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.

Interhospital transportation of patients with severe lung failure on pumpless extracorporeal lung assist.

BACKGROUND: To describe the use of pumpless extracorporeal interventional lung assist (iLA) for transportation of patients with severe life-threatening acute lung failure from tertiary hospitals to a specialized centre. METHODS: Retrospective analysis in eight patients with severe lung failure requiring interhospital transport, in whom implementation of an iLA system at a tertiary hospital for air/ground transportation was performed. RESULTS: After implementation of iLA, a rapid increase in CO2-elimination (Pa(CO2) before iLA: 8.92+/-2.9 kPa, immediately after implementation: 5.06+/-0.93 kPa, 24 h after implementation: 4.53+/-1.20 kPa [mean+/-SD], P<0.05) was observed and a significant improvement in oxygenation (Pa(O2) before iLA: 6.66+/-2.26 kPa, immediately after implementation: 10.39+/-3.33 kPa, 24 h after implementation: 10.25+/-5.46 kPa, P<0.05) was noted. During transport, no severe complications occurred. Four patients died during further treatment due to multiple trauma or multiple organ failure. CONCLUSIONS: Due to ease of handling, high effectiveness and relatively low costs, iLA seems to be a useful system for treatment and transportation of patients with severe acute lung injury or ARDS suffering from life-threatening hypoxia and/or hypercapnia.

Mitral prolapse caused by lymphangioma.

A 62-year-old female patient with known mitral-valve prolapse for the previous five years presented with progressive dyspnea and intermittent palpitations. This clinical presentation was investigated by two-dimensional echocardiography which revealed moderate mitral regurgitation due to a pedunculated mass oscillating between the left atrium and the left ventricle. Successful operative treatment consisted of en-bloc resection of the tumor from the anterior mitral valve leaflet and its primary cords and subsequent reconstruction of the mitral valve. Pathohistological examination revealed a cystic lymphangioma originating from mitral-valve tissue. To our knowledge this is the first reported case in the literature.

[Pumpless extracorporeal lung assist using arterio-venous shunt in severe ARDS. Experience with 30 cases]. / Pumpenfreie extrakorporale Lungenunterstützung mit arteriovenösem Shunt beim schweren akuten Lungenversagen des Erwachsenen. Bericht über 30 Einsätze.

BACKGROUND: Extracorporeal lung assist has been proposed as an invasive measure in patients with acute respiratory distress syndrome (ARDS) when oxygenation is critically impaired. However, this technique generally requires high personnel and technical resources. We report on a new system, which is characterised by a short circuit arterio-venous shunt using arterio-venous pressure gradient as driving force (pumpless extracorporeal lung assist [pECLA]). PATIENTS AND METHODS: In 30 patients with ARDS due to multitrauma, pneumonia or after surgery (p(a)O(2)/F(I)O(2)-ratio 67+/-23 mmHg) pECLA was established by insertion of cannulae to the femoral artery and vein followed by connection with a membrane gas exchanger. For this system, only "low dose" continuous heparin infusion is required. RESULTS: Arterial oxygenation was acutely and significantly increased by pECLA (p(a)O(2)/F(I)O(2)=103+/-56 mmHg 2 h after begin) and carbon dioxide removal was markedly enhanced in 25 out of 30 patients (87%) allowing a lung protective ventilation strategy. The mean duration of pECLA therapy was 6.5 days, 15 patients (50%) died due to ARDS or non-ARDS related reasons. CONCLUSION: pECLA represents a feasable and effective treatment in patients with severe ARDS. Compared with pump-driven systems pECLA is characterised by low costs and reduced personnel requirements. However, mortality remains high in patients suffering from severe ARDS despite newer treatment modalities.

Adequacy of perfusion during hypothermia: regional distribution of cardiopulmonary bypass flow, mixed venous and regional venous oxygen saturation - hypothermia and distribution of flow and oxygen.

BACKGROUND: Hypothermia during CPB is used to reduce metabolic activity, thus protecting organs and tissues. The aim of this study was to investigate the relationship between regional and mixed venous oxygen saturation and distribution of pump flow with respect to hypothermia. METHODS: Twenty-five patients undergoing a Ross procedure were included in a prospective, controlled study. During standard CPB, temperature was reduced stepwise to 28 degrees C. Blood gases (a-stat regimen) were analysed in samples from the inferior (IVC) and the superior vena cava (SVC), arterial and mixed venous blood. Flow was detected separately in the SVC, IVC, arterial, and collecting venous line. Samples were taken, and flows were measured before CPB, during hypothermia, during rewarming, and 30 min after CPB discontinuation. RESULTS: Oxygen saturation in the IVC was lower than in the SVC and in mixed venous blood at all times (max. difference - 17.3 +/- 3.0 % during hypothermia, - 23.8 +/- 2.9 % during rewarming, p < 0.01). There was a statistical correlation of mixed and IVC venous oxygen saturation (r = 0.79, p < 0.001) but not of SVC venous blood. Hypothermia had a major influence on pump flow distribution as backflow from the SVC decreased significantly in favour of IVC flow with increasing degree of hypothermia (increase of flow difference from 1.15 +/- 0.23 l/min to 1.49 +/- 0.36 l/min, p < 0.01). Temperature profiles were similar when detected in aorta, pulmonary artery, tympanum and nasopharygeum, but differed significantly from other sites. CONCLUSIONS: During hypothermic CPB, regional deoxygenation occurs in spite of normal mixed venous saturation. The level of hypothermia has a major impact on bypass flow distribution with cerebral perfusion reduction. Methods of regional oxygenation assessment are needed, and altered strategies during hypothermia have to be taken into consideration.

The Fontan-operation: from intra- to extracardiac procedure.

PURPOSE: For treatment of univentricular heart, the Fontan operation has been established as the definitive palliation. The current controversy is mainly based on the high incidence of arrhythmias after an intra-atrial lateral tunnel Fontan operation. METHODS: From January 1995 until April 2002, 46 children underwent a Fontan-type operation with or without a small fenestration. In 33 patients (group I) an intracardiac tunnel and in 13 patients (group II) an extracardiac conduit procedure was performed. PRINCIPAL FINDINGS: There was no perioperative mortality. All patients showed postoperative a significant increase of arterial oxygen saturation, from 76 to 86% after surgery with fenestration, or to 90.5% without fenestration respectively. In patients with fenestration procedure, the saturation rose to 90% after closure of fenestrations 9 to 12 months after operation. CONCLUSIONS: Modified Fontan operations can be performed in normothermia on the beating heart with acceptable mortality. The extracardiac conduit Fontan procedure has the benefits of less surgical injury and a higher intraoperative flexibility.

[Extracorporeal lung support and endovascular stent in traumatic aortic rupture and severe lung failure]. / Pumpenlose extrakorporale Lungenunterstützung und endovaskuläres Stenting bei traumatischer Aortenruptur und schwerem Lungenversagen.

Blunt thoracic injury in association with aortic rupture represents a life-threatening situation. Surgical repair used to be the preferred method of treatment. Because most patients are multiple trauma patients including head injuries, bone fractures and respiratory failure, urgent surgical procedures portend excessively high morbidity and mortality rates. Delay in operative management bears the risk of exsanguinating hemorrhage, secondary complications, prolonged hospital stay with increased costs. We present here an alternative treatment protocol including pumpless extracorporal lung assist and endovascular aortic stent graft placement in a 20-year old traffic accident victim. This procedure may be an especially useful treatment option in managing patients with complex lung and aortic pathology primarily not suitable for transportation or surgery.

Pumpless extracorporeal lung assist using an arterio-venous shunt. Applications and limitations.

We report the use of a pumpless extracorporeal lung assist (PECLA) in 70 patients with severe pulmonary failure of various causes. The device was used under rescue conditions in patients with preserved cardiac function. By establishing a shunt between femoral artery and vein using the arterio-venous pressure gradient as the driving force for the blood flow through the oxygenator, PECLA proved to be extremely effective in terms of oxygenation and carbon dioxide removal.

Role of the chain termini for the folding transition state of the cold shock protein.

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Dynamic association of trigger factor with protein substrates.

Trigger factor is a ribosome-bound folding helper, which, apparently, combines two functions, chaperoning of nascent proteins and catalyzing prolyl isomerization in their folding. Immediate chaperone binding at the ribosome might interfere with rapid protein folding reactions, and we find that trigger factor indeed retards the in vitro folding of a protein with native prolyl isomers. The kinetic analysis of trigger factor binding to a refolding protein reveals that the adverse effects of trigger factor on conformational folding are minimized by rapid binding and release. The complex between trigger factor and a substrate protein is thus very short-lived, and fast-folding proteins can escape efficiently from an accidental interaction with trigger factor. Protein chains with incorrect prolyl isomers cannot complete folding and therefore can rebind for further rounds of catalysis. Unlike DnaK, trigger factor interacts with substrate proteins in a nucleotide-independent binding reaction, which seems to be optimized for high catalytic activity rather than for chaperone function. The synthetic lethality, observed when the genes for both DnaK and trigger factor are disrupted, might result from an indirect linkage. In the absence of trigger factor, folding is retarded and more aggregates form, which can neither be prevented nor disposed of when DnaK is lacking as well.

[Treatment of supravalvular pulmonary stenosis after arterial switch operations (ASO)]. / Die Behandlung supravalvulärer Pulmonalstenosen nach Arteriellen Switch-Operationen (ASO).

The aim of this study was to evaluate the incidence of postoperative pulmonary supravalvular stenosis in patients with d-TGA and to assess the rate of success or failure of balloon angioplasty. Out of 70 patients with d-TGA 67 patients underwent successful arterial switch operation. Twelve children developed severe supravalvular pulmonary stenosis with a peak gradient above 50 mmHg (range: 50-120 mmHg). In these patients 19 high pressure dilatations were performed up to a diameter of 130% of the native valve dimension. The mean age at angioplasty was 17 months (range: 3-36 months). Successful intervention was defined as a > 50% decrease of predilatation peak pressure gradient or right ventricular pressure < 50 mmHg. Dilatations were performed without complications. Complete resolution was primarily achieved in 1 patient. In 7 patients the pressure gradients could be reduced to 10-45 mmHg (mean: 25 mmHg). In another two patients a palliative stent-implantation into the pulmonary trunk was necessary to reduce the pressure gradient. Because unsuccessful intervention, two patients needed subsequent operation. During follow-up of 6-9 months after intervention severe restenosis occurred in 3 patients (2 after stent-implantation; 1 after re-re-dilatation) who then also needed operation. Balloon dilatation should be the first treatment in patients with pulmonary stenosis after ASO in TGA owing to the low complication rate and the potential benefit of this procedure. Recurrent and combined stenoses with narrow pulmonary valve annulus should be treated surgically.

Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms.

Spinach starch debranching enzyme, a limit dextrinase or pullulanase (EC 3.2.1.41), is a monomeric protein of 100 kDa that produces up to seven coexisting and mutually interconvertible isomers of different specific activity, a phenomenon that has been termed microheterogeneity and for which a structural explanation has not yet been presented. The enzyme can be activated by reduction, in particular by thiol reagents, and inactivated by oxidation and the concomitant change of the patterns of its isomeric forms could be quantified by chromatofocusing. The hypothesis was examined that reduction of the enzyme's thiol groups shifts the isomer pattern towards the forms with a higher specific activity while oxidation favours the less active forms. Using TCEP as reductant only the form with the highest specific activity was obtained. This form was almost inaccessible for proteolysis by trypsin while the oxidized and GSH-activated enzyme yielded four peptides when treated with trypsin. Their sequence indicated cleavage predominantly of loops connecting the beta-strands and alpha-helices of the (beta/alpha)(8)-barrel which forms the catalytic site of the pullulanase. Formation of various disulphide bridges between the loops connecting the barrel structures -- predominantly on one side -- may be the reason for the microheterogeneity of the spinach pullulanase. In vivo, the enzyme maintains its activated state due to the high concentration of GSH in the chloroplast. However, the chloroplast's pH shifts from day (pH 8) to night (pH 7) and thus could also alter the activity of the protein in accordance with the required function in starch metabolism.

In-vitro selection of highly stabilized protein variants with optimized surface.

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity.

The Escherichia coli periplasmic peptidyl-prolyl isomerase (PPIase) SurA is involved in the maturation of outer membrane porins. SurA consists of a substantial N-terminal region, two iterative parvulin-like domains and a C-terminal tail. Here we show that a variant of SurA lacking both parvulin-like domains exhibits a PPIase-independent chaperone-like activity in vitro and almost completely complements the in vivo function of intact SurA. SurA interacts preferentially (>50-fold) with in vitro synthesized porins over other similarly sized proteins, leading us to suggest that the chaperone-like function of SurA preferentially facilitates maturation of outer membrane proteins.

Surgical treatment of pulmonary artery sarcoma.

OBJECTIVE: Pulmonary artery sarcomas are rare and usually fatal tumors. The diagnosis is difficult and delayed in most cases. Newer imaging techniques could allow early diagnosis in patients with symptoms of pulmonary vascular obstruction. Surgical resection improves clinical symptoms and offers the only chance of cure. We report the case histories of 7 patients with primary pulmonary artery sarcomas treated by surgical resection with or without adjuvant therapy. METHODS: Seven patients (3 women and 4 men; mean age, 52.3 years; preoperative New York Heart Association functional class III/IV, n = 5/2) underwent operations. Malignancy was preoperatively suspected in 5 patients, and 2 patients had a presumptive diagnosis of chronic pulmonary embolism. Tumor resection with partial or total prosthetic replacement (n = 2), reconstruction (n = 5), or both, of central parts of the pulmonary arteries was performed in 6 patients. Thromboendarterectomy was necessary in 4 patients, and pneumonectomy was necessary in 2 patients. Six patients received adjuvant therapy. RESULTS: There was no perioperative mortality. All patients had a substantial improvement in exercise tolerance and hemodynamics 3 months after their operations. Four patients died 7, 9, 18, and 19 months after their operations because of recurrent tumor or pulmonary metastases. Two patients are alive 21 and 35 months after primary surgical repair, with pulmonary metastases detected by computed tomographic scans. One patient is alive 62 months after resection without clinical or radiologic signs of tumor recurrence or metastasis. CONCLUSIONS: Early diagnosis of primary pulmonary artery sarcomas can be improved by computed tomography and magnetic resonance scanning. Radical surgical resection probably presents the only chance for cure. The role of neoadjuvant or adjuvant treatment modalities has to be defined. Pulmonary artery sarcoma need not necessarily be a fatal diagnosis.

Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.

Crystal structures of mutant forms of the Bacillus caldolyticus cold shock protein differing in thermal stability.

The cold shock proteins Bc-Csp from the thermophile Bacillus caldolyticus and Bs-CspB from the mesophile Bacillus subtilis differ significantly in their conformational stability, although the two proteins differ by only 12 out of 67 amino acid residues. The three-dimensional structure of these small and compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors is very similar. Previous work has shown that Bc-Csp displays a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB, and indicated that electrostatic interactions are, in part, responsible for this effect. It was further described that the stability difference is almost exclusively due to surface-exposed charged residues at sequence positions 3 and 66 of Bc-Csp and Bs-CspB, whereas all other amino acid changes between both proteins have no net effect on stability. To investigate how two surface residues determine the stability of Bc-Csp, Arg3 and Leu66 were replaced by glutamic acid, corresponding to the Bs-CspB sequence. The crystal structures of the resultant protein variants, Bc-Csp R3E and Bc-Csp L66E, were determined at 1.4 A and 1.27 A resolution, and refined to R values of 13.9 % and 15.8 %, respectively. Both structures closely resemble Bc-Csp in their global fold and show different hydrogen bonding and salt-bridge patterns when two independent molecules in the asymmetric unit of the crystal are compared. To extend the study to neighbored residues that help determine the surface charge around Arg3 and Leu66, the mutant proteins Bc-Csp E46A, Bc-Csp R3E/E46A/L66E and Bc-Csp V64T/L66E/67A were crystallized. Their structures were determined at resolutions of 1.8 A, 1.32 A and 1.8 A and refined to R values of 18.5 %, 13.8 % and 19.3 %, respectively. A systematic comparison of the crystal structures of all forms of the B. caldolyticus cold shock protein shows varying patterns of hydrogen bonds and electrostatic interactions around residues 3 and 66. Thermal destabilization of the protein by mutation appears to correlate with the extent of an acidic surface patch near the C-terminal carboxylate group.

Complete atrioventricular septal defect associated with tetralogy of Fallot. Favourable outcome of transatrial transpulmonary repair.

BACKGROUND: Complete correction of atrioventricular septal defect (AVSD) associated with tetralogy of Fallot (TOF) has been reported to account for an increased surgical risk. Impaired right ventricular function after classic transventricular repair, residual outflow tract stenosis, and incompetence of the pulmonary or atrioventricular valves are considered to be essential factors affecting the results. METHODS: From 3/95 to 6/98 six consecutive patients with AVSD and TOF underwent repair (age 18 months to 7.3 years) using a combined transatrial-transpulmonary approach. RV outflow tract balloon dilatation preceded transatrial correction in 4 patients. Pulmonary annulotomy but not transanular patching was necessary in 4 cases. The septal defects were closed by two separate patches using a Dacron patch with short depth and anterior extension for the ventricular component. RESULTS: All patients survived and had stable sinus rhythm. Echocardiography demonstrated mild, but hemodynamically insignificant mitral regurgitation in two and tricuspid regurgitation in four patients. Right ventricle to pulmonary artery gradients ranged from 5 to 35 mmHg (mean 24.2 mmHg) without progression. During follow-up ranging from 4 months to 3.5 years (mean 16.8 months) no reoperation was necessary. CONCLUSIONS: The transatrial-transpulmonary approach for correction of AVSD with TOF contributes to improved results after repair of this rare combination of defects.