The current role of surgery in the treatment of cardiac metastases from malignant melanoma: an educational presentation

Abstract A 71 year-old male with a history of multiple excisions of an initial Clark's level V melanoma of the breast followed by combined radiation and interferon treatment, as well as a recurrence, 3 years later, of a BRAF-positive tumor of the shoulder, with subsequent therapy with dabrafenib and trametinib, presented again with progressive intracardiac masses causing significant right ventricular outflow obstruction. Additionally, the patient complained of dyspnea and fatigue on exertion, thus he was scheduled for surgical resection.

The Current Role of Surgery in the Treatment of Cardiac Metastases from Malignant Melanoma: an Educational Presentation.

A 71 year-old male with a history of multiple excisions of an initial Clark's level V melanoma of the breast followed by combined radiation and interferon treatment, as well as a recurrence, 3 years later, of a BRAF-positive tumor of the shoulder, with subsequent therapy with dabrafenib and trametinib, presented again with progressive intracardiac masses causing significant right ventricular outflow obstruction. Additionally, the patient complained of dyspnea and fatigue on exertion, thus he was scheduled for surgical resection.

A technique to resect the Edwards SAPIEN 3 Transcatheter Heart Valve 18 months after implantation in case of surgical aortic valve replacement.

Transcatheter aortic valve implantation (TAVI) evolved to an established treatment for meanwhile moderate-risk surgical patients suffering from severe aortic stenosis. Due to its less invasiveness, avoiding the use of cardiopulmonary bypass, the procedure demonstrated to be an efficient and safe treatment option. However, long-term results regarding these new valve prostheses are still lacking. Potential prosthesis failure and/ or dysfunction would require either, if feasible, a transcatheter "valve in valve" procedure, or a conventional surgical valve replacement. In the literature, anecdotal reports are describing surgical removal techniques of the currently transcatheter-implanted prostheses. We herein propose a modified surgical removal technique of an infected Edwards SAPIEN 3 Transcatheter Heart Valve 18 months after its implantation.

Cardiovascular magnetic resonance assessment of the aortic valve stenosis: an in vivo and ex vivo study.

BACKGROUND: Aortic valve area (AVA) estimation in patients with aortic stenosis may be obtained using several methods. This study was undertaken to verify the cardiovascular magnetic resonance (CMR) planimetry of aortic stenosis by comparing the findings with invasive catheterization, transthoracic (TTE) as well as tranesophageal echocardiography (TEE) and anatomic CMR examination of autopsy specimens. METHODS: Our study was performed in eight patients with aortic valve stenosis. Aortic stenosis was determined by TTE and TEE as well as catheterization and CMR. Especially, after aortic valve replacement, the explanted aortic valves were examined again with CMR ex vivo model. RESULTS: The mean AVA determined in vivo by CMR was 0.75 ± 0.09 cm(2) and ex vivo by CMR was 0.65 ± 0.09 cm(2) and was closely correlated (r = 0.91, p < 0.001). The mean absolute difference between AVA derived by CMR ex vivo and in vivo was -0.10 ± 0.04 cm(2). The mean AVA using TTE was 0.69 ± 0.07 with a significant correlation between CMR ex vivo (r = 0.85, p < 0.007) and CMR in vivo (r = 0.86, p < 0.008). CMR ex vivo and in vivo had no significant correlation with AVA using Gorlin formula by invasive catheterization or using planimetry by TEE. CONCLUSION: In this small study using an ex vivo aortic valve stenosis model, the aortic valve area can be reliably planimetered by CMR in vivo and ex vivo with a well correlation between geometric AVA by CMR and the effective AVA calculated by TTE.

Combination of the human prolyl isomerase FKBP12 with unrelated chaperone domains leads to chimeric folding enzymes with high activity.

Folding enzymes often use distinct domains for the binding of substrate proteins ("chaperone domains") and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.

A kinetic approach to determining the conformational stability of a protein that dimerizes after folding.

A strongly stabilized form of the ß1 domain of the streptococcal protein G, termed Gß1-M2, was previously obtained by an in vitro selection method for stabilized protein variants. It contains four substitutions, but how they contribute to the Gibbs free energy of denaturation (ΔG(D)) could not be determined, because, unlike the wild-type protein, Gß1-M2 dimerizes in a spectroscopically silent reaction. Here we determined the ΔG(D) of the folded Gß1-M2 monomer by using a kinetic approach that uncouples the folding of the monomer from dimerization. The conformational equilibration of the monomer is faster than dimer formation, and therefore, its stability constant could be determined from the ratio of the rate constants for monomer unfolding and refolding. In this approach, double-mixing experiments were essential for uncovering the unfolding kinetics of the transient Gß1-M2 monomer and the association of the monomers after their folding. The analysis revealed that the selected substitutions stabilize the Gß1-M2 monomer by 15 kJ mol(-1) in an additive fashion. The combination of single- and double-mixing kinetic experiments thus allowed us to determine the thermodynamic stability of a transient species that is inaccessible in equilibrium experiments. It can be applied for proteins in which monomer folding and oligomerization are kinetically uncoupled.

Folding mechanism of an extremely thermostable (ßα)(8)-barrel enzyme: a high kinetic barrier protects the protein from denaturation.

HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (ßα)(8)-barrel protein. We elucidated the unfolding and refolding mechanism of HisF. Its unfolding transition is reversible and adequately described by the two-state model, but 6 weeks is necessary to reach equilibrium (at 25 °C). During refolding, initially a burst-phase off-pathway intermediate is formed. The subsequent productive folding occurs in two kinetic phases with time constants of ~3 and ~20 s. They reflect a sequential process via an on-pathway intermediate, as revealed by stopped-flow double-mixing experiments. The final step leads to native HisF, which associates with the glutaminase subunit HisH to form the functional ImGPS complex. The conversion of the on-pathway intermediate to the native protein results in a 10(6)-fold increase of the time constant for unfolding from 89 ms to 35 h (at 4.0 M GdmCl) and thus establishes a high energy barrier to denaturation. We conclude that the extra stability of HisF is used for kinetic protection against unfolding. In its refolding mechanism, HisF resembles other (ßα)(8)-barrel proteins.

Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation.

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG(D) = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG(D) = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.

Semisynthesis of a homogeneous glycoprotein enzyme: ribonuclease C: part 2.

Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.

Semisynthesis of a homogeneous glycoprotein enzyme: ribonuclease C: part 1.

Seven in one blow: The efficient formation of mixed disulfides on the thiol-rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.

Recellularization of biological heart valves with human vascular cells: in vitro hemocompatibility assessment.

Coverage of cardiovascular bioprostheses with autologous endothelium is used for the purpose of improving blood compatibility. The aim of our study was to analyze endothelialization potential of glutaraldehyde-fixed heart valves, cellular functions of seeded endothelial cells (EC), and the impact of a two-stage seeding protocol using human vascular fibroblasts (FB) and EC from saphenous veins (HSVEC) on cellular functional properties in vitro. Adherence and morphology of adhered cells were assessed by scanning electronic microscopy and immunohistochemistry. Reproducible, complete surface coverage with EC was established on decellularized and glutaraldehyde-fixed bovine pericardium. Analyzing functional properties of cells directly adhered to biomaterial revealed nonproliferative cells, which were capable of inflammatory stimulation in terms of TNF-induced increase in interleukin-6 secretion and adhesion of inflammatory cells. Furthermore, EC showed sustained antithrombotic properties quantified by platelet adhesion onto EC and prostacyclin secretion by EC. Preseeding with vascular fibroblasts using a two-stage seeding protocol induced EC proliferation and improved inflammatory and anti-thrombotic functions. Cardiovascular biomaterials differ significantly in their potential to allow for adhesion of human EC. Successfully endothelialized biomaterial, however, revealed cellular properties which are likely to be favorable to improving performance of biomaterials. Two-stage seeding adds regenerative potential and improves cell functions of adherent EC.

Dilatation of the ascending aorta in bicuspid aortic valve disease: a magnetic resonance imaging study.

BACKGROUND: Bicuspid aortic valve disease (BAV) is increasingly recognized as a disease of the entire proximal aorta including both valvular and vascular complications. The aim of our study was to assess the dimensions of the thoracic aorta using MRI in a broad spectrum of BAV and tricuspid aortic valve disease (TAV) and to define the prevalence of the dilatation of the ascending aorta (AA) >or= 4.5 cm in severe BAV disease. METHODS AND RESULTS: MRI studies were performed on a 1.5 T scanner in a total of 195 consecutive patients with aortic valve disease. Eighty-four aortic valves were classified as BAV and 103 as TAV. In 8 patients, classification of the aortic valve was not possible due to poor image quality. Mean diameters of the AA were significantly greater in BAV compared to TAV (4.39+/-0.85 Vs. 3.55+/-0.47 cm, P<0.0001), whereas no differences were observed in the mean diameters of the aortic arch. Diameters of the descending aorta were slightly smaller in BAV compared to TAV (2.45+/-0.43 Vs. 2.58+/-0.31 cm, P<0.05). In BAV, AA dilatation was independent of the severity of valve dysfunction. In TAV, aortic regurgitation but not stenosis correlated weakly with AA dilatation. Prevalence of AA dilatation >or= 4.5 cm in BAV with severe aortic stenosis and regurgitation was 38% and 41%, respectively. CONCLUSION: Dilatation of the proximal aorta is a frequent finding in BAV and independent of the severity of valve dysfunction. With respect to the high prevalence of AA dilatation >or= 4.5 cm in BAV with severe valve dysfunction, careful assessment of the dimensions of the AA is crucial to identify patients in whom concomitant AA replacement is indicated according to current guidelines.

A folded and functional protein domain in an amyloid-like fibril.

The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.

Bridge to lung transplantation through a pulmonary artery to left atrial oxygenator circuit.

BACKGROUND: There is no mechanical device available to support patients with end-stage lung failure for weeks and months until appropriate donor organs for lung transplantation are available. METHODS: In a 38-year-old female patient with primary pulmonary hypertension a paracorporeal artificial lung (PAL) system was placed parallel to the pulmonary circulation with connections to the pulmonary artery and to the left atrium. The key component of the PAL was a low-resistance membrane oxygenator. RESULTS: After institution, the PAL had a blood flow of 3.5 L/min and created a PaO(2)/fraction of inspired oxygen ratio of 270, while the oxygenator was provided with oxygen 3 L/min. The pulmonary artery pressure declined by almost 50%. The PAL worked well over 62 days until appropriate donor lungs were available. With resuming more physical activity, an increased flow through the native lung augmented the fraction of unsaturated blood arriving at the left atrium, which mandated increasing oxygen flow to the PAL. CONCLUSIONS: The data obtained with this case encourage further research into PAL systems, which may hopefully serve as a bridge to lung transplant device in appropriate patients in the future.

Influence of inflow cannula length in axial-flow pumps on neurologic adverse event rate: results from a multi-center analysis.

BACKGROUND: The application of axial-flow pumps in patients with end-stage heart failure reveals a significantly reduced infectious complication rate as compared with rates observed with pulsatile devices. The remaining adverse event rate relates mainly to thromboembolic complications with neurologic consequences. We investigated the dependence of the neurologic adverse event rate on the length of the inflow cannula. METHODS: A total of 216 consecutive patients with an axial-flow pump (INCOR; Berlin Heart GmbH, Berlin, Germany) were included in a retrospective multi-center analysis. In 138 patients, a short inflow cannula (24-mm tip length into the left ventricle), and in 78 patients a long inflow cannula (tip length 34 mm) was applied. RESULTS: Patients with a long inflow cannula (LC) demonstrated a better survival rate than those with a short inflow cannula (SC) at the end of the observation period (LC, 63.4%; SC, 52.9%; p = 0.05). The thromboembolic adverse event rate was also significantly lower. Only 3 of the 78 patients (3.8%) with an LC had a thromboembolic adverse event (thromboembolic events per patient-year = 0.11) as compared with 32 (23.2%) of SC patients (thromboembolic events per patient-year = 0.50, p < 0.001). CONCLUSIONS: Patients with a long inflow cannula had a better survival rate and a lower incidence of cerebrovascular adverse events than patients with a short inflow cannula.

Chaperone-aided in vitro renaturation of an engineered E1 envelope protein for detection of anti-Rubella virus IgG antibodies.

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.

Advantages of human umbilical vein scaffolds derived from cesarean section vs. vaginal delivery for vascular tissue engineering.

The current study investigated whether the mode of delivery and the mode of sample collection affect the functional properties of umbilical veins as scaffolds for vascular tissue engineering purposes. Human umbilical vein (HUV) from planned cesarean-sections (PCS) showed a 1.7-fold higher maximum contraction with potassium chloride compared to spontaneous vaginal deliveries (VDs, p=0.029). The maximum contractions with histamine were 2.0- and 2.9-fold higher in the PCS and emergency c-section (ECS) groups, respectively, compared to the VD group (p=0.003). The dose-response curves of serotonin were shifted to the right approx. 6- and 5-fold in the VD group compared to PCS and ECS, respectively (p=0.009). There were no differences between the birth groups in terms of tetrazolium dye reduction, platelet adhesion, and the structural integrity. The release of the antithrombotic compound prostacyclin from vessels of the PCS and ECS groups was 6.6- and 3.5-fold higher, respectively, than in the VD group (p<0.001). There was no correlation between the duration of ischemia and any of the functional parameters. This study provides evidence that vessels obtained from PCS are to be preferred for tissue engineering purposes, as they can be harvested in a sterile fashion and show superior vasoconstrictor responses and antithrombotic properties. The data also support a once-per-day pickup schedule for umbilical cords without a deterioration of the functional properties.

From Baghdad to Germany: use of a new pumpless extracorporeal lung assist system in two severely injured US soldiers.

The authors describe a new extracorporeal pumpless interventional lung assist system (iLA) that was implemented in two US soldiers with severe acute respiratory distress syndrome received from enemy action in Iraq, who were at risk for critical hypoxemia/hypercapnia. The system is characterized by a new low-resistance gas exchange membrane that is integrated in an arterial-venous bypass established by cannulation of the femoral artery and vein. Cardiovascular stability is essential to produce sufficiently high blood flow rates over the gas exchange unit. After implantation of the interventional lung assist, oxygenation increased and carbon dioxide elimination improved rapidly. Ventilator settings were able to be adjusted to the decreased pulmonary gas exchange needs, making protective lung strategies possible. Air transport of both patients with the running iLA system was uneventful. The iLA was removed after 15 and 8 days of continuous operation, respectively, and both soldiers were successfully weaned from mechanical ventilation. Interventional, extracorporeal pump-free pulmonary support opens up new possibilities for pulmonary protection due to ease of use, effectiveness, and low costs; however, there is concern of distal limb ischemia. Experiences to date are encouraging, although randomized studies are lacking, and the procedure carries significant risks.