attached to a solid matrix.

Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

Pressure-induced dissociation of ribosomes and elongation cycle intermediates. Stabilizing conditions and identification of the most sensitive functional state.

Pressure-induced dissociation of ribosomes has been considered a major reason for the inhibition of protein biosynthesis and, hence, bacterial growth at high hydrostatic pressure [Jaenicke, R. (1981) Annu. Rev. Biophys. Bioeng. 10, 1-67]. We reexamined the issue, using a buffer system with polyamines that has been optimized to reproduce in-vivo-like performance of protein biosynthesis in vitro. By slightly modifying this buffer, we were able to find conditions that stabilize functional ribosomal complexes against the dissociating effect of pressure up to 100 MPa and uncharged tight couples up to 60 MPa. Approaching the physiological conditions by reducing the Mg2+ concentration down to 4 mM, one finds a significant destabilization of the post-translocational complex, which represents the most pressure-sensitive intermediate of the elongation cycle and is possibly the limiting factor for the pressure-induced block of protein biosynthesis and bacterial growth.