Publications by authors named "Paul Stephens"

34 Publications

Echocardiographic Screening of Cardiovascular Status in Pediatric Sickle Cell Disease.

Pediatr Cardiol 2019 Dec 21;40(8):1670-1678. Epub 2019 Sep 21.

Division of Cardiology, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 34th St. and Civic Center Blvd, Philadelphia, PA, 19104, USA.

Although elevated right ventricular pressure and left ventricular diastolic dysfunction measured by echocardiogram are independent predictors of death in adults with sickle cell disease (SCD), the utility of routine echocardiographic screening in the pediatric population is controversial. We performed a 3-year retrospective review of children ≥ 10 years of age with SCD who underwent an outpatient transthoracic echocardiogram as part of a screening program. Of 172 patients referred for screening, 105 (61%) had a measurable tricuspid regurgitation jet velocity (TRV): median 2.4 m/s (IQR 2.3-2.5). Elevated right ventricular (RV) pressure (TRV ≥ 2.5 m/s, 25 mmHg), documented in 30% (32/105), was significantly associated with chronic transfusion therapy and elevated lactate dehydrogenase. Left ventricle (LV) dilation, documented in 25% (44/172), was significantly associated with lower hemoglobin, and higher reticulocyte count, lactate dehydrogenase level, and bilirubin level. There was no association between elevated right ventricular pressure or left ventricle dilation and indices of biventricular systolic or diastolic function. The one death in the cohort during the study period had normal echocardiographic findings. In conclusion, mild RV pressure elevation and LV dilation in children with SCD is associated with abnormal laboratory markers of disease severity, but not with ventricular dysfunction over the 3-year study period.
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http://dx.doi.org/10.1007/s00246-019-02202-3DOI Listing
December 2019

Repair of Total Anomalous Pulmonary Venous Connection: Risk Factors for Postoperative Obstruction.

Ann Thorac Surg 2019 07 16;108(1):122-129. Epub 2019 Mar 16.

Division of Pediatric Cardiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Background: Pulmonary venous obstruction after repair of total anomalous pulmonary venous connection (TAPVC) results in substantial morbidity and mortality. Risk factors for postoperative obstruction remain ambiguous. In addition, the existing literature has no standard definition for preoperative obstruction, making patient counseling difficult.

Methods: All patients undergoing repair of TAPVC at our institution from January 1, 2006, to October 23, 2017, were identified. The primary outcome was the development of postoperative obstruction, analyzed as a time-to-event outcome. Clinical information was extracted to assess risk factors. Degrees of preoperative obstruction were defined based on echocardiographic, catheterization, and clinical findings. Univariable and multivariable Cox proportional hazard regression methods were used to identify factors associated with the primary outcome.

Results: During the study interval, 119 patients underwent repair of TAPVC (40% single ventricle), and postoperative obstruction developed in 25 patients (21%). Risk factors associated with obstruction were heterotaxy syndrome, single-ventricle heart disease, additional procedures at the time of vein repair, mixed-type TAPVC, and preoperative obstruction. Having even mild preoperative obstruction (≥1.2 m/s by Doppler echocardiography) was predictive of postoperative obstruction. A multivariable model showed mixed-type TAPVC and the presence of preoperative obstruction were associated with a more than twofold greater hazard of obstruction.

Conclusions: TAPVC in the setting of heterotaxy and a single ventricle remains challenging, with high rates of postoperative obstruction. Mixed-type TAPVC is an independent risk factor for postoperative obstruction, particularly in patients with isolated TAPVC. Even mild preoperative obstruction is a risk factor for postoperative obstruction. These results may help risk-stratify TAPVC patients.
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http://dx.doi.org/10.1016/j.athoracsur.2019.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591098PMC
July 2019

Postoperative Obstruction of the Pulmonary Veins in Mixed Total Anomalous Pulmonary Venous Connection.

Pediatr Cardiol 2018 Oct 5;39(7):1489-1495. Epub 2018 Jun 5.

Division of Cardiology, The Children's Hospital of Philadelphia, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA.

Total anomalous pulmonary venous connection (TAPVC) is a rare form of congenital heart disease in which the pulmonary veins drain by various pathways to the right atrium instead of the left atrium. Postoperative obstruction of the pulmonary veins is a known complication. Identifying risk factors for morbidity and mortality is important for counseling and monitoring. We describe a pattern of postoperative obstruction in a specific arrangement of mixed TAPVC. Five patients with a type of mixed TAPVC, namely, three pulmonary veins connecting to the coronary sinus and the left upper pulmonary vein (LUPV) connecting to the innominate vein, were identified over an 11-year period at our institution. Two additional patients with this TAPVC arrangement were cared for at our institution after having surgery at other institutions. Of these, one patient received only comfort care at birth due to other clinical issues. The six other patients underwent surgical unroofing of the coronary sinus. The anomalous LUPV was not addressed during the initial surgery in any of these cases. Following repair, one patient died from non-cardiac reasons. The remaining five patients all developed obstruction of the repaired pulmonary veins with decompression through the unrepaired LUPV, requiring surgical revision. Three patients underwent a second reoperation as well. Three of the six repaired patients also developed refractory atrial arrhythmias. This cohort suggests that this mixed TAPVC pattern predisposes patients to obstruction after surgical repair. Further investigation may aid pediatric cardiologists in risk-stratifying and counseling these patients. Alternative surgical approaches may need to be considered.
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http://dx.doi.org/10.1007/s00246-018-1921-9DOI Listing
October 2018

Pretibial lacerations.

Br J Hosp Med (Lond) 2017 Nov;78(11):C162-C166

Consultant Plastic and Reconstructive Surgeon, Department of Plastic Surgery, Salisbury District Hospital, Salisbury.

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http://dx.doi.org/10.12968/hmed.2017.78.11.C162DOI Listing
November 2017

IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design.

Biochem J 2017 09 7;474(18):3179-3188. Epub 2017 Sep 7.

Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Davidson Building, Glasgow G12 8QQ, U.K.

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here, we show that some humanised heavy chains (HCs) can fold, form dimers and be secreted even in the absence of a light chain (LC). Quality control of recombinant antibody assembly is thought to rely upon folding of the HC C1 domain. This domain acts as a switch for secretion, only folding upon interaction with the LC C domain. We show that the secreted heavy-chain dimers contain folded C1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent on the HC variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will have an impact on the efficient production of both humanised antibodies and the design of novel antibody formats.
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http://dx.doi.org/10.1042/BCJ20170342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590090PMC
September 2017

Backpack palsy with Horner's syndrome.

BMJ Case Rep 2017 May 22;2017. Epub 2017 May 22.

Department of Plastic Surgery, Salisbury NHS Foundation Trust, Salisbury, UK.

Traumatic injuries to the brachial plexus are typically high impact and can be debilitating, life-changing injuries. Backpack palsy is a rare but well-established cause of brachial plexus injury, arising as a result of heavy backpack use. We present an unusual case of backpack palsy with Horner's syndrome.
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http://dx.doi.org/10.1136/bcr-2017-219402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753723PMC
May 2017

Abnormalities in serum biomarkers correlate with lower cardiac index in the Fontan population.

Cardiol Young 2017 Jan;27(1):59-68

7Department of Pediatrics,Division of Cardiology,The Children's Hospital of Philadelphia,Philadelphia,PennsylvaniaUnited States of America.

Background: Fontan survivors have depressed cardiac index that worsens over time. Serum biomarker measurement is minimally invasive, rapid, widely available, and may be useful for serial monitoring. The purpose of this study was to identify biomarkers that correlate with lower cardiac index in Fontan patients. Methods and results This study was a multi-centre case series assessing the correlations between biomarkers and cardiac magnetic resonance-derived cardiac index in Fontan patients ⩾6 years of age with biochemical and haematopoietic biomarkers obtained ±12 months from cardiac magnetic resonance. Medical history and biomarker values were obtained by chart review. Spearman's Rank correlation assessed associations between biomarker z-scores and cardiac index. Biomarkers with significant correlations had receiver operating characteristic curves and area under the curve estimated. In total, 97 cardiac magnetic resonances in 87 patients met inclusion criteria: median age at cardiac magnetic resonance was 15 (6-33) years. Significant correlations were found between cardiac index and total alkaline phosphatase (-0.26, p=0.04), estimated creatinine clearance (0.26, p=0.02), and mean corpuscular volume (-0.32, p<0.01). Area under the curve for the three individual biomarkers was 0.63-0.69. Area under the curve for the three-biomarker panel was 0.75. Comparison of cardiac index above and below the receiver operating characteristic curve-identified cut-off points revealed significant differences for each biomarker (p<0.01) and for the composite panel [median cardiac index for higher-risk group=2.17 L/minute/m2 versus lower-risk group=2.96 L/minute/m2, (p<0.01)].

Conclusions: Higher total alkaline phosphatase and mean corpuscular volume as well as lower estimated creatinine clearance identify Fontan patients with lower cardiac index. Using biomarkers to monitor haemodynamics and organ-specific effects warrants prospective investigation.
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http://dx.doi.org/10.1017/S1047951116000093DOI Listing
January 2017

Sudden cardiac death in the young: the value of exercise testing.

Authors:
Paul Stephens

Cardiol Young 2017 Jan;27(S1):S10-S18

Division of Cardiology,The Children's Hospital of Philadelphia,Philadelphia,Pennsylvania,United States of America.

Paediatric exercise stress testing has historically been used to assess the functional status of patients after repair of CHDs and to assess the efficacy of medical or device therapy in patients with arrhythmias. Exercise stress testing is one of very few hospital- or clinic-based tests that can assess the response of the cardiopulmonary system in an environment that simulates the body's response to vigorous play and competitive sport. Exercise stress testing is therefore a useful modality in the assessment of child and athletes at risk for sudden cardiac death. The author discusses some cardiovascular maladies that can cause sudden cardiac death by utilising case illustrations as a learning tool.
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http://dx.doi.org/10.1017/S1047951116002171DOI Listing
January 2017

Implementation of a Clinical Pathway for Chest Pain in a Pediatric Emergency Department.

Pediatr Emerg Care 2018 Nov;34(11):778-782

Division of General Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA.

Objective: To evaluate the impact of a pediatric emergency department (ED) chest pain clinical pathway on resource utilization.

Methods: Motivated by perceived overuse of cardiology consultation for non-cardiac chest pain in the ED, clinicians from the Divisions of Cardiology and Emergency Medicine collaboratively developed a chest pain clinical pathway, educated staff, and implemented the pathway on March 1, 2014. We reviewed records of children aged 3 to 18 years without prior diagnoses of heart disease who presented to the ED with chest pain between March 1, 2013, and April 22, 2015. We compared diagnostic testing rates, ED length of stay, and cardiology consults before and after implementation of the pathway.

Results: A total of 1687 patients were pathway eligible (675 patients preimplementation and 1012 postimplementation). Resource utilization was lower than expected before pathway implementation and remained low after implementation. There was a statistically significant reduction in rates of chest x-ray ordering after pathway implementation and ED length of stay but no change in other diagnostic testing or cardiology consultation. Follow-up in our health care system for pediatric chest pain increased from 15% to 29% with implementation, but none of these visits resulted in the diagnosis of a new cardiac condition. There were no instances identified where use of the pathway resulted in missed cardiac disease.

Conclusions: Implementation of a clinical pathway for pediatric chest pain did lead to a reduction in chest x-ray ordering in the ED and was associated with a higher rate of outpatient follow up for non-pathologic chest pain. Preimplementation utilization was lower than the prepathway perceptions of overuse suggested.
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http://dx.doi.org/10.1097/PEC.0000000000000861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359077PMC
November 2018

Chromatin function modifying elements in an industrial antibody production platform--comparison of UCOE, MAR, STAR and cHS4 elements.

PLoS One 2015 7;10(4):e0120096. Epub 2015 Apr 7.

Protein Expression and Purification Group, UCB, Slough, Berkshire, United Kingdom.

The isolation of stably transfected cell lines suitable for the manufacture of biotherapeutic protein products can be an arduous process relying on the identification of a high expressing clone; this frequently involves transgene amplification and maintenance of the clones' expression over at least 60 generations. Maintenance of expression, or cell line stability, is highly dependent upon the nature of the genomic environment at the site of transgene integration, where epigenetic mechanisms lead to variable expression and silencing in the vast majority of cases. We have assessed four chromatin function modifying elements (A2UCOE, MAR X_S29, STAR40 and cHS4) for their ability to negate chromatin insertion site position effects and their ability to express and maintain monoclonal antibody expression. Each element was analysed by insertion into different positions within a vector, either flanking or between heavy chain (HC) and light chain (LC) antibody expression cassettes. Our results clearly show that the A2UCOE is the most beneficial element in this system, with stable cell pools and clones increasing antibody yields 6.5-fold and 6.75-fold respectively. Stability analysis demonstrated that the reduction in antibody expression, seen with cells transfected with the control vector over 120 generations, was mitigated in the clones containing A2UCOE-augmented transgenes. Analysis also showed that the A2UCOE reduced the amount of transgene promoter DNA methylation, which contributed to the maintenance of starting levels of expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120096PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4388700PMC
April 2016

Reduced Culture Temperature Differentially Affects Expression and Biophysical Properties of Monoclonal Antibody Variants.

Antibodies (Basel) 2014 Sep 29;3(3):253-271. Epub 2014 Aug 29.

SUNY College of Nanoscale Science and Engineering, Albany, NY 12203, USA.

Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.
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http://dx.doi.org/10.3390/antib3030253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583600PMC
September 2014

Authors' reply.

J Am Soc Echocardiogr 2014 Mar;27(3):341-2

Department of Pediatric Cardiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1016/j.echo.2013.12.019DOI Listing
March 2014

The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.

MAbs 2014 Jan-Feb;6(1):143-59

UCB Pharma; Berkshire, UK.

Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.
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http://dx.doi.org/10.4161/mabs.27044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929438PMC
October 2014

Elevated left ventricular outflow tract velocities on exercise stress echocardiography may be a normal physiologic response in healthy youth.

J Am Soc Echocardiogr 2013 Dec 15;26(12):1372-8. Epub 2013 Oct 15.

Division of Cardiology, The Children's Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Background: Children with heart disease are at risk for sudden death during exercise, yet decisions regarding sports participation are often based on resting data. Acceleration across the left ventricular outflow tract (LVOT) assessed on stress echocardiography may suggest a diagnosis of hypertrophic cardiomyopathy in patients in whom it is not otherwise obvious. However, the range of peak velocities across the LVOT in healthy youth is unknown. The aim of this study was to describe LVOT velocities with maximal exercise in this age group.

Methods: Subjects up to 18 years old were prospectively enrolled if they had normal results on resting echocardiography and were undergoing exercise testing for other reasons. Subjects with significant comorbidities, suspected cardiomyopathy, or family histories of cardiomyopathy were excluded. Peak LVOT velocities were measured in the upright position using continuous-wave Doppler immediately after maximal exercise.

Results: Fifty subjects (mean age, 13.8 ± 2.8 years) were included. Twenty-eight (56%) were male, and 40 (80%) were Caucasian. The median peak LVOT velocity measured immediately after exercise was 2.5 m/sec (range, 1.3-5.9 m/sec). Sixteen subjects (32%) developed peak LVOT velocities of ≥3 m/sec. Twelve of the 16 (75%) with elevated velocities had a dynamic outflow tract Doppler pattern, of whom eight had evidence of intracavitary narrowing on two-dimensional echocardiography.

Conclusions: The development of significant exercise-induced LVOT velocities may be a normal physiologic finding in healthy youth. The measurement of LVOT velocities alone with maximal exercise may not help distinguish patients with hypertrophic cardiomyopathy from healthy children.
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http://dx.doi.org/10.1016/j.echo.2013.08.020DOI Listing
December 2013

A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression.

Biotechnol Prog 2013 May-Jun;29(3):697-706. Epub 2013 Mar 20.

Protein Expression and Purification Group, UCB, Slough, Berkshire, SL1 4EN, England.

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells but reported yields from Chinese hamster ovary (CHO) cells are lower at ∼300 mg/L. We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X-box binding protein (XBP-1S) and endoplasmic reticulum oxidoreductase (ERO1-Lα) and has been named CHOS-XE. CHOS-XE cells produced increased antibody (MAb) yields (5.3- 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L.
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http://dx.doi.org/10.1002/btpr.1693DOI Listing
January 2014

A family with a complex clinical presentation characterized by arrhythmogenic right ventricular dysplasia/cardiomyopathy and features of branchio-oculo-facial syndrome.

Am J Med Genet A 2013 Feb 10;161A(2):371-6. Epub 2013 Jan 10.

Division of Cardiology, Johns Hopkins University, Baltimore, Maryland, USA.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a familial form of cardiomyopathy typically caused by mutations in genes that encode an element of the cardiac desmosome. Branchio-oculo-facial syndrome (BOFS) is a craniofacial disorder caused by TFAP2A mutations. In a family segregating ARVD/C, some members also had features of BOFS. Genetic testing for ARVD/C identified a mutation in PKP2, encoding plakophilin-2, a component of the cardiac desmosome. Evaluation of dysmorphology by chromosome microarray (CMA) identified a 4.4 Mb deletion at chromosome 6p24 that included both TFAP2A and DSP, encoding desmoplakin, an additional component of the cardiac desmosome implicated in ARVD/C. A family member with both the 6p24 deletion and PKP2 mutation had more severe cardiac dysfunction. These findings suggest that this contiguous gene deletion contributes to both ARVD/C and BOFS, and that DSP haploinsufficiency may contribute to cardiomyopathy. This family provides a clinical example that underscores the need for careful evaluation in clinical scenarios where genetic heterogeneity is known to exist. Finally, it suggests that individuals with unexplained cardiomyopathy and dysmorphic facial features may benefit from CMA analysis.
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http://dx.doi.org/10.1002/ajmg.a.35733DOI Listing
February 2013

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability.

J Biol Chem 2012 Jul 18;287(29):24525-33. Epub 2012 May 18.

UCB Pharma Slough, 208 Bath Road, Slough SL1 3WE, United Kingdom.

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.
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http://dx.doi.org/10.1074/jbc.M112.369744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397877PMC
July 2012

Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in Chinese hamster ovary cells.

Biotechnol Prog 2012 May-Jun;28(3):846-55. Epub 2012 May 21.

Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamental understanding of how mammalian cells handle high levels of transgene expression and of the relationship between sequence and expression are vital to the development of new antibodies and for increasing recombinant antibody titers. In this work, we analyzed a pair of mutants that vary by a single amino acid at Kabat position 49 (heavy-chain framework), resulting in differential transient and stable titers with no apparent loss of antigen affinity. Through analysis of mRNA, gene copy number, intracellular antibody content, and secreted antibody, we found that while translational/post-translational mechanisms are limiting in transient systems, it appears that the amount of available transgenic mRNA becomes the limiting event on stable integration of the recombinant genes. We also show that amino acid substitution at residue 49 results in production of a non-secreted HC variant and postulate that stable antibody expression is maintained at a level which prevents toxic accumulation of this HC-related protein. This study highlights the need for proper sequence engineering strategies when developing therapeutic antibodies and alludes to the early analysis of transient expression systems to identify the potential for aberrant stable expression behavior.
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http://dx.doi.org/10.1002/btpr.1542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394691PMC
November 2012

[Effectiveness of histopathologic examination in a series of 400 forensic autopsies].

Ann Pathol 2012 Feb 20;32(1):4-13. Epub 2012 Jan 20.

Service d'anatomie pathologique, CHU d'Amiens, place Victor-Pauchet, 80054 Amiens cedex 01, France.

Objectives: To assess the usefulness of histopathologic examination in forensic autopsies.

Material And Methods: All consecutive pathological reports and slides from forensic autopsies performed in our department since 2006 have been reviewed.

Results: Four hundred forensic necropsies were reviewed. In only 150 cases (38%), pathologists had data about manner of death and gross autopsy findings. Major diagnoses, related to death, and unsuspected by forensic pathologists, were discovered in 83 cases (21%): in 48 cases (12%) gross examination of the heart, lungs and liver showed gross diagnoses missed by the forensic pathologists, and in only 35 cases (9%) microscopic examination revealed a major unsuspected diagnosis (in brain, heart, lungs, liver, kidney and pancreas specimens). In 213 cases (53%), histopathologic examination confirmed gross autopsy findings and allowed to date some wounds. In 104 cases (26%), microscopic examination was not contributory.

Conclusion: Microscopic examination revealed major diagnoses in less than 10% of forensic autopsies. Its effectiveness is limited for homicides and suicides. Systematic microscopic examination of numerous organs is often useless and should be limited to cases with no anatomic causes of death. Our study emphasizes the need for a better communication between forensic pathologists and histopathologists, and for a better training of some forensic pathologists for gross examination.
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http://dx.doi.org/10.1016/j.annpat.2011.10.011DOI Listing
February 2012

Ventricular tachycardia in infants with structurally normal heart: a benign disorder.

Cardiol Young 2010 Dec 20;20(6):641-7. Epub 2010 Aug 20.

The Children's Hospital of Philadelphia, Pennsylvania, USA.

We evaluated the presentation, treatment, and outcome of infants who present with ventricular tachycardia in the first year of life. Seventy-six infants were admitted to our institution with a diagnosis of ventricular tachycardia between January, 1987 and May, 2006. Forty-five infants were excluded from the study because of additional confounding diagnoses including accelerated idioventricular rhythm, Wolff-Parkinson-White syndrome, supraventricular tachycardia with aberrancy, long QT syndrome, cardiac rhabdomyoma, myocarditis, congenital lesions, or incomplete data. The remaining 31 included infants who had a median age at presentation of 1 day, with a range from 1 to 255 days, and a mean ventricular tachycardia rate of 213 beats per minute, with a range from 171 to 280, at presentation. The infants were treated chronically with propranolol (38.7%), amiodarone (12.9%), mexiletine (3.2%), propranolol and mexiletine (9.7%), or propranolol and procainamide (6.5%). The median duration of treatment was 13 months, with a range from 3 to 105 months. Ventricular tachycardia resolved spontaneously in all infants. No patient died, or received catheter ablation or device therapy. Median age at last ventricular tachycardia was 59 days, with a range from 1 to 836 days. Mean follow-up was 45 months, with a range from 5 to 164 months, with a mean ventricular tachycardia-free period of 40 months. Infants with asymptomatic ventricular tachycardia, a structurally normal heart, and no additional electrophysiological diagnosis all had spontaneous resolution of tachycardia. Furthermore, log-rank analysis of the time to ventricular tachycardia resolution showed no difference between children who received chronic outpatient anti-arrhythmic treatment and those who had no such therapy. While indications for therapy cannot be determined from this study, lack of symptoms or myocardial dysfunction suggests that therapy may not be necessary.
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http://dx.doi.org/10.1017/S1047951110000867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751393PMC
December 2010

Conducting rehabilitation groups for people suffering from chronic pain.

Int J Nurs Pract 2010 Jun;16(3):233-40

Department of Health Studies, Faculty of Social Sciences, University of Stavanger, Stavanger, Norway.

The aim of this study was to offer guidelines for counsellors who work with rehabilitation groups of patients with chronic pain. The sample involved nine counsellors engaged in a multidisciplinary pain management programme. Two focus group interviews were conducted. Data were analysed using qualitative content analysis. These indicate that main challenges facing counsellors were related to maintaining constructive group processes and being mentally prepared. The counsellors reported that knowledge concerning self-awareness, theoretical frameworks and counselling techniques was important. Personal learning included: group leadership, teamwork, grasping the inside story and obtaining supervision. The results show how important it is to have trained counsellors that are well prepared to prevent and deal with challenging group processes. Counsellors need to understand the concept of pain and be acquainted with cognitive behavioural framework and group processes. The results indicate that counsellors perceive regular supervision as supportive and is likely to promote good team functioning.
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http://dx.doi.org/10.1111/j.1440-172X.2010.01825.xDOI Listing
June 2010

A comparison of the thermal properties of 2- and 3-fluted drills and the effects on bone cell viability and screw pull-out strength in an ovine model.

Clin Biomech (Bristol, Avon) 2010 Jul 1;25(6):613-7. Epub 2010 Apr 1.

Surgical and Orthopaedic Research Laboratories, University of New South Wales, Sydney, Australia.

Background: Drilling of bone is associated with an increase in temperature of the surrounding bone which may result in osteonecrosis.

Methods: In this study, cutting efficiency and thermal properties of one 2-fluted drill and two 3-fluted drills were determined in vitro using a porcine model. Drills were then used to create pilot holes in an in vivo ovine model to facilitate implantation of pedicle screws. The effect of the characteristic thermal profiles of each drill on cortical bone cell viability and screw pull-out strength was then assessed.

Findings: Cutting efficiencies of both 3-fluted designs were found to be greater than that of the 2-fluted drill, but this did not translate into a decrease in the maximum temperatures during drilling for both drills. Histologically, no empty osteocyte lacunae were seen at 2 or 4 weeks, suggesting that temperatures were not sufficiently high enough to induce thermonecrosis in the ovine tibia. No differences were found in the pull-out strength of the screws.

Interpretation: Both 2- and 3-fluted drills are currently in clinical use. Despite the theoretical advantage that 3-fluted drills possess over their 2-fluted counterparts, there is a lack of evidence in the literature in support of their use. In this study the observed increases in cutting efficiency of the 3-fluted drills tested did not translate into a reduction in heat generation or improvement in bone healing or screw fixation.
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http://dx.doi.org/10.1016/j.clinbiomech.2010.02.007DOI Listing
July 2010

High resolution NMR-based model for the structure of a scFv-IL-1beta complex: potential for NMR as a key tool in therapeutic antibody design and development.

J Biol Chem 2009 Nov 23;284(46):31928-35. Epub 2009 Sep 23.

Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom.

Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1beta. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.
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http://dx.doi.org/10.1074/jbc.M109.025304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797264PMC
November 2009

Asymptomatic anomalous right coronary artery from the pulmonary trunk.

Cardiol Young 2009 Aug 3;19(4):391-2. Epub 2009 Jun 3.

Division of Cardiology at The Children's Hospital of Philadelphia, PA, USA.

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http://dx.doi.org/10.1017/S1047951109990217DOI Listing
August 2009

A short treatment with an antibody to sclerostin can inhibit bone loss in an ongoing model of colitis.

J Bone Miner Res 2009 Oct;24(10):1662-71

Department of Inflammation Biology, UCB Celltech, Slough, United Kingdom.

Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl-AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation-induced bone loss. At the end of the experiment, Scl-AbI-treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl-Ab)-treated animals. In a second experiment, treatment with Scl-AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age-matched control mice (maximum load, -26%, p < 0.05; energy, -37%, p < 0.05; ultimate strength, -33%, p < 0.05; elastic modulus, -17%, p < 0.05). A short treatment with Scl-AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl-AbI-treated animals were not significantly different from those of noncolitic age-matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclast-mediated bone resorption.
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http://dx.doi.org/10.1359/jbmr.090403DOI Listing
October 2009

Anomalous aortic origin of a coronary artery with an interarterial course: should family screening be routine?

J Am Coll Cardiol 2008 May;51(21):2062-4

Division of Cardiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

Objectives: We sought to present cases of familial occurrence of anomalous aortic origin of a coronary artery with an interarterial course (AAOCA) to determine if it would alter our current screening and management recommendations.

Background: Anomalous aortic origin of a coronary artery with an interarterial course is a rare congenital anomaly that carries an increased risk of sudden death in children and young adults. There are no reports in the literature of familial AAOCA in the pediatric population.

Methods: In preparation for a multi-institutional prospective study evaluating patient management and surgical outcomes in children and young adults with AAOCA, a questionnaire was sent to multiple pediatric institutions in North and South America. Several respondents indicated caring for families with more than 1 member with AAOCA. These patients were identified and charts were retrospectively reviewed.

Results: We identified 5 families in which a child was diagnosed with AAOCA and another family member was subsequently identified through screening with echocardiography. The odds of this occurring are significantly greater than what would be expected by chance. All identified by screening were asymptomatic and had anomalous right coronary artery despite 2 of the 5 index cases having anomalous left coronary artery.

Conclusions: It is possible that there is a genetic link for AAOCA. Future research into this is warranted. Due to the potential risk of myocardial ischemia and sudden death associated with AAOCA, screening first-degree relatives for AAOCA using transthoracic echocardiography would be the prudent approach to potentially prevent a sudden catastrophic event.
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http://dx.doi.org/10.1016/j.jacc.2008.01.052DOI Listing
May 2008

Identification, imaging, functional assessment and management of congenital coronary arterial abnormalities in children.

Cardiol Young 2007 Sep;17 Suppl 2:56-67

Section of Pediatric Cardiology, Yale University and the Yale New Haven Children's Hospital, New Haven, Connecticut 06520-8064, United States of America.

The coronary arteries, the vessels through which both substrate and oxygen are provided to the cardiac muscle, normally arise from paired stems, right and left, each arising from a separate and distinct sinus of the aortic valve. The right coronary artery runs through the right atrioventricular groove, terminating in the majority of instances in the inferior interventricular groove. The main stem of the left coronary artery bifurcates into the anterior descending, or interventricular, and the circumflex branches. Origin of the anterior descending and circumflex arteries from separate orifices from the left sinus of Valsalva occurs in about 1% of the population, while it is also frequent to find the infundibular artery arising as a separate branch from the right sinus of Valsalva. Anomalies of the coronary arteries can result from rudimentary persistence of an embryologic coronary arterial structure, failure of normal development or normal atrophy as part of development, or misplacement of connection of a an otherwise normal coronary artery. Anomalies, therefore, can be summarized in terms of abnormal origin or course, abnormal number of coronary arteries, lack of patency of the orifice of coronary artery, or abnormal connections of the arteries. Anomalous origin of the left coronary artery from the pulmonary trunk occurs with an incidence of approximately 1 in 300,000 children. The degree of left ventricular dysfunction produced likely relates to the development of collateral vessels that arise from the right coronary artery, and provide flow into the left system. Anomalous origin of either the right or the left coronary artery from the opposite sinus of Valsalva can be relatively innocuous, but if the anomalous artery takes an interarterial course between the pulmonary trunk and the aorta, this can underlie sudden death, almost invariably during or immediately following strenuous exercise or competitive sporting events. Distal anomalies of the coronary arteries most commonly involve abnormal connections, or fistulas, between the right or left coronary arterial systems and a chamber or vessel. We discuss the current techniques available for imaging these various lesions, along with their functional assessment, concluding with a summary of current strategies for management.
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http://dx.doi.org/10.1017/S1047951107001163DOI Listing
September 2007

Effect of low intensity pulsed ultrasound on healing of an ulna defect filled with a bone graft substitute.

J Biomed Mater Res B Appl Biomater 2008 Jul;86(1):74-81

Surgical and Orthopaedic Research Laboratories, University of New South Wales, Division of Surgery, Prince of Wales Hospital, Randwick, New South Wales, Australia.

A 1.5 cm unilateral rabbit ulna defect model was performed in 18 adult NZ white rabbits. The defects were filled with a beta-tricalcium phosphate bone graft substitute (JAX TCP). The surgical site in half the animals was treated daily with 20 min of low intensity pulsed ultrasound (LIPUS). Animals were sacrificed at 4 weeks (n = 3 per group) or 12 weeks (n = 6 per group) following surgery for radiographic and histologic endpoints. Radiography revealed some resorption of the JAX TCP by 12 weeks in the control and LIPUS treated groups. LIPUS treatment did not accelerate this resorption. Some new bone formation was noted in the control groups at the defect margins while little bone formed in the center of the defect at 4 and 12 weeks. In contrast, radiographs revealed more new bone at 4 and 12 weeks in the LIPUS treated animals throughout the section. Bone mineral density (DEXA) revealed a statistically significant difference at 4 weeks with LIPUS while no differences were found at 12 weeks. Histology of the LIPUS treated sections demonstrated new woven bone formation on and between the JAX TCP bone graft substitute particles across the defect. VEGF expression was increased with LIPUS treatment at 4 weeks and remained elevated at 12 weeks compared with controls. CBFA-1 expression levels were elevated with LIPUS treatment at both time points. LIPUS treatment increased bone formation in ulna defect healing with a beta-tricalcium phosphate bone graft substitute.
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http://dx.doi.org/10.1002/jbm.b.30989DOI Listing
July 2008

Comparison of poly-L-lactide and polylactide carbonate interference screws in an ovine anterior cruciate ligament reconstruction model.

Arthroscopy 2007 Jul;23(7):757-65, 765.e1-2

Surgical & Orthopaedic Research Laboratories, University of New South Wales, Prince of Wales Hospital, Sydney, Australia.

Purpose: The purpose of this study was to compare polylactide carbonate (PLC) interference screws with poly-L-lactide (PLLA) screws in an ovine anterior cruciate ligament reconstruction model.

Methods: A PLC screw or PLLA screw was placed in the center of a 4-strand soft-tissue autograft fixating the graft within the tibial tunnel. Assessments were made at 6 and 12 weeks for fixation strength and at time points of 6, 12, 26, and 52 weeks via computed tomography and histology.

Results: No adverse or inflammatory reactions were noted for either material at any time point. Mechanical fixation strength increased from 6 to 12 weeks for both the PLC and PLLA screws, with no significant differences in fixation strength being found between the 2 groups. By 26 weeks, the PLC screw was partially replaced by new bone, a process that was completed by 52 weeks. The PLLA screws were intact and surrounded by a fibrous layer at 52 weeks with no obvious resorption. New bone formation within the tendon construct located in the bone tunnel proximal to the interference screw was also noted in the PLC screw group but was not observed in the PLLA group.

Conclusions: This study has supported the hypothesis that this bioabsorbable composite has sufficient mechanical properties and strength retention to function successfully as an interference screw but also stimulates a biologic healing response, enabling replacement by bone and tunnel healing.

Clinical Relevance: This study shows both the satisfactory mechanical characteristics and osteoconductive nature of PLC used in an interference screw in an ovine anterior cruciate ligament reconstruction model.
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http://dx.doi.org/10.1016/j.arthro.2007.01.030DOI Listing
July 2007

Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.

Inflamm Bowel Dis 2007 Nov;13(11):1323-32

Inflammation Discovery, UCB Celltech, Slough, UK.

Background: Inhibitors of tumor necrosis factor alpha (TNFalpha) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti-TNFalpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems.

Methods: The ability of each anti-TNFalpha agent to neutralize soluble and membrane-bound TNFalpha; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)-induced interleukin (IL)-1beta production by human monocytes was measured in vitro.

Results: All 4 agents neutralized soluble TNFalpha and bound to and neutralized membrane TNFalpha. Infliximab and adalimumab were comparable in their ability to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS-induced IL-1beta production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept.

Conclusions: In contrast to the other anti-TNFalpha agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti-TNFalpha agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFalpha agents in CD.
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http://dx.doi.org/10.1002/ibd.20225DOI Listing
November 2007