Examining the application of the IDEAL framework in the reporting and evaluation of innovative invasive procedures: secondary qualitative analysis of a systematic review.

OBJECTIVES: The development of new surgical procedures is fundamental to advancing patient care. The Idea, Developments, Exploration, Assessment and Long-term (IDEAL) framework describes study designs for stages of innovation. It can be difficult to apply due to challenges in defining and identifying innovative procedures. This study examined how the IDEAL framework is operationalised in real-world settings; specifically, the types of innovations evaluated using the framework and how authors justify their choice of IDEAL study design. DESIGN: Secondary qualitative analysis of a systematic review. DATA SOURCES: Citation searches (Web of Science and Scopus) identified studies following the IDEAL framework and citing any of the ten key IDEAL/IDEAL_D papers. ELIGIBILITY CRITERIA: Studies of invasive procedures/devices of any design citing any of the ten key IDEAL/IDEAL_D papers. DATA EXTRACTION AND SYNTHESIS: All relevant text was extracted. Three frameworks were developed, namely: (1) type of innovation under evaluation; (2) terminology used to describe stage of innovation and (3) reported rationale for IDEAL stage. RESULTS: 48 articles were included. 19/48 described entirely new procedures, including those used for the first time in a different clinical context (n=15/48), reported as IDEAL stage 2a (n=8, 53%). Terminology describing stage of innovation was varied, inconsistent and ambiguous and was not defined. Authors justified their choice of IDEAL study design based on limitations in published evidence (n=36) and unknown feasibility and safety (n=32) outcomes. CONCLUSION: Identifying stage of innovation is crucial to inform appropriate study design and governance decisions. Authors' rationale for choice of IDEAL stage related to the existing evidence base or lack of sufficient outcome data for procedures. Stage of innovation was poorly defined with inconsistent descriptions. Further work is needed to develop methods to identify innovation to inform practical application of the IDEAL framework. Defining the concept of innovation in terms of uncertainty, risk and degree of evidence may help to inform decision-making.

Evolution of Food Protein-Induced Enterocolitis Syndrome (FPIES) Index Trigger Foods and Subsequent Reactions After Initial Diagnosis.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy treated by trigger food avoidance and supportive care. Whether the prevalence of different trigger foods is changing with evolving food introduction patterns is unknown. The rate and nature of subsequent reactions after initial diagnosis have not been fully studied. OBJECTIVE: We sought to characterize how trigger foods have changed over time and investigate the nature of subsequent reactions after initial diagnosis. METHODS: We collected data regarding patients' FPIES reactions from 347 patients seen in the University of Michigan Allergy and Immunology clinic for FPIES from 2010 to 2022. Inclusion criteria consisted of pediatric patients diagnosed with FPIES by an allergist based on international consensus guidelines. RESULTS: Most foods including less commonly cited FPIES triggers increased in frequency over time. The most common index trigger was oat. A total of 32.9% (114 of 347) patients experienced a subsequent reaction after education on trigger avoidance and safe home introduction of new foods, with 34.2% (41 of 120) of subsequent reactions to new triggers at home and 45% (54 of 120) to known triggers at home. Of patients reacting subsequently, 28% (32 of 114) experienced a subsequent reaction necessitating an emergency department visit. The most common new subsequent reaction triggers were egg and potato, whereas peanut most commonly triggered reactions on oral food challenge. CONCLUSIONS: The risk profile of FPIES triggers may be evolving over time, though high-risk FPIES foods remain common. The subsequent reaction rate after counseling indicates that home food introduction poses risk. This study highlights the need for improved safety of new food introduction and/or prediction methods for FPIES to help prevent potentially dangerous home FPIES reactions.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

The Assembled Genome of the Stroke-Prone Spontaneously Hypertensive Rat.

BACKGROUND: We report the creation and evaluation of a de novo assembly of the genome of the spontaneously hypertensive rat, the most widely used model of human cardiovascular disease. METHODS: The genome is assembled from long read sequencing (PacBio HiFi and continuous long read data [CLR]) and scaffolded with long-range structural information obtained from Bionano optical maps and proximity ligation sequencing proximity analysis of the genome. The genome assembly was polished with Illumina short reads. Completeness of the assembly was investigated using Benchmarking Universal Single Copy Orthologs analysis. The genome assembly was also evaluated with the rat reference gene set, using NCBI automated protocols. We also generated orthogonal single molecule transcript sequence reads (Iso-Seq) from 8 tissues and used them to validate the coding assembly, to annotate the assembly with RNA transcripts representing unique full length transcript isoforms for each gene and to determine whether divergences between RefSeq sequences and the assembly were attributable to assembly errors or polymorphisms. RESULTS: The assembly analysis indicates that this assembly is comparable in contiguity and completeness to the current rat reference assembly, while the use of HiFi sequencing yields an assembly that is more correct at the single base level. Synteny analysis was performed to uncover the extent of synteny and the presence and distribution of chromosomal rearrangements between the reference and this assembly. CONCLUSION: The resulting genome assembly is reference quality and captures significant structural variation.

Reference long-read isoform-aware transcriptomes of 4 human peripheral blood lymphocyte subsets.

Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.

mRatBN7.2: familiar and unfamiliar features of a new rat genome reference assembly.

Rat genomic tools have been slower to emerge than for those of humans and mice and have remained less thorough and comprehensive. The arrival of a new and improved rat reference genome, mRatBN7.2, in late 2020 is a welcome event. This assembly, like predecessor rat reference assemblies, is derived from an inbred Brown Norway rat. In this "user" survey we hope to provide other users of this assembly some insight into its characteristics and some assessment of its improvements as well as a few caveats that arise from the unique aspects of this assembly. mRatBN7.2 was generated by the Wellcome Sanger Institute as part of the large Vertebrate Genomes Project. This rat assembly has now joined human, mouse, chicken, and zebrafish in the National Center for Biotechnology Information (NCBI)'s Genome Reference Consortium, which provides ongoing curation of the assembly. Here we examine the technical procedures by which the assembly was created and assess how this assembly constitutes an improvement over its predecessor. We also indicate the technical limitations affecting the assembly, providing illustrations of how these limitations arise and the impact that results for this reference assembly.

Identifying barriers to utilization of a medication access program among referred patients surveyed after discharge from an acute care hospital.

BACKGROUND: For uninsured residents of select counties in North Carolina, the Cumberland County Medication Access Program (CCMAP) provides prescriptions at no cost. Uninsured patients hospitalized at Cape Fear Valley Medical Center are referred to CCMAP at discharge by Cape Fear Valley Health System employees, primarily coordination of care personnel and outpatient pharmacy personnel. The purpose of this study was to describe the most frequently reported utilization barriers among surveyed patients referred to CCMAP after discharge from Cape Fear Valley Medical Center. METHODS: This was a single-center, survey-based, descriptive research study. Referring Cape Fear Valley Health System employees collected the medical record number of patients referred to CCMAP at discharge between October 22, 2020 and December 31, 2020. These patients were contacted via a research team member by telephone at least 30 days after discharge to voluntarily participate in a survey regarding their ability to receive prescriptions from CCMAP after discharge. Patient-reported utilization barriers and demographics were recorded. A similar survey was voluntarily completed by referring health system employees. Employee-reported utilization barriers were collected to identify discrepancies in perceived utilization barriers among discharged patients and referring health system employees. RESULTS: There were 69 patients referred to CCMAP at discharge by outpatient pharmacy personnel. A total of 17 patients met inclusion criteria and completed the survey. Of these, 35.29% of the patients reported their greatest utilization barrier to be uncertainty about how to apply for CCMAP. In addition, 25 surveys were completed by referring outpatient pharmacy personnel. Of these, 56% of the participants reported they believe the greatest utilization barrier to be patient uncertainty about how to apply for CCMAP. CONCLUSIONS: Uninsured patients discharged from Cape Fear Valley Medical Center could benefit from increased assistance with completing CCMAP applications and enrollment with the program before discharge to improve continuity of care.

Retrospective assessment of antimicrobial stewardship initiative in outpatient use of ertapenem for uncomplicated extended spectrum beta lactamase Enterobacteriaceae urinary tract infections.

BACKGROUND: Urinary tract infections (UTI) are often over-diagnosed and over-treated, which can induce and select for resistant pathogens. After observing wide-spread outpatient use of ertapenem, a broad-spectrum antibiotic, a structured antimicrobial stewardship initiative (ASI) to improve appropriate antimicrobial prescribing was undertaken. ASI objectives were to achieve a goal of reducing ertapenem utilization for extended spectrum beta lactamase Enterobacteriaceae (ESBL-EB) UTI by 10% and evaluate the clinical outcomes associated with the ASI. METHODS: A pre-to-post cohort study was conducted at a single-center integrated healthcare system between November 1, 2014 and February 26, 2017. An intensive, 90-day, pharmacist-driven, structured ASI was implemented between November 1, 2015 and January 29, 2016. Female patients aged ≥18 years who were treated for an uncomplicated, ESBL-EB urinary tract infection (UTI) were included. Primary outcome was clinical resolution defined as cure, persistence, relapse and recurrence. Secondary outcome measured was monthly ertapenem use expressed as number of days of therapy (DOT)/1000 adjusted patient days (APD). Segmented regression analysis for interrupted time series was performed to estimate ASI intervention effect. RESULTS: A total of 184 patients were included in the study. Ertapenem utilization decreased from 0.0145 DOT/1000 APD in Nov. 2014 to 0.0078 DOT/1000 APD Feb. 2017(p < 0.01). The mean ertapenem DOT declined 19% overall from the pre vs. post intervention periods (32 vs 26, p < 0.01). Frequency of recurrent UTIs between treatments did not significantly differ and no adverse effects were reported in patients treated with aminoglycosides. CONCLUSIONS: A structured ASI for uncomplicated ESBL-EB UTI was associated with a clinically meaningful decrease in ertapenem utilization and once-daily, 5-day aminoglycoside treatment was well-tolerated.

Cohort Generation and Characterization of Patient-Specific Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Homozygous familial hypercholesterolemia (hoFH) is a rare disorder caused primarily by pathological mutations in the low-density lipoprotein receptor (LDLR), which disrupts LDL-cholesterol (LDL-C) metabolism homeostasis. hoFH patients are at extremely high risk for cardiovascular disease and are resistant to standard therapies. LDLR knockout animals and in vitro cell models overexpressing different mutations have proved useful, but may not fully recapitulate human LDLR mutation biology. We and others have generated induced pluripotent stem cells (iPSC) from hoFH patient's fibroblasts and T cells and demonstrated their ability to recapitulate hoFH biology. In this study, we present the generation and characterization of a cohort of seven hoFH-iPSC lines derived from peripheral blood mononuclear cells (PBMC) collected from four homozygous and three compound heterozygous patients. The hoFH-iPSC cohort demonstrated a wide range of LDLR expression and LDL-C internalization in response to rosuvastatin that correlated with the predicted pathogenicity of the mutation. We were able to confirm that hoFH-iPSC cohort were pluripotent by differentiation toward all three germ layers and specifically to hepatocyte-like cells (HLC), the cell with primary LDL-C metabolic regulatory control, by expression of hepatocyte markers. hoFH patient PBMC-derived iPSC recapitulate the LDLR dysfunction of their specific mutation. They were capable of differentiating to HLC and could be useful for early developmental studies, pharmacology/toxicology, and potentially autologous cell therapy.

A Stabilizing Agent, PCA/DTPA, Improves Plasma Storage Life for the Chromsystems Vitamin C Assay up to Six Months.

The commonly used Chromsystems vitamin C (ascorbate) assay (Munich, Germany) has a sample storage life of five days at -20°C. Stabilizing agents have been successfully used to increase longevity; however, their suitability with this commercial assay is unclear. We investigated the compatibility of a stabilizing agent, perchloric acid/diethylenetriaminepentaacetic acid (PCA/DTPA), with the Chromsystems assay. Plasma was stored at -80°C, with or without PCA/DTPA. Storage up to six months was assessed through baseline and repeat analyses, stability was assessed by comparing paired non-stabilized and PCA/DTPA-stabilized plasma, and performance was assessed using allowable performance specifications of an external quality assurance program. Ascorbate concentration was significantly lower in non-stabilized plasma than in paired PCA/DTPA-stabilized plasma, with a proportional difference of 11% (P=0.01). All storage analysis results were within the allowable performance specifications. Storage at -80°C prevented plasma ascorbate oxidation; however, substantial oxidation occurred during sample processing. In conclusion, PCA/DTPA significantly reduces ascorbate oxidation, and PCA/DTPA-stabilized ascorbate plasma is compatible with the Chromsystems assay and stable for up to six months, when stored at -80°C.

Impact of health system protocol on patient qualification for influenza point-of-care testing.

BACKGROUND: Point-of-care testing (POCT) is a service that community pharmacies are implementing to increase patient access to care. Many pharmacies develop protocols with physicians to maximize patient qualification for POCT, while maintaining patient safety. OBJECTIVE: To determine the number of patients seen for influenza in the emergency department (ED) during the 2018-2019 season who would qualify for protocol-driven influenza testing. METHODS: This was a retrospective review of medical records. Patients seen in this 92 bed ED, level III trauma center between October 1, 2018 and May 1, 2019 were included if their age was older than 11 years or younger than 71 years with an influenza-related diagnosis. Patients were excluded if they were pregnant or breastfeeding, were allergic to oseltamivir, were recently diagnosed with pneumonia, or recently received a live influenza vaccine. Patient information collected included: sex, age, height, weight, pulse, blood pressure, respiratory rate, temperature, oxygen saturation, mental status, symptoms, time since onset of symptoms, immune system status, and history of respiratory illness or respiratory disease. These data points were used to determine eligibility for POCT based on a prespecified protocol that included criteria such as vital signs, symptom presentation, and other health conditions. The primary end point was the number of patients eligible for institutional protocol-driven POCT. RESULTS: There were 1955 ED visits with a primary diagnosis of influenza; 451 were eligible for study inclusion, and 49 (11%) qualified for POCT. The most common reason that patients did not qualify was temperature. If required temperature had been removed from the protocol, 155 patients (34%) would have qualified for POCT. CONCLUSION: On the basis of the institutional protocol, a small proportion of patients qualified for POCT. Without the protocol temperature requirement, the number of patients who qualified for POCT would have greatly increased. This study identified opportunities for improvement in the institutional protocol. Future research is needed to reassess the number of patients who qualify once revisions are made.

Effect of Vitamin C, Hydrocortisone, and Thiamine vs Hydrocortisone Alone on Time Alive and Free of Vasopressor Support Among Patients With Septic Shock: The VITAMINS Randomized Clinical Trial.

Importance: It is unclear whether vitamin C, hydrocortisone, and thiamine are more effective than hydrocortisone alone in expediting resolution of septic shock. Objective: To determine whether the combination of vitamin C, hydrocortisone, and thiamine, compared with hydrocortisone alone, improves the duration of time alive and free of vasopressor administration in patients with septic shock. Design, Setting, and Participants: Multicenter, open-label, randomized clinical trial conducted in 10 intensive care units in Australia, New Zealand, and Brazil that recruited 216 patients fulfilling the Sepsis-3 definition of septic shock. The first patient was enrolled on May 8, 2018, and the last on July 9, 2019. The final date of follow-up was October 6, 2019. Interventions: Patients were randomized to the intervention group (n = 109), consisting of intravenous vitamin C (1.5 g every 6 hours), hydrocortisone (50 mg every 6 hours), and thiamine (200 mg every 12 hours), or to the control group (n = 107), consisting of intravenous hydrocortisone (50 mg every 6 hours) alone until shock resolution or up to 10 days. Main Outcomes and Measures: The primary trial outcome was duration of time alive and free of vasopressor administration up to day 7. Ten secondary outcomes were prespecified, including 90-day mortality. Results: Among 216 patients who were randomized, 211 provided consent and completed the primary outcome measurement (mean age, 61.7 years [SD, 15.0]; 133 men [63%]). Time alive and vasopressor free up to day 7 was 122.1 hours (interquartile range [IQR], 76.3-145.4 hours) in the intervention group and 124.6 hours (IQR, 82.1-147.0 hours) in the control group; the median of all paired differences was -0.6 hours (95% CI, -8.3 to 7.2 hours; P = .83). Of 10 prespecified secondary outcomes, 9 showed no statistically significant difference. Ninety-day mortality was 30/105 (28.6%) in the intervention group and 25/102 (24.5%) in the control group (hazard ratio, 1.18; 95% CI, 0.69-2.00). No serious adverse events were reported. Conclusions and Relevance: In patients with septic shock, treatment with intravenous vitamin C, hydrocortisone, and thiamine, compared with intravenous hydrocortisone alone, did not significantly improve the duration of time alive and free of vasopressor administration over 7 days. The finding suggests that treatment with intravenous vitamin C, hydrocortisone, and thiamine does not lead to a more rapid resolution of septic shock compared with intravenous hydrocortisone alone. Trial Registration: ClinicalTrials.gov Identifier: NCT03333278.

Pharmacokinetic data support 6-hourly dosing of intravenous vitamin C to critically ill patients with septic shock.

OBJECTIVES: To study vitamin C pharmacokinetics in septic shock. DESIGN: Prospective pharmacokinetic study. SETTING: Two intensive care units. PARTICIPANTS: Twenty-one patients with septic shock enrolled in a randomised trial of high dose vitamin C therapy in septic shock. INTERVENTION: Patients received 1.5 g intravenous vitamin C every 6 hours. Plasma samples were obtained before and at 1, 4 and 6 hours after drug administration, and vitamin C concentrations were measured by high performance liquid chromatography. MAIN OUTCOME MEASURES: Clearance, volume of distribution, and half-life were calculated using noncompartmental analysis. Data are presented as median (interquartile range [IQR]). RESULTS: Of the 11 participants who had plasma collected before any intravenous vitamin C administration, two (18%) were deficient (concentrations < 11 µmol/L) and three (27%) had hypovitaminosis C (concentrations between 11 and 23 µmol/L), with a median concentration 28 µmol/L (IQR, 11-44 µmol/L). Volume of distribution was 23.3 L (IQR, 21.9-27.8 L), clearance 5.2 L/h (IQR, 3.3-5.4 L/h), and half-life 4.3 h (IQR, 2.6-7.5 h). For the participants who had received at least one dose of intravenous vitamin C before sampling, T0 concentration was 258 µmol/L (IQR, 162- 301 µmol/L). Pharmacokinetic parameters for subsequent doses were a median volume of distribution 39.9 L (IQR, 31.4-44.4 L), clearance 3.6 L/h (IQR, 2.6-6.5 L/h), and half-life 6.9 h (IQR, 5.7-8.5 h). CONCLUSION: Intravenous vitamin C (1.5 g every 6 hours) corrects vitamin C deficiency and hypovitaminosis C and provides an appropriate dosing schedule to achieve and maintain normal or elevated vitamin C levels in septic shock.

CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high-quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte-like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH-HLCs need to be corrected. Genome editing technology clustered-regularly-interspaced-short-palindromic-repeats/CRISPR-associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. CONCLUSION: We used CRISPR/Cas9 genome editing to permanently correct a 3-base pair homozygous deletion in LDLR exon 4 of patient-derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

Safety assessment of genetically modified plants with deliberately altered composition.

The development and marketing of 'novel' genetically modified (GM) crops in which composition has been deliberately altered poses a challenge to the European Union (EU)'s risk assessment processes, which are based on the concept of substantial equivalence with a non-GM comparator. This article gives some examples of these novel GM crops and summarizes the conclusions of a report that was commissioned by the European Food Safety Authority on how the EU's risk assessment processes could be adapted to enable their safety to be assessed.

Ongoing requirement for pacing post-transcatheter aortic valve implantation and surgical aortic valve replacement.

OBJECTIVES: Transcatheter aortic valve implantation (TAVI) is an established intervention for aortic stenosis. While it is known that the requirement for permanent pacing is higher following CoreValve (Medtronic, Inc., Minneapolis, MN, USA) TAVI than after surgical aortic valve replacement (SAVR), it remains uncertain whether pacing is required in the medium-to-long term. We hypothesized that complete heart block following TAVI is more likely to resolve than that following SAVR. METHODS: A retrospective analysis of prospectively collated data on 528 patients undergoing TAVI or SAVR from May 2008 to December 2010 at a cardiac tertiary referral hospital. Demographic data, timing and indication for pacing post-procedure plus follow-up were recorded. Paced patients were compared and analysed by existing initial indication for pacing. RESULTS: In total, 31 (5.9%) patients received a pacemaker, and there were limited differences between not paced and paced patient characteristics by procedure type. Of these, a greater proportion were implanted post-TAVI compared with SAVR (17 vs 3.2%, P<0.001). The mean time to pacemaker follow-up for TAVI and SAVR was 234 and 188 days, P=0.32, respectively. Fewer patients compared with pacing indication remained in complete heart block at latest follow-up for TAVI (76.5 vs 33.3%, P=0.02) and SAVR (92.9 vs 58.3%, P=0.04). Although, there was a trend towards a greater magnitude of TAVI patients regaining atrioventricular nodal conduction, this did not differ significantly from that seen in SAVR patients. CONCLUSIONS: In keeping with previous reports, this single-centre experience demonstrates that patients undergoing TAVI have higher rates of pacemaker implantation than those following SAVR. However, pacing indication in the short-to-medium term may not persist for all paced patients post-TAVI and -SAVR with the suggestion that a significant proportion recover atrioventricular conduction, which tended to be greatest in TAVI paced patients.

VAMP8 is a vesicle SNARE that regulates mucin secretion in airway goblet cells.

Mucin secretion is an innate defence mechanism, which is noxiously upregulated in obstructive lung diseases (e.g. chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma). Mucin granule exocytosis is regulated by specific protein complexes, but the SNARE exocytotic core has not been defined in airway goblet cells. In this study, we identify VAMP8 as one of the SNAREs regulating mucin granule exocytosis. VAMP8 mRNA was present in human airway and lung epithelial cells, and deep-sequencing and expression analyses of airway epithelial cells revealed that VAMP8 transcripts were expressed at 10 times higher levels than other VAMP mRNAs. In human airway epithelial cell cultures and freshly excised tissues, VAMP8 immunolocalised mainly to goblet cell mucin granules. The function of VAMP8 in airway mucin secretion was tested by RNA interference techniques. Both VAMP8 short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) reduced mucin secretion induced by PAR agonists, neutrophil elastase and ATP in two airway epithelial cell culture models. Notably, basal (non-agonist elicited) mucin secretion was also reduced in these experiments. VAMP8 knockdown was also effective in decreasing mucin secretion in airway epithelial cell cultures with induced mucous metaplasia/mucin hypersecretion. Unlike VAMP8 silencing, knockdown of VAMP2 or VAMP3 did not affect mucin secretion. Importantly, in VAMP8 knock-out (KO) mice with IL-13-induced mucous metaplasia, mucin content in the bronchoalveolar lavage (BAL) and ATP-stimulated mucin secretion in the trachea were reduced compared to WT-matched littermates. Our data indicate that VAMP8 is an essential SNARE in airway mucin granule exocytosis. Reduction of VAMP8 activity/expression may provide a novel therapeutic target to ameliorate airway mucus obstruction in lung diseases.

Genotyping three SNPs affecting warfarin drug response by isothermal real-time HDA assays.

BACKGROUND: The response to the anticoagulant drug warfarin is greatly affected by genetic polymorphisms in the VKORC1 and CYP2C9 genes. Genotyping these polymorphisms has been shown to be important in reducing the time of the trial and error process for finding the maintenance dose of warfarin thus reducing the risk of adverse effects of the drug. METHOD: We developed a real-time isothermal DNA amplification system for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. For each SNP, real-time isothermal Helicase Dependent Amplification (HDA) reactions were performed to amplify a DNA fragment containing the SNP. Amplicons were detected by fluorescently labeled allele specific probes during real-time HDA amplification. RESULTS: Fifty clinical samples were analyzed by the HDA-based method, generating a total of 150 results. Of these, 148 were consistent between the HDA-based assays and a reference method. The two samples with unresolved HDA-based test results were repeated and found to be consistent with the reference method. CONCLUSION: The HDA-based assays demonstrated a clinically acceptable performance for genotyping the VKORC1 -1639G>A SNP and two SNPs (430C>T and 1075A>C) for the CYP2C9 enzyme (CYP2C9*2 and CYP2C9*3), all of which are relevant in warfarin pharmacogenentics.

Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.

Overexpression of the epithelial Na(+) channel beta subunit (Scnn1b gene, betaENaC protein) in transgenic (Tg) mouse airways dehydrates mucosal surfaces, producing mucus obstruction, inflammation, and neonatal mortality. Airway inflammation includes macrophage activation, neutrophil and eosinophil recruitment, and elevated KC, TNF-alpha, and chitinase levels. These changes recapitulate aspects of complex human obstructive airway diseases, but their molecular mechanisms are poorly understood. We used genetic and pharmacologic approaches to identify pathways relevant to the development of Scnn1b-Tg mouse lung pathology. Genetic deletion of TNF-alpha or its receptor, TNFR1, had no measurable effect on the phenotype. Deletion of IL-4Ralpha abolished transient mucous secretory cell (MuSC) abundance and eosinophilia normally observed in neonatal wild-type mice. Similarly, IL-4Ralpha deficiency decreased MuSC and eosinophils in neonatal Scnn1b-Tg mice, which correlated with improved neonatal survival. However, chronic lung pathology in adult Scnn1b-Tg mice was not affected by IL-4Ralpha status. Prednisolone treatment ablated eosinophilia and MuSC in adult Scnn1b-Tg mice, but did not decrease mucus plugging or neutrophilia. These studies demonstrate that: 1) normal neonatal mouse airway development entails an IL-4Ralpha-dependent, transient abundance of MuSC and eosinophils; 2) absence of IL-4Ralpha improved neonatal survival of Scnn1b-Tg mice, likely reflecting decreased formation of asphyxiating mucus plugs; and 3) in Scnn1b-Tg mice, neutrophilia, mucus obstruction, and airspace enlargement are IL-4Ralpha- and TNF-alpha-independent, and only MuSC and eosinophilia are sensitive to glucocorticoids. Thus, manipulation of multiple pathways will likely be required to treat the complex pathogenesis caused by airway surface dehydration.