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OBJECTIVES: To determine the long-term outcomes among a cohort of patients with Kawasaki disease (KD) and a history of giant coronary artery aneurysms (CAAs) at a single US center. STUDY DESIGN: Medical records for all patients with KD and giant CAAs at a pediatric academic institution were reviewed. Primary outcomes included major adverse cardiovascular events (MACE) and normalization of CA luminal diameter, using Kaplan-Meier analyses. RESULTS: There were 60 patients with KD and giant CAAs identified between 1989 and 2023. The majority of patients were male (71.7%) with a median age at diagnosis of 0.9 years (range, 0.2-13.3 years). Patients were followed for a median of 11 years, up to 34.5 years. MACE occurred in 13 patients (21.7%) at a median of 1.4 years (range, 0.04-22.6 years) after KD diagnosis. The 10-, 20-, and 30-year MACE-free rates were 75%, 75%, and 60%. Patients with maximal CA z scores of ≥20 or bilateral CAA were more likely to have MACE. During follow-up, 26.7% of CAA regressed to a normal luminal diameter at a median of 3.6 years (range, 0.6-12.0 years). The 10-, 20- and 30-year likelihood of CA regression to normal luminal diameter was 36%, 46%, and 46%. CONCLUSIONS: Over 30 years, MACE occurred in nearly 22% of patients, more often in those with bilateral CAA or CA z scores of ≥20. Despite regression to a normal luminal diameter in >25% of CAAs, patients with a history of KD-associated giant CAA require ongoing surveillance for cardiac complications, even years after the initial disease.
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OBJECTIVE: To retrospectively report the historical and clinical findings, diagnostics, treatment, and outcome of horses with penetrating wood foreign bodies (PWFBs) of the coronary band. ANIMALS: 15 client-owned horses. CLINICAL PRESENTATION: Horses had varying degrees of lameness and soft tissue swelling of the coronary band and pastern region. A defect in the coronary band was identified, but the actual wood foreign body was not always readily visualized. RESULTS: Medical records of horses diagnosed with PWFBs of the coronary band between 2004 and 2023 were reviewed. Information retrieved from the medical records included history, signalment, diagnostics, treatment, and outcome. Thirteen of 15 horses that sustained a PWFB to the coronary band were participating in foxhunting. Penetrating wood foreign bodies occurred more frequently near the central axis or toe region (11/15) and more commonly in the forelimbs (11/15). Removal of PWFBs can be performed with the horse standing and sedated with regional anesthesia. Complete removal of the PWFB required partial removal of the adjacent hoof wall. CLINICAL RELEVANCE: Penetrating wood foreign bodies occurred in the coronary band and lodged distally in the hoof wall of horses. Foxhunting may be a risk factor for this type of injury. Penetrating wood foreign bodies occurred most commonly in the front feet, near the central axis of the coronary band. Complete removal of the PWFB required removing a section of the adjacent hoof wall. The prognosis for return to the previous level of activity following treatment was favorable.
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Corpos Estranhos , Madeira , Animais , Cavalos , Corpos Estranhos/veterinária , Corpos Estranhos/cirurgia , Masculino , Estudos Retrospectivos , Feminino , Casco e Garras/patologia , Doenças dos Cavalos/cirurgiaRESUMO
In children with hypertrophic cardiomyopathy (HCM), the genotype-phenotype association of abnormal electrocardiographic (ECG) features in the backdrop of gene positivity has not been well described. This study aimed to describe the abnormal ECG findings in children with HCM harboring who have genetic variants and determine the association with major adverse cardiac events (MACE). We retrospectively analyzed 81 variants-positive, phenotype-positive (V+P+), 66 variant-positive, phenotype-negative (V+P-), and 85 non-sarcomeric subjects. We analyzed ECG findings and clinical outcomes in the three groups of subjects. Repolarization abnormalities (ST and T wave changes) and pathologic Q waves were the most common abnormalities in variant and non-sarcomeric subjects. The V+P+ group showed higher occurrence of ST segment changes and T wave abnormalities compared to V+P- group. Independent predictors of MACE included ST segment changes (OR 3.54, CI 1.20-10.47, p = 0.022). T wave changes alone did not predict outcome (OR 2.13, CI 0.75-6.07, p = 0.157), but combined repolarization abnormalities (ST+T changes) were strong predictors of MACE (OR 5.84, CI 1.43-23.7, p = 0.014) than ST segment changes alone. Maximal wall z score by echocardiography was a predictor of MACE (OR 1.21, CI 1.07-1.37, p = 0.002). Despite the presence of significant myocardial hypertrophy (z score > 4.7), voltage criteria for LVH were much less predictive. In the non-sarcomeric group, RVH was significantly associated with MACE (OR 3.85, CI 1.08-13.73, p = 0.038). These abnormal ECG findings described on the platform of known genetic status and known myocardial hypertrophy may add incremental value to the diagnosis and surveillance of disease progression in children with HCM. Select ECG findings, particularly repolarization abnormalities, may serve as predictors of MACE in children.
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Background: Exercise stress testing (EST) in pediatric hypertrophic cardiomyopathy (HCM) patients has not well described in a large heterogenous cohort. Objectives: The objective of the study was to determine the clinical utility of EST in pediatric HCM. Methods: This was a retrospective single-center analysis of HCM patients younger than 21 years who had EST between January 1, 2000, and January 1, 2019. Clinical, demographic characteristics, and EST data were analyzed, using the last EST during the study or prior to the event in subjects with a primary outcome. The primary composite endpoint included cardiac death, transplant, or arrhythmia requiring implantable cardioverter-defibrillator placement. Outcome analysis was performed using Cox proportional hazard modeling. Results: The study cohort included 140 patients, 52% with a recognized genetic variant. There were 2 tests aborted due to safety concerns (ST-segment changes, ventricular ectopy). The median age at first EST was 13.6 years. Ninety percent of patients were tested using cycle ergometry, and 44% were on a beta-blocker. The median peak oxygen consumption was 37.1 mL/kg/min (IQR: 12.5 mL/kg/min) or 81.2% predicted, the mean anaerobic threshold was 21.8 Ml (IQR: 8.3 mL), and the median peak power was 2.6 ± 1.1 W/kg or 73.7% predicted. Ectopy during EST was seen in 44% of patients, and 8% had an abnormal blood pressure response to exercise. The endpoint was reached in 12 patients. The presence of any degree of ectopy was a predictor of the composite endpoint (hazard ratio: 5.8; 95% CI: 1.3-26.7). Conclusions: EST is clinically useful in select pediatric patients with HCM. Ectopy on EST is a risk factor for cardiac death, cardiac transplant, and arrhythmias requiring implantable cardioverter-defibrillator.
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Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.
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Fragmentos Fab das Imunoglobulinas , Imunoglobulina G , Proteínas Recombinantes , Animais , Células CHO , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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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Anemia Falciforme/fisiopatologia , Função Ventricular Esquerda/fisiologia , Função Ventricular Direita/fisiologia , Adolescente , Anemia Falciforme/complicações , Criança , Progressão da Doença , Ecocardiografia , Feminino , Humanos , Masculino , Estudos Retrospectivos , Medição de RiscoRESUMO
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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Procedimentos Cirúrgicos Cardiovasculares/efeitos adversos , Complicações Pós-Operatórias/etiologia , Síndrome de Cimitarra/cirurgia , Estenose de Veia Pulmonar/etiologia , Ecocardiografia Doppler , Feminino , Seguimentos , Átrios do Coração/cirurgia , Humanos , Lactente , Recém-Nascido , Masculino , Veias Pulmonares/anormalidades , Veias Pulmonares/diagnóstico por imagem , Veias Pulmonares/patologia , Veias Pulmonares/cirurgia , Estudos Retrospectivos , Fatores de Risco , Síndrome de Cimitarra/diagnóstico por imagem , Grau de Desobstrução VascularRESUMO
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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Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Complicações Pós-Operatórias/etiologia , Veias Pulmonares/cirurgia , Síndrome de Cimitarra/cirurgia , Angiografia/métodos , Criança , Humanos , Lactente , Recém-Nascido , Masculino , Veias Pulmonares/patologia , Reoperação/estatística & dados numéricos , Estudos RetrospectivosRESUMO
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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Dor no Peito/diagnóstico , Procedimentos Clínicos/estatística & dados numéricos , Serviço Hospitalar de Emergência/estatística & dados numéricos , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Adolescente , Criança , Pré-Escolar , Feminino , Recursos em Saúde/estatística & dados numéricos , Humanos , Tempo de Internação/estatística & dados numéricos , Masculino , Ensaios Clínicos Controlados não Aleatórios como Assunto , Encaminhamento e Consulta/estatística & dados numéricosRESUMO
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 in vivo is thought to rely upon folding of the HC CH1 domain. This domain acts as a switch for secretion, only folding upon interaction with the LC CL domain. We show that the secreted heavy-chain dimers contain folded CH1 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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Anticorpos Monoclonais/biossíntese , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Células CHO , Cricetulus , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Dobramento de Proteína , Proteínas Recombinantes/químicaRESUMO
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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Neuropatias do Plexo Braquial/etiologia , Plexo Braquial/lesões , Síndrome de Horner/complicações , Militares , Paralisia/etiologia , Adulto , Humanos , Masculino , Suporte de CargaRESUMO
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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Biomarcadores/sangue , Débito Cardíaco/fisiologia , Técnica de Fontan/métodos , Cardiopatias Congênitas/sangue , Monitorização Fisiológica/métodos , Adolescente , Adulto , Criança , Feminino , Seguimentos , Cardiopatias Congênitas/fisiopatologia , Cardiopatias Congênitas/cirurgia , Humanos , Masculino , Prognóstico , Estudos Prospectivos , Curva ROC , Adulto JovemRESUMO
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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Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiologia , Morte Súbita Cardíaca/etiologia , Teste de Esforço , Exercício Físico/fisiologia , Adolescente , Atletas , Criança , Ecocardiografia , Eletrocardiografia , Humanos , EsportesRESUMO
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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Formação de Anticorpos/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Elementos Reguladores de Transcrição/genética , Transgenes/fisiologia , Animais , Células CHO , Cricetulus , Metilação de DNA , Epigênese Genética/genética , Inativação Gênica , Vetores Genéticos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Regiões Promotoras Genéticas/genéticaAssuntos
Adolescente/fisiologia , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Erros de Diagnóstico/prevenção & controle , Ecocardiografia Doppler/métodos , Teste de Esforço , Ventrículos do Coração/diagnóstico por imagem , Obstrução do Fluxo Ventricular Externo/diagnóstico por imagem , Feminino , Humanos , MasculinoRESUMO
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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Anticorpos Monoclonais , Células da Medula Óssea/imunologia , Imunoglobulina G , Plasmócitos/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Fluorescência , Células HEK293 , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Plasmócitos/citologia , Coelhos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Fatores de TempoRESUMO
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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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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Adolescente/fisiologia , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Erros de Diagnóstico/prevenção & controle , Ecocardiografia Doppler/métodos , Teste de Esforço , Ventrículos do Coração/diagnóstico por imagem , Obstrução do Fluxo Ventricular Externo/diagnóstico por imagem , Reações Falso-Positivas , Feminino , Humanos , Masculino , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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 â¼300 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.