RyR2 C-terminal truncating variants identified in patients with arrhythmic phenotypes exert a dominant negative effect through formation of wildtype-truncation heteromers.

Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex.

An International Multicenter Evaluation of Type 5 Long QT Syndrome: A Low Penetrant Primary Arrhythmic Condition.

BACKGROUND: Insight into type 5 long QT syndrome (LQT5) has been limited to case reports and small family series. Improved understanding of the clinical phenotype and genetic features associated with rare KCNE1 variants implicated in LQT5 was sought through an international multicenter collaboration. METHODS: Patients with either presumed autosomal dominant LQT5 (N = 229) or the recessive Type 2 Jervell and Lange-Nielsen syndrome (N = 19) were enrolled from 22 genetic arrhythmia clinics and 4 registries from 9 countries. KCNE1 variants were evaluated for ECG penetrance (defined as QTc >460 ms on presenting ECG) and genotype-phenotype segregation. Multivariable Cox regression was used to compare the associations between clinical and genetic variables with a composite primary outcome of definite arrhythmic events, including appropriate implantable cardioverter-defibrillator shocks, aborted cardiac arrest, and sudden cardiac death. RESULTS: A total of 32 distinct KCNE1 rare variants were identified in 89 probands and 140 genotype positive family members with presumed LQT5 and an additional 19 Type 2 Jervell and Lange-Nielsen syndrome patients. Among presumed LQT5 patients, the mean QTc on presenting ECG was significantly longer in probands (476.9±38.6 ms) compared with genotype positive family members (441.8±30.9 ms, P<0.001). ECG penetrance for heterozygous genotype positive family members was 20.7% (29/140). A definite arrhythmic event was experienced in 16.9% (15/89) of heterozygous probands in comparison with 1.4% (2/140) of family members (adjusted hazard ratio [HR] 11.6 [95% CI, 2.6-52.2]; P=0.001). Event incidence did not differ significantly for Type 2 Jervell and Lange-Nielsen syndrome patients relative to the overall heterozygous cohort (10.5% [2/19]; HR 1.7 [95% CI, 0.3-10.8], P=0.590). The cumulative prevalence of the 32 KCNE1 variants in the Genome Aggregation Database, which is a human database of exome and genome sequencing data from now over 140 000 individuals, was 238-fold greater than the anticipated prevalence of all LQT5 combined (0.238% vs 0.001%). CONCLUSIONS: The present study suggests that putative/confirmed loss-of-function KCNE1 variants predispose to QT prolongation, however, the low ECG penetrance observed suggests they do not manifest clinically in the majority of individuals, aligning with the mild phenotype observed for Type 2 Jervell and Lange-Nielsen syndrome patients.

An International Multicenter Evaluation of Inheritance Patterns, Arrhythmic Risks, and Underlying Mechanisms of CASQ2-Catecholaminergic Polymorphic Ventricular Tachycardia.

BACKGROUND: Genetic variants in calsequestrin-2 (CASQ2) cause an autosomal recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVT), although isolated reports have identified arrhythmic phenotypes among heterozygotes. Improved insight into the inheritance patterns, arrhythmic risks, and molecular mechanisms of CASQ2-CPVT was sought through an international multicenter collaboration. METHODS: Genotype-phenotype segregation in CASQ2-CPVT families was assessed, and the impact of genotype on arrhythmic risk was evaluated using Cox regression models. Putative dominant CASQ2 missense variants and the established recessive CASQ2-p.R33Q variant were evaluated using oligomerization assays and their locations mapped to a recent CASQ2 filament structure. RESULTS: A total of 112 individuals, including 36 CPVT probands (24 homozygotes/compound heterozygotes and 12 heterozygotes) and 76 family members possessing at least 1 presumed pathogenic CASQ2 variant, were identified. Among CASQ2 homozygotes and compound heterozygotes, clinical penetrance was 97.1% and 26 of 34 (76.5%) individuals had experienced a potentially fatal arrhythmic event with a median age of onset of 7 years (95% CI, 6-11). Fifty-one of 66 CASQ2 heterozygous family members had undergone clinical evaluation, and 17 of 51 (33.3%) met diagnostic criteria for CPVT. Relative to CASQ2 heterozygotes, CASQ2 homozygote/compound heterozygote genotype status in probands was associated with a 3.2-fold (95% CI, 1.3-8.0; P=0.013) increased hazard of a composite of cardiac syncope, aborted cardiac arrest, and sudden cardiac death, but a 38.8-fold (95% CI, 5.6-269.1; P<0.001) increased hazard in genotype-positive family members. In vitro turbidity assays revealed that p.R33Q and all 6 candidate dominant CASQ2 missense variants evaluated exhibited filamentation defects, but only p.R33Q convincingly failed to dimerize. Structural analysis revealed that 3 of these 6 putative dominant negative missense variants localized to an electronegative pocket considered critical for back-to-back binding of dimers. CONCLUSIONS: This international multicenter study of CASQ2-CPVT redefines its heritability and confirms that pathogenic heterozygous CASQ2 variants may manifest with a CPVT phenotype, indicating a need to clinically screen these individuals. A dominant mode of inheritance appears intrinsic to certain missense variants because of their location and function within the CASQ2 filament structure.

Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice.

BACKGROUND & AIMS: Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. METHODS: Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. RESULTS: Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. CONCLUSIONS: Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Genetic Testing in Pediatric Cardiomyopathy.

Genetic testing is recommended in patients with dilated cardiomyopathy (DCM); however, limited studies demonstrate high yields of genetic testing in non-hypertrophic (HCM) patients. Furthermore, there is sparse genotype-phenotype data in pediatric DCM patients. We performed a retrospective review of 70 consecutive probands with cardiomyopathy (non-HCM) who underwent genetic evaluation. Mean age at presentation was 5.48 years. Echocardiography revealed mean ejection fraction of 32.4%. The LVEDd z score ranged from - 5.7 to + 15.9. Cardiomyopathy was classified as dilated in 56, 10 with non-compaction, 2 with restrictive, and 2 with ARVC. TTN gene mutations were the most common gene involved. Genetic testing was negative in 16/70 (23%) giving a yield of 77% including VUS. 33% (23/70) of probands had a positive family history among whom the diagnostic yield was 57% (13/23) for pathogenic mutations. Yield for positive genetic testing in the DCM with positive family history group was 9/18 (50%). There were 6 deaths (9%) and 26/70 (37%) underwent transplantation. More frequent cardiac transplantations (48 vs. 34%) and deaths (17 vs. 2%) were seen in mutation-positive vs. mutation-negative subgroups. This study demonstrates an increasing yield of genetic testing in DCM although with a high rate of VUS detection. Use of genetic information for better management and prognostication will require big data analysis.

Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury.

Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-δ (PKCδ) as a critical regulator of the acute inflammatory response and demonstrated that PKCδ inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCδ is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)-conjugated PKCδ inhibitory peptide (PKCδ-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [<1% initial dose (ID)], whereas IT administration resulted in lung retention of >65% ID with minimal uptake in liver or kidney (<2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCδ-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCδ-TAT at the time of CLP surgery significantly reduced PKCδ activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide.

Pulmonary endothelial protein kinase C-delta (PKCδ) regulates neutrophil migration in acute lung inflammation.

Excessive neutrophil migration across the pulmonary endothelium into the lung and release of oxidants and proteases are key elements in pathogenesis of acute lung injury. Previously, we identified protein kinase C-delta (PKCδ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that intratracheal instillation of a TAT-conjugated PKCδ inhibitory peptide (PKCδ-TAT) is lung protective in a rat model of sepsis-induced indirect pulmonary injury (cecal ligation and puncture). In the present study, intratracheal instillation of this PKCδ inhibitor resulted in peptide distribution throughout the lung parenchyma and pulmonary endothelium and decreased neutrophil influx, with concomitant attenuation of sepsis-induced endothelial ICAM-1 and VCAM-1 expression in this model. To further delineate the role of PKCδ in regulating neutrophil migration, we used an in vitro transmigration model with human pulmonary microvascular endothelial cells (PMVECs). Consistent with in vivo findings, inhibition of PMVEC PKCδ decreased IL-1ß-mediated neutrophil transmigration. PKCδ regulation was stimulus-dependent; PKCδ was required for transmigration mediated by IL-1ß and fMLP (integrin-dependent), but not IL-8 (integrin-independent). PKCδ was essential for IL-1ß-mediated neutrophil adherence and NF-κB-dependent expression of ICAM-1 and VCAM-1. In PMVECs, IL-1ß-mediated production of ROS and activation of redox-sensitive NF-κB were PKCδ dependent, suggesting an upstream signaling role. Thus, PKCδ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the inflamed lung.

An international multicenter cohort study on implantable cardioverter-defibrillators for the treatment of symptomatic children with catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) may cause sudden cardiac death (SCD) despite medical therapy. Therefore, implantable cardioverter-defibrillators (ICDs) are commonly advised. However, there is limited data on the outcomes of ICD use in children. OBJECTIVE: The purpose of this study was to compare the risk of arrhythmic events in pediatric patients with CPVT with and without an ICD. METHODS: We compared the risk of SCD in patients with RYR2 (ryanodine receptor 2) variants and phenotype-positive symptomatic CPVT patients with and without an ICD who were younger than 19 years and had no history of sudden cardiac arrest at phenotype diagnosis. The primary outcome was SCD; secondary outcomes were composite end points of SCD, sudden cardiac arrest, or appropriate ICD shocks with or without arrhythmic syncope. RESULTS: The study included 235 patients, 73 with an ICD (31.1%) and 162 without an ICD (68.9%). Over a median follow-up of 8.0 years (interquartile range 4.3-13.4 years), SCD occurred in 7 patients (3.0%), of whom 4 (57.1%) were noncompliant with medications and none had an ICD. Patients with ICD had a higher risk of both secondary composite outcomes (without syncope: hazard ratio 5.85; 95% confidence interval 3.40-10.09; P < .0001; with syncope: hazard ratio 2.55; 95% confidence interval 1.50-4.34; P = .0005). Thirty-one patients with ICD (42.5%) experienced appropriate shocks, 18 (24.7%) inappropriate shocks, and 21 (28.8%) device-related complications. CONCLUSION: SCD events occurred only in patients without an ICD and mostly in those not on optimal medical therapy. Patients with an ICD had a high risk of appropriate and inappropriate shocks, which may be reduced with appropriate device programming. Severe ICD complications were common, and risks vs benefits of ICDs need to be considered.

Use of a high caloric liquid meal as an alternative to a solid meal for gastric emptying scintigraphy.

BACKGROUND: Gastric emptying scintigraphy (GES) using a solid meal is often recommended for the evaluation of gastroparesis. However, some patients cannot tolerate the standardized egg-white sandwich (EWS) solid meal and an alternative meal is needed. AIM: The aim of this study was to compare GES, regional gastric emptying, and gastric contractility using a liquid nutrient meal (LNM; Ensure Plus(®)) to those using EWS. METHODS: Twenty healthy volunteers underwent GES using EWS and LNM on separate days. Gastric emptying was measured using scintigraphy and with a wireless motility capsule (WMC; SmartPill(®)). RESULTS: The gastric emptying half-time with LNM was similar to that with EWS (1.41 ± 0.11 vs 1.52 ± 0.08 h; P = 0.28) and the two were significantly correlated (r = 0.53; P = 0.017). There were time-related differences in gastric emptying of the LNM compared to EWS: in the first hour, gastric retention of EWS was slightly greater than that of LNM, whereas at 3 and 4 h, gastric retention of EWS was slightly less than that of LNM. Regionally, the percentage retention of the meal in the proximal stomach was greater for EWS than for LNM at 0.5 h. WMC gastric emptying times and gastric contractility for the two meals were not significantly different. CONCLUSIONS: Overall gastric emptying of the LNM was similar to that of the EWS meal. The LNM empties without a lag phase and takes slightly longer to empty from the distal stomach, likely due to its higher fat content. These differences are likely due to early accommodation with retention of solids in the proximal stomach and the need for trituration of solids. We conclude that this LNM can serve as an alternative to the conventional solid EWS for GES.

Evaluation of age at symptom onset, proband status, and sex as predictors of disease severity in pediatric catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Children with catecholaminergic polymorphic ventricular tachycardia (CPVT) are at risk for sudden death, and a risk stratification tool does not exist. OBJECTIVE: The purpose of this study was to determine whether proband status, age at symptom onset, and/or sex are independent predictors of cardiac events. METHODS: A multicenter, ambispective, cohort of pediatric CPVT patients was categorized by sex, proband status, and age at symptom onset (D1: first decade of life [symptom onset <10 years] or D2: second decade of life [symptom onset 10-18 years, inclusive]). Demographics, therapy, genetics, and outcomes were compared between groups. RESULTS: A total of 133 patients were included and stratified into 58 D1 and 75 D2 patients (68 female and 65 male; 106 probands and 27 relatives). Localization of RYR2 variants to hotspots differed based on proband status and age at symptom onset. The cardiac event rate was 33% (n = 44/133), inclusive of a 3% (n = 4/133) mortality rate, over a median of 6 years (interquartile range 3-11) after time of symptom onset. Proband status, rather than age at of symptom onset or sex, was an independent predictor of time to first cardiac event (P = .008; hazard ratio = 4.4). The 5-, 10- and 15-year event-free survival rates for probands were 77%, 56%, and 46%, respectively, and for relatives were 96%, 91%, and 86%, respectively. Event risk after diagnosis was 48% (32/67) in patients on ß-blocker or flecainide alone vs 10% (5/48) in patients on ß-blocker plus flecainide and/or left cardiac sympathetic denervation (P <.001). CONCLUSION: Proband status, but not age at symptom onset or male sex, independently predicted an earlier onset of cardiac events. A larger sample size would enable a comprehensive investigation of other risk factors.

Genetic testing and cascade screening in pediatric long QT syndrome and hypertrophic cardiomyopathy.

BACKGROUND: The efficacy of cascade screening for the inherited heart conditions long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM) is incompletely characterized. OBJECTIVE: The purpose of this study was to examine the use of genetic testing and yield of cascade screening across diverse regions in the United States and to evaluate obstacles to screening in multipayer systems. METHODS: An institutional review board-approved 6 United States pediatric center retrospective chart review of LQTS and HCM patients from 2008-2014 was conducted for (1) genetic test completion and results and (2) family cascade screening acceptance, methods, results, and barriers. RESULTS: The families of 315 index patients (mean age 9.0 ± 5.8 years) demonstrated a 75% (254) acceptance of cascade screening. The yield of relative screening was 39% (232/601), an average of 0.91 detected per family. Genetic testing was less utilized in HCM index patients and relatives. Screening participation was greater in families of gene-positive index patients (88%) (P <.001) compared to gene-negative patients (53%). Cascade method utilization: Cardiology-only 45%, combined genetic and cardiology 39%, and genetic only 16%. Screening yield by method: combined 57%, genetic-only 29%, and cardiology-only 20%. Family decisions were the leading barriers to cascade screening (26% lack of followthrough and 26% declined), whereas insurance (6%) was the least cited barrier. CONCLUSION: Family participation in cascade screening is high, but the greatest barriers are family mediated (declined, lack of followthrough). Positive proband genetic testing led to greater participation. Cardiology-only screening was the most utilized method, but combined cardiology and genetic screening had the highest detection.

Radiation-guided targeting of combretastatin encapsulated immunoliposomes to mammary tumors.

PURPOSE: Radiation upregulates expression of endothelial cell adhesion molecules providing a potential avenue for targeting drugs to irradiated tissue. Induced upregulation of E-selectin can be used to target immunoliposomes to solid tumors. The effects of targeting immunoliposomes containing the antivascular drug combretastatin disodium phosphate (CA4P) to irradiated mammary tumors were investigated in this study. METHODS: Mice bearing transplanted MCa-4 mouse mammary tumors were assigned to one of the factorial treatments permuting the administration of free CA4P, tumor irradiation, CA4P encapsulated liposomes, and CA4P encapsulated immunoliposomes (conjugated with anti-E-selectin). Single and fractionated dosing of radiation and/or CA4P was evaluated. RESULTS: For single dose treatments the group that received a single dose of radiation plus a single dose of immunoliposomes showed a significant delay in tumor growth compared to all other treatment groups. Fractionated radiation plus fractionated doses of immunoliposomes resulted in further tumor growth delay; however, it was not significantly different from other fractionated dose treatment groups that combined radiation and CA4P. CONCLUSIONS: Targeting of antivascular drugs to irradiated tumors via ligand-bearing liposomes results in significant tumor growth delay. This effect can be further potentiated using a fractionated irradiation dosing schedule combined with fractionated immunoliposome treatments.

Transsphincteric pH profile at the gastroesophageal junction.

BACKGROUND & AIMS: Esophageal acid exposure conventionally is measured 5 cm above the lower esophageal sphincter (LES). The aim of this study was to compare pH profiles at sites within the LES, the distal esophagus, and the proximal stomach. METHODS: Ten normal subjects underwent esophageal manometry followed by 24-hour esophagogastric pH monitoring using an 8-channel pH probe recording at 5 and 1.5 cm above and at 0, 1.5, 3.0, 4.5, 6.0, and 9.5 cm below the proximal LES border. During pH recording, a 4-hour gastric emptying test with an egg sandwich meal was performed. RESULTS: The LES was 3.2 +/- 0.4 cm in length. There was a progressive increase in acid exposure from the esophageal to the gastric pH sensors. pH was less than 4 for 3.4% +/- 1.6%, 12.7% +/- 8.5%, 26.5% +/- 10.2%, 48.1% +/- 11.3%, 66.5% +/- 9.9%, 80.8% +/- 5.6%, 89.2% +/- 3.0%, and 96.7% +/- 1.1% of the total time for pH probes at 5 and 1.5 cm above and 0, 1.5, 3, 4.5, 6.0, and 9.5 cm below the proximal LES border, respectively. Percentage acid exposures correlated significantly with the position of the probe (r = -0.95; P < .01). Intrasphincteric acidity increased postprandially. Gastric emptying was correlated inversely with the intragastric hydrogen ion concentration (r = -0.82). CONCLUSIONS: The percentage of recording time that pH was less than 4 was significantly higher in the intrasphincteric area and 1.5 cm above the proximal LES compared with the traditional site 5 cm above the proximal manometric LES border. High acid exposure in the intrasphincteric region might explain the susceptibility of the distal esophagus to erosions, strictures, and Barrett's esophagus.

Framework to Enhance Nurses' Use of EMR.

The ability of nurses to adopt and successfully use EMR is expected to have a significant impact on achieving benefits such as reduction in healthcare costs and improvement in healthcare quality. A review of the current research literature reveals issues and concerns relating to the adoption and use of EMR by nurses in hospital environments. This article presents a literature review of such issues and concerns, and suggests a framework for enhancing the adoption and use of EMR by nurses and hospitals.

Egg labeling methods for gastric emptying scintigraphy are not equivalent in producing a stable solid meal.

UNLABELLED: A wide range of radiolabeled test meals have been used for gastric emptying scintigraphy. The purpose of this study was to test whether (99m)Tc-sulfur colloid-labeled liquid egg white is as stable as 2 fresh whole eggs labeled with (99m)Tc-sulfur colloid and whether the cooking method is important. METHODS: Whole eggs and liquid egg white were mixed with (99m)Tc-sulfur colloid and cooked by either microwaving or frying on a griddle. The cooked eggs were tested for breakdown after 2 and 4 h of incubation in gastric fluid or HCl. RESULTS: Labeled liquid egg white, prepared by either method of cooking, exhibited less breakdown in gastric fluid than whole eggs. Whole eggs cooked in the microwave exhibited significantly more breakdown than liquid egg white. CONCLUSION: (99m)Tc-Sulfur colloid binds better to egg whites compared with whole eggs. These results emphasize the need to evaluate the stability of new radiolabeled test meal preparations, including the method of cooking.

Differences in binding of (99m)Tc-disintegrins to integrin alphavbeta3 on tumor and vascular cells.

UNLABELLED: Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as alphavbeta3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing alphavbeta3 and to cultured tumor cells which express alphavbeta3. METHODS: Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with (99m)Tc and tested for binding to cells in vitro. RESULTS: (99m)Tc-Kistrin, flavoridin and VLO4 had the highest binding, (99m)Tc-echistatin had moderate binding, and (99m)Tc-bitistatin and (99m)Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to alphavbeta3 to various extents: echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, (99m)Tc-kistrin was not displaced by competing peptide or plasma proteins. CONCLUSIONS: These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting alphavbeta3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting alphavbeta3 on vasculature because they bind strongly to surface receptors and are not readily displaced.

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans.

INTRODUCTION: 99mTc recombinant bitistatin (rBitistatin) is a radioligand for alphaIIbbeta3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [99mTc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. METHODS: [99mTc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [99mTc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [99mTc]rBitistatin as part of a Phase I clinical trial. RESULTS: The main organs that accumulated [99mTc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [99mTc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [99mTc]rBitistatin did not affect platelet counts in humans or dogs. CONCLUSIONS: [99mTc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [99mTc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute thrombi.

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes.

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.

Simultaneous assessment of gastric accommodation and emptying: studies with liquid and solid meals.

UNLABELLED: The aim of this study was to develop a scintigraphic test to measure gastric emptying and accommodation simultaneously. METHODS: Gastric emptying and accommodation were measured in healthy subjects. To determine gastric accommodation, the stomach was imaged with SPECT 20 min after intravenous administration of 185 MBq (5 mCi) (99m)Tc-pertechnetate. After ingestion of 11 MBq (300 micro Ci) (111)In-diethylenetriaminepentaacertic acid in a liquid nutrient drink or an (111)In-oxine-labeled egg sandwich, dual-isotope imaging assessed SPECT gastric dimensions and gastric emptying every 20 min up to 240 min. Gastric accommodation was calculated as the percentage change in planar (2-dimensional) gastric cross-sectional area (CSA) using a left anterior oblique planar projection and the percentage change in total SPECT gastric voxel counts (3-dimensional) compared with the baseline image. RESULTS: With the liquid nutrient drink (9 subjects), maximal mean CSA (158% +/- 12% of baseline; P < 0.05) occurred 40 min after meal ingestion, when only 69% +/- 3% of the radiolabeled liquid nutrient drink remained in the stomach. At 120 min, mean CSA was 125% +/- 8% of baseline, but only 35% +/- 3% of the liquid nutrient drink remained in the stomach. Using SPECT to measure 3-dimensional volumes, maximal gastric volume occurred 20 min after meal ingestion (189% +/- 25% of baseline). With the solid egg meal (10 subjects), maximal total CSA (159% +/- 13% of baseline) occurred immediately after meal ingestion; total CSA remained significantly increased above baseline for the first 3 h after ingestion of the egg meal, despite only 12% +/- 4% gastric retention at 3 h. Using SPECT to measure 3-dimensional volumes, maximal gastric volume occurred immediately after the meal (184% +/- 19% of baseline). CONCLUSION: This method permits simultaneous measurement of gastric emptying and accommodation. In healthy subjects, the gastric accommodation response is prolonged and persists despite nearly complete emptying of a liquid or solid meal.