Gunter Kerst

Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

BackgroundHuman multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.ResultsAfter transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.ConclusionPhysiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Permanent Cardiac Pacing in Children: Choosing the Optimal Pacing Site A Multicenter Study

Background— We evaluated the effects of the site of ventricular pacing on left ventricular (LV) synchrony and function in children requiring permanent pacing. Methods and Results— One hundred seventy-eight children (aged <18 years) from 21 centers with atrioventricular block and a structurally normal heart undergoing permanent pacing were studied cross-sectionally. Median age at evaluation was 11.2 (interquartile range, 6.3–15.0) years. Median pacing duration was 5.4 (interquartile range, 3.1–8.8) years. Pacing sites were the free wall of the right ventricular (RV) outflow tract (n=8), lateral RV (n=44), RV apex (n=61), RV septum (n=29), LV apex (n=12), LV midlateral wall (n=17), and LV base (n=7). LV synchrony, pump function, and contraction efficiency were significantly affected by pacing site and were superior in children paced at the LV apex/LV midlateral wall. LV dyssynchrony correlated inversely with LV ejection fraction (R=0.80, P=0.031). Pacing from the RV outflow tract/lateral RV predicted significantly decreased LV function (LV ejection fraction <45%; odds ratio, 10.72; confidence interval, 2.07–55.60; P=0.005), whereas LV apex/LV midlateral wall pacing was associated with preserved LV function (LV ejection fraction ≥55%; odds ratio, 8.26; confidence interval, 1.46–47.62; P=0.018). Presence of maternal autoantibodies, gender, age at implantation, duration of pacing, DDD mode, and QRS duration had no significant impact on LV ejection fraction. Conclusions— The site of ventricular pacing has a major impact on LV mechanical synchrony, efficiency, and pump function in children who require lifelong pacing. Of the sites studied, LV apex/LV midlateral wall pacing has the greatest potential to prevent pacing-induced reduction of cardiac pump function.

High Proportion of Leukemic Stem Cells at Diagnosis Is Correlated with Unfavorable Prognosis in Childhood Acute Myeloid Leukemia

In acute myeloid leukemia (AML), the leukemia-initiating cell is found within the CD34+/CD38− cell compartment. Over the last years evidence grew that AML is initiated and propagated by leukemic stem cells (LSCs). Conceivably, these most immature leukemia cells are more resistant to therapy and subsequently initiate relapse. The authors studied 17 patients with childhood AML treated according to the AML-BFM 98/04 protocol. At diagnosis, the authors determined the characteristic immunophenotype of the leukemic cells by flow cytometry and investigated the expression of CD34, CD38, and CD45 to define a population of immunophenotypically immature cells (CD34+/CD38−/CD45−/low) enriched for LSCs in many cases of AML. The authors compared the fraction of this population of all myeloid cells at diagnosis with event-free survival. Kaplan-Meier analysis revealed significant higher event free survival of patients with low CD34+/CD38−/CD45−/low cell proportion (<0.68%) compared to patients with high burden of this population (>0.83%; log-rank P < .04). This correlation was not found for the total number of CD34+ cells. This is the first study to show that a higher proportion of immature CD34+/CD38−/CD45−/low blasts at diagnosis correlates with unfavorable prognosis in childhood AML. The results suggest that a large CD34+/CD38−/CD45−/low population reflects a higher fraction of LSCs, leading to increased chemotherapy resistance and elevated relapse rate. Thus the initial frequency of CD34+/CD38−/CD45−/low cells may serve as a prognostic marker in pediatric AML. Future treatment in childhood AML should specifically target this immature population as well as the mature blast population.

High frequency of immature cells at diagnosis predicts high minimal residual disease level in childhood acute lymphoblastic leukemia

Prognosis for children with acute lymphoblastic leukemia (ALL) has considerably improved, yet relapse still occurs in a significant proportion of patients. Conceivably, the most immature leukemia cells may be more resistant to therapy and initiate relapse. We studied 42 patients with childhood ALL treated according to the ALL-BFM 2000 protocol. At diagnosis, we determined the characteristic immunophenotype of the leukemic cells by flow cytometry and also investigated the expression of CD34 and CD38 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)). We then studied levels of minimal residual disease (MRD) after induction therapy (day 33) and after consolidation therapy (week 12). We found a significant, increasing correlation between the prevalence of CD34(+)/CD38(-) cells at diagnosis and MRD levels at day 33 and week 12. Our results suggest that the initial frequency of CD34(+)/CD38(-) cells may serve as a prognostic marker in pediatric ALL.

Patterns of monocyte subpopulations and their surface expression of HLA-DR during adverse events after hematopoietic stem cell transplantation

Human leukocyte antigen DR surface expression in “classical” CD14++CD16− (M1), “intermediate” CD14++CD16+ (M2), and “non-classical” CD14+CD16++ (M3) monocytes reflects the activation state of these cells. The full spectrum of monocyte and its function is still unknown. The present pilot study describes the monocyte subpopulations and their human leukocyte antigen DR expression during the post-transplant period as well as during transplant-related adverse events of 30 pediatric patients and young adults with hemato-oncological malignancies and immunodeficiency disorders in comparison to healthy children and young adults. A significant change of the human leukocyte antigen DR expression in all three monocyte subpopulations during the period after bone marrow transplantation depending on the time after transplantation and adverse events could be recognized. Prior to and during sepsis or bacterial infection, a significant decrease in human leukocyte antigen DR expression occurred. A significant increase on CD14++CD16− monocytes could be observed during graft-versus-host disease. The alterations of human leukocyte antigen DR expression on the monocyte subpopulations during adverse events after hematopoietic stem cell transplantation may be a sign of changes in the capacity of these subpopulations. Moreover, human leukocyte antigen DR expression in monocyte subpopulations may be used to monitor treatment responses in these entities.

Transcatheter creation of a de novo communication across an extracardiac Fontan conduit for catheter ablation of a “left-sided” accessory pathway

Extracardiac polytetrafluoroethylene (PTFE) conduits are often used in modified Fontan procedures for separating systemic and pulmonary circulations in morphological and functional single ventricles [1]. However, most arrhythmia substrates of congenital and acquired supraventricular tachycardias will then be located ‘‘left-sided’’ across the extracardiac conduit. Remote magnetic navigation not only may facilitate a retrograde approach, but may also fail to reach the arrhythmia substrate [2]. Hybrid intervention consisting of transcatheter ablation via a sternotomy approach and atriotomy incision [3] as well as catheter ablation via direct transthoracic percutaneous access [4] have been described, but appear to be associated with frequent complications. An antegrade approach via femoral access requires special techniques for traversing the stiff PTFE material, the pericardial space and the atrial muscular wall. This is the first report that describes a modified transseptal puncture technique using no more than mechanical force [5] for successful catheter ablation across an extracardiac PTFE Fontan conduit. A 23-year-old male patient with double-outlet right ventricle, mitral atresia, severe left ventricular hypoplasia, subpulmonary ventricular septal defect, valvular pulmonary stenosis and D-transposition of the great arteries after modified Fontan anastomosis presented with a 15-year history of recurrent supraventricular tachycardia. Bidirectional superior cavopulmonary anastomosis (Glenn) had been performed at the age of 6 years, modified Fontan anastomosis with an extracardiac non-fenestrated 19 mm PTFE conduit connecting the inferior vena cava and the pulmonary arteries 2 years later. Tricuspid valve (TV) repair employing an anuloplasty ring was carried out for tricuspid regurgitation at the age of 17 years. Despite various oral antiarrhythmic medications [propafenone (up to 600 mg per day, 13 mg/kg per day); propafenone (up to 600 mg per day, 13 mg/kg per day) plus bisoprolol (2,5 mg per day) plus digoxin (aiming at serum levels of 1–2 ng/ ml); sotalol (up to 320 mg per day, 5 mg/kg per day); sotalol (160 mg per day) plus digoxin (aiming at serum levels of 1–2 ng/ml)], the patient continued to experience recurrent adenosine-sensitive supraventricular tachycardias (cycle length 340 ms). After discontinuation of antiarrhythmic medication, electrophysiology testing was performed with one steerable quadripolar catheter placed retrograde across the aortic valve in a right ventricular (RV) position and with one steerable decapolar catheter at the junction of the left pulmonary artery and the calcified extracardiac conduit where atrial sensing and pacing was possible. A supraventricular tachycardia (cycle length 350 ms) characteristic for orthodromic atrioventricular reciprocating tachycardia (AVRT) was easily induced by programmed ventricular pacing (Fig. 1a, b). Electroanatomical mapping of earliest retrograde atrial activation during tachycardia was performed using the EnSite Velocity Cardiac Mapping System (EnSite NavX, St. Jude Medical, St. Paul, MN, USA). Using the steerable quadripolar catheter placed retrograde across the aortic valve, the G. Kerst (&) D. Schranz M. B. Gonzalez y Gonzalez Department of Pediatric Cardiology, Pediatric Heart Center Giessen, University Children’s Hospital, Feulgenstr. 10-12, 35392 Giessen, Germany e-mail: gunter.kerst@paediat.med.uni-giessen.de

A novel technique for zero-fluoroscopy catheter ablation used to manage Wolff-Parkinson-White syndrome with a left-sided accessory pathway.

Conventional catheter ablation of cardiac arrhythmias is associated with the potential adverse effects of low-dose ionizing radiation on both patients and laboratory personnel. Due to the greater radiation sensitivity and the longer life expectancy of children, reduction of radiation exposure for them is of particular importance. A novel technique for zero-fluoroscopy catheter ablation is described using real-time tissue-tip contact force measurements for a 10-year-old boy who had Wolff–Parkinson–White syndrome with a left-sided accessory pathway.

The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.

Restrictive ventricular septal defect and critical subaortic stenosis in tetralogy of Fallot

IntroductionTetralogy of Fallot is characterized by a non-restrictive malalignment ventricular septal defect (VSD), an overriding aorta and right ventricular outflow tract obstruction resulting from anterior deviation of the infundibular septum. Due to the large VSD, systolic pressures are equal in both ventricles. In rare cases, redundant tricuspid valve tissue may prolapse into the VSD resulting in restriction of the defect size and causing suprasystemic pressure in the right ventricle. Subaortic obstruction by prolapse of the redundant tricuspid tissue into the left ventricular outflow tract, although theoretically possible in this situation, has not been described yet in the literature.Case reportWe report on a newborn with tetralogy of Fallot presenting with cyanosis and severe arterial hypotension a few hours after birth. Echocardiography revealed redundant hammock-like accessory tricuspid valve tissue almost completely occluding the originally large VSD. Suprasystemic pressure in the right ventricle resulted in protrusion of tricuspid valve tissue across the VSD and thereby caused severe left ventricular outflow tract obstruction. Emergency pulmonary balloon valvuloplasty performed for decompression of the right ventricle, reduced prolapse of tricuspid tissue into the left ventricular outflow tract and resulted in significant improvement of pulmonary and systemic blood flow.ConclusionIn tetralogy of Fallot, accessory tricuspid valve tissue may obstruct the VSD as well as the left ventricular outflow tract resulting in a life threatening condition in newborns shortly after birth.

Ebstein’s Malformation With Imperforate Tricuspid Valve

A neonate was admitted at the age of 8 days with a history of increasing cyanosis and tachydyspnea after a normal pregnancy and delivery at 42 weeks’ gestation. A transthoracic echocardiography appeared to indicate pulmonary atresia with an intact ventricular septum. The child was intubated, intravenous prostaglandin E1 was started, and the patient was transferred to our hospital. The newborn presented with central cyanosis while being ventilated with 100% oxygen. No heart murmur was perceptible. Transthoracic echocardiography revealed marked apical displacement of the tricuspid valve leaflets (Figure 1 and Movie I). Linear attachments of the anterior and the mural leaflets resulted in complete separation of the inlet portion from the apical trabecular and outlet portions of the right ventricle (Figure 2 and Movie II). On color Doppler, we were unable to demonstrate either antegrade or regurgitant flow across the valve …