The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP.

Pediatric immune thrombocytopenia (ITP) is usually self-limited. However, approximately 20% of children develop chronic ITP, which can be associated with significant morbidity because of long-term immunosuppression and splenectomy in refractory cases. To explore the molecular mechanism of chronic ITP compared with acute ITP, we studied 63 pediatric patients with ITP. Gene expression analysis of whole blood revealed distinct signatures for acute and chronic ITP. Oxidative stress-related pathways were among the most significant chronic ITP-associated pathways. Overexpression of VNN1, an oxidative stress sensor in epithelial cells, was most strongly associated with progression to chronic ITP. Studies of normal persons demonstrated VNN1 expression in a variety of blood cells. Exposure of blood mononuclear cells to oxidative stress inducers elicited dramatic up-regulation of VNN1 and down-regulation of PPARγ, indicating a role for VNN1 as a peripheral blood oxidative stress sensor. Assessment of redox state by tandem mass spectrometry demonstrated statistically significant lower glutathione ratios in patients with ITP versus healthy controls; lower glutathione ratios were also seen in untreated patients with ITP compared with recently treated patients. Our work demonstrates distinct patterns of gene expression in acute and chronic ITP and implicates oxidative stress pathways in the pathogenesis of chronic pediatric ITP.

Biocompatibility assessment of the first generation PediaFlow pediatric ventricular assist device.

The PediaFlow pediatric ventricular assist device is a miniature magnetically levitated mixed flow pump under development for circulatory support of newborns and infants (3-15 kg) with a targeted flow range of 0.3-1.5 L/min. The first generation design of the PediaFlow (PF1) was manufactured with a weight of approximately 100 g, priming volume less than 2 mL, length of 51 mm, outer diameter of 28 mm, and with 5-mm blood ports. PF1 was evaluated in an in vitro flow loop for 6 h and implanted in ovines for three chronic experiments of 6, 17, and 10 days. In the in vitro test, normalized index of hemolysis was 0.0087 ± 0.0024 g/100L. Hemodynamic performance and blood biocompatibility of PF1 were characterized in vivo by measurements of plasma free hemoglobin, plasma fibrinogen, total plasma protein, and with novel flow cytometric assays to quantify circulating activated ovine platelets. The mean plasma free hemoglobin values for the three chronic studies were 4.6 ± 2.7, 13.3 ± 7.9, and 8.8 ± 3.3 mg/dL, respectively. Platelet activation was low for portions of several studies but consistently rose along with observed animal and pump complications. The PF1 prototype generated promising results in terms of low hemolysis and platelet activation in the absence of complications. Hemodynamic results validated the magnetic bearing design and provided the platform for design iterations to meet the objective of providing circulatory support for young children with exceptional biocompatibility.

Preclinical performance of a pediatric mechanical circulatory support device: The PediaFlow ventricular assist device.

OBJECTIVES: The PediaFlow (HeartWare International, Inc, Framingham, Mass) is a miniature, implantable, rotodynamic, fully magnetically levitated, continuous-flow pediatric ventricular assist device. The fourth-generation PediaFlow was evaluated in vitro and in vivo to characterize performance and biocompatibility. METHODS: Supported by 2 National Heart, Lung, and Blood Institute contract initiatives to address the limited options available for pediatric patients with congenital or acquired cardiac disease, the PediaFlow was developed with the intent to provide chronic cardiac support for infants as small as 3 kg. The University of Pittsburgh-led Consortium evaluated fourth-generation PediaFlow prototypes both in vitro and within a preclinical ovine model (n = 11). The latter experiments led to multiple redesigns of the inflow cannula and outflow graft, resulting in the implantable design represented in the most recent implants (n = 2). RESULTS: With more than a decade of extensive computational and experimental efforts spanning 4 device iterations, the AA battery-sized fourth-generation PediaFlow has an operating range of 0.5 to 1.5 L/min with minimal hemolysis in vitro and excellent hemocompatibility (eg, minimal hemolysis and platelet activation) in vivo. The pump and finalized accompanying implantable components demonstrated preclinical hemodynamics suitable for the intended pediatric application for up to 60 days. CONCLUSIONS: Designated a Humanitarian Use Device for "mechanical circulatory support in neonates, infants, and toddlers weighing up to 20 kg as a bridge to transplant, a bridge to other therapeutic intervention such as surgery, or as a bridge to recovery" by the Food and Drug Administration, these initial results document the biocompatibility and potential of the fourth-generation PediaFlow design to provide chronic pediatric cardiac support.

Towards the development of a pediatric ventricular assist device.

The very limited options available to treat ventricular failure in children with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device at the University of Pittsburgh (UoP) and University of Pittsburgh Medical Center (UPMC). Our effort involves a consortium consisting of UoP, Children's Hospital of Pittsburgh (CHP), Carnegie Mellon University, World Heart Corporation, and LaunchPoint Technologies, Inc. The overall aim of our program is to develop a highly reliable, biocompatible ventricular assist device (VAD) for chronic support (6 months) of the unique and high-risk population of children between 3 and 15 kg (patients from birth to 2 years of age). The innovative pediatric ventricular assist device we are developing is based on a miniature mixed flow turbodynamic pump featuring magnetic levitation, to assure minimal blood trauma and risk of thrombosis. This review article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

In vitro evaluation of multiobjective hemodynamic control of a heart-assist pump.

Ventricular assist devices now clinically used for treatment of end-stage heart failure require responsive and reliable hemodynamic control to accommodate the continually changing demands of the body. This is an essential ingredient to maintaining a high quality of life. To satisfy this need, a control algorithm involving a trade-off between optimal perfusion and avoidance of ventricular collapse has been developed. An optimal control strategy has been implemented in vitro that combines two competing indices: representing venous return and prevalence of suction. The former is derived from the first derivative of diastolic flow with speed, and the latter derived from the harmonic spectra of the flow signal. The responsiveness of the controller to change in preload and afterload were evaluated in a mock circulatory simulator using a HeartQuest centrifugal blood pump (CF4b, MedQuest Products, Salt Lake City, UT). To avoid the need for flow sensors, a state estimator was used, based on the back-EMF of the actuator. The multiobjective algorithm has demonstrated more robust performance as compared with controllers relying on individual indices.

In Vitro and In Vivo Performance Evaluation of the Second Developmental Version of the PediaFlow Pediatric Ventricular Assist Device.

Ventricular assist devices (VADs) have significantly impacted the treatment of adult cardiac failure, but few options exist for pediatric patients. This has motivated our group to develop an implantable magnetically levitated rotodynamic VAD (PediaFlow®) for 3-20 kg patients. The second prototype design of the PediaFlow (PF2) is 56% smaller than earlier prototypes, and achieves 0.5-1.5 L/min blood flow rates. In vitro hemodynamic performance and hemolysis testing were performed with analog blood and whole ovine blood, respectively. In vivo evaluation was performed in an ovine model to evaluate hemocompatibility and end-organ function. The in vitro normalized index of hemolysis was 0.05-0.14 g/L over the specified operating range. In vivo performance was satisfactory for two of the three implanted animals. A mechanical defect caused early termination at 17 days of the first in vivo study, but two subsequent implants proceeded without complication and electively terminated at 30 and 70 days. Serum chemistries and plasma free hemoglobin were within normal limits. Gross necropsy revealed small, subclinical infarctions in the kidneys of the 30 and 70 day animals (confirmed by histopathology). The results of these experiments, particularly the biocompatibility demonstrated in vivo encourage further development of a miniature magnetically levitated VAD for the pediatric population. Ongoing work including further reduction of size will lead to a design freeze in preparation for of clinical trials.

PediaFlow™ Maglev Ventricular Assist Device: A Prescriptive Design Approach.

This report describes a multi-disciplinary program to develop a pediatric blood pump, motivated by the critical need to treat infants and young children with congenital and acquired heart diseases. The unique challenges of this patient population require a device with exceptional biocompatibility, miniaturized for implantation up to 6 months. This program implemented a collaborative, prescriptive design process, whereby mathematical models of the governing physics were coupled with numerical optimization to achieve a favorable compromise among several competing design objectives. Computational simulations of fluid dynamics, electromagnetics, and rotordynamics were performed in two stages: first using reduced-order formulations to permit rapid optimization of the key design parameters; followed by rigorous CFD and FEA simulations for calibration, validation, and detailed optimization. Over 20 design configurations were initially considered, leading to three pump topologies, judged on the basis of a multi-component analysis including criteria for anatomic fit, performance, biocompatibility, reliability, and manufacturability. This led to fabrication of a mixed-flow magnetically levitated pump, the PF3, having a displaced volume of 16.6 cc, approximating the size of a AA battery and producing a flow capacity of 0.3-1.5 L/min. Initial in vivo evaluation demonstrated excellent hemocompatibility after 72 days of implantation in an ovine. In summary, combination of prescriptive and heuristic design principles have proven effective in developing a miniature magnetically levitated blood pump with excellent performance and biocompatibility, suitable for integration into chronic circulatory support system for infants and young children; aiming for a clinical trial within 3 years.

G4-forming sequences in the non-transcribed DNA strand pose blocks to T7 RNA polymerase and mammalian RNA polymerase II.

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure.

Desmosomes are intercellular junctions in which cadherin cell adhesion molecules are linked to the intermediate filament (IF) system. Desmoplakin is a member of the plakin family of IF-binding proteins. The C-terminal domain of desmoplakin (DPCT) mediates binding to IFs in desmosomes. The DPCT sequence contains three regions, termed A, B and C, consisting of 4.5 copies of a 38-amino acid repeat motif. We demonstrate that these regions form discrete subdomains that bind to IFs and report the crystal structures of domains B and C. In contrast to the elongated structures formed by other kinds of repeat motifs, the plakin repeats form a globular structure with a unique fold. A conserved basic groove found on the domain may represent an IF-binding site.

Orientation of bound ligands in mannose-binding proteins. Implications for multivalent ligand recognition.

Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.