Plasma Progerin in Patients With Hutchinson-Gilford Progeria Syndrome: Immunoassay Development and Clinical Evaluation.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare, fatal, premature aging disease caused by a toxic protein called progerin. Circulating progerin has not been previously detected, precluding research using readily available biological samples. This study aimed to develop a plasma progerin assay to evaluate progerin's quantity, response to progerin-targeted therapy, and relationship to patient survival. METHODS: Biological samples were collected by The Progeria Research Foundation Cell and Tissue Bank from a non-HGPS cohort cross-sectionally and a HGPS cohort longitudinally. HGPS donations occurred at baseline and intermittently while treated with farnesylation inhibitors lonafarnib±pravastatin and zoledronate, within 3 sequential open-label clinical trials at Boston Children's Hospital totaling >10 years of treatment. An ultrasensitive single-molecule counting progerin immunoassay was developed with prespecified performance parameters. Intra- and interpatient group statistics were descriptive. The relationship between progerin and survival was assessed by using joint modeling with time-dependent slopes parameterization. RESULTS: The assay's dynamic detection range was 59 to 30 000 pg/mL (R2=0.9987). There was no lamin A cross-reactivity. Mean plasma progerin in non-HGPS participants (n=69; 39 male, 30 female; age, 0.2-71.3 years) was 351±251 pg/mL, and in drug-naive participants with HGPS (n=74; 37 female, 37 male; age, 2.1-17.5 years) was 33 261±12 346 pg/mL, reflecting a 95-fold increase in affected children (P<0.0001). Progerin levels did not differ by sex (P=0.99). Lonafarnib treatment resulted in an average per-visit progerin decrease from baseline of between 35% to 62% (all P<0.005); effects were not augmented by adding pravastatin and zoledronate. Progerin levels fell within 4 months of therapy and remained lower for up to 10 years. The magnitude of progerin decrease positively associated with patient survival (P<0.0001; ie, 15 000 pg/mL decrease yields a 63.9% decreased risk of death). For any given decrease in progerin, life expectancy incrementally increased with longer treatment duration. CONCLUSIONS: A sensitive, quantitative immunoassay for progerin was developed and used to demonstrate high progerin levels in HGPS plasma that decreased with lonafarnib therapy. The extent of improved survival was associated with both the magnitude of progerin decrease and duration at lower levels. Thus, plasma progerin is a biomarker for HGPS whose reduction enables short- and long-term assessment of progerin-targeted treatment efficacy. REGISTRATION: URL: https://www. CLINICALTRIALS: gov. Unique identifiers: NCT00879034 and NCT00916747.

Quantifying the Release of Biomarkers of Myocardial Necrosis from Cardiac Myocytes and Intact Myocardium.

BACKGROUND: Myocardial infarction is diagnosed when biomarkers of cardiac necrosis exceed the 99th centile, although guidelines advocate even lower concentrations for early rule-out. We examined how many myocytes and how much myocardium these concentrations represent. We also examined if dietary troponin can confound the rule-out algorithm. METHODS: Individual rat cardiac myocytes, rat myocardium, ovine myocardium, or human myocardium were spiked into 400-µL aliquots of human serum. Blood was drawn from a volunteer after ingestion of ovine myocardium. High-sensitivity assays were used to measure cardiac troponin T (cTnT; Roche, Elecsys), cTnI (Abbott, Architect), and cardiac myosin-binding protein C (cMyC; EMD Millipore, Erenna®). RESULTS: The cMyC assay could only detect the human protein. For each rat cardiac myocyte added to 400 µL of human serum, cTnT and cTnI increased by 19.0 ng/L (95% CI, 16.8-21.2) and 18.9 ng/L (95% CI, 14.7-23.1), respectively. Under identical conditions cTnT, cTnI, and cMyC increased by 3.9 ng/L (95% CI, 3.6-4.3), 4.3 ng/L (95% CI, 3.8-4.7), and 41.0 ng/L (95% CI, 38.0-44.0) per µg of human myocardium. There was no detectable change in cTnI or cTnT concentration after ingestion of sufficient ovine myocardium to increase cTnT and cTnI to approximately 1 × 108 times their lower limits of quantification. CONCLUSIONS: Based on pragmatic assumptions regarding cTn and cMyC release efficiency, circulating species, and volume of distribution, 99th centile concentrations may be exceeded by necrosis of 40 mg of myocardium. This volume is much too small to detect by noninvasive imaging.

Biomarkers in fasting serum to estimate glucose tolerance, insulin sensitivity, and insulin secretion.

BACKGROUND: Biomarkers for estimating reduced glucose tolerance, insulin sensitivity, or impaired insulin secretion would be clinically useful, since these physiologic measures are important in the pathogenesis of type 2 diabetes mellitus. METHODS: We conducted a cross-sectional study in which 94 individuals, of whom 84 had 1 or more risk factors and 10 had no known risk factors for diabetes, underwent oral glucose tolerance testing. We measured 34 protein biomarkers associated with diabetes risk in 250-µL fasting serum samples. We applied multiple regression selection techniques to identify the most informative biomarkers and develop multivariate models to estimate glucose tolerance, insulin sensitivity, and insulin secretion. The ability of the glucose tolerance model to discriminate between diabetic individuals and those with impaired or normal glucose tolerance was evaluated by area under the ROC curve (AUC) analysis. RESULTS: Of the at-risk participants, 25 (30%) were found to have impaired glucose tolerance, and 11 (13%) diabetes. Using molecular counting technology, we assessed multiple biomarkers with high accuracy in small volume samples. Multivariate biomarker models derived from fasting samples correlated strongly with 2-h postload glucose tolerance (R(2) = 0.45, P < 0.0001), composite insulin sensitivity index (R(2) = 0.91, P < 0.0001), and insulin secretion (R(2) = 0.45, P < 0.0001). Additionally, the glucose tolerance model provided strong discrimination between diabetes vs impaired or normal glucose tolerance (AUC 0.89) and between diabetes and impaired glucose tolerance vs normal tolerance (AUC 0.78). CONCLUSIONS: Biomarkers in fasting blood samples may be useful in estimating glucose tolerance, insulin sensitivity, and insulin secretion.

Generation of PEGylated VPAC1-selective antagonists that inhibit proliferation of a lung cancer cell line.

Vasoactive intestinal peptide (VIP) binds to two receptors, VPAC1 and VPAC2. Non-selective VIP antagonists have been shown to inhibit human cancer cell proliferation and reduce tumor growth in mice. Many human cancers over-express VPAC1 but not VPAC2. We show that VPAC1-selective antagonists can inhibit human cancer cell proliferation and identify five positions in the VPAC1-selective antagonist PG 97-269 that may be responsible for VPAC1 selectivity. Position 16 appears to be particularly critical for selectivity, as demonstrated in the replacement of Arg16 of PG 97-269 with the native VIP amino acid; this single change results in greatly reduced VPAC1 binding and selectivity. Finally, we show that site-specific conjugation with a 22kDa polyethylene glycol (PEG) at the C-terminus of VPAC1-selective antagonists further improves VPAC1-selective binding and has minimal effect on antagonistic activity. Our studies have further solidified VPAC1 as a cancer target and offer the possibility of generating highly potent VPAC1-selective antagonists with minimal number of mutations to reduce the risk of immunogenicity and potentially prolonged duration of action to allow more efficient treatment regimen.

A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes.

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K(d)) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.

Clinically significant novel biomarkers for prediction of first ever myocardial infarction: the Tromsø Study.

BACKGROUND: Identification of individuals with high risk for first-ever myocardial infarction (MI) can be improved. The objectives of the study were to survey multiple protein biomarkers for association with the 10-year risk of incident MI and identify a clinically significant risk model that adds information to current common risk models. METHODS AND RESULTS: We used an immunoassay platform that uses a sensitive, sample-efficient molecular counting technology to measure 51 proteins in samples from the fourth survey (1994) in the Tromsø Study, a longitudinal study of men and women in Tromsø, Norway. A case control design was used with 419 first-ever MI cases (169 females/250 males) and 398 controls (244 females/154 males). Of the proteins measured, 17 were predictors of MI when considered individually after adjustment for traditional risk factors either in men, women, or both. The 6 biomarkers adjusted for traditional risk factors that were selected in a multivariable model (odds ratios [OR] per standard deviation) using a stepwise procedure were apolipoprotein B/apolipoprotein A1 ratio (1.40), kallikrein (0.73), lipoprotein a (1.29), matrix metalloproteinase 9 (1.30), the interaction term IP-10/CXCL10×women (0.69), and the interaction term thrombospondin 4×men (1.38). The composite risk of these biomarkers added significantly to the traditional risk factor model with a net reclassification improvement of 14% (P=0.0002), whereas the receiver operating characteristic area increased from 0.757 to 0.791, P=0.0004. CONCLUSIONS: Novel protein biomarker models improve identification of 10-year MI risk above and beyond traditional risk factors with 14% better allocation to either high or low risk group.

Microfluidic strategies applied to biomarker discovery and validation for multivariate diagnostics.

Complex diseases are caused by combinatorial genetic, environmental and lifestyle factors. The emergence of multibiomarker tests to define these diseases and to identify the early, presymptomatic stages offers several advantages to the conventional use of single marker tests. The development of multibiomarker protein-based tests remains constrained by technological and operational limitations in assaying hundreds to thousands of proteins in thousands of samples. In order to develop a multibiomarker test that stratifies risk for Type 2 diabetes, we took a candidate-driven immunoassay approach utilizing a microfluidics platform to analyze 89 candidate proteins in thousands of samples, which allowed us to move from discovery to a commercial test in 2 years. Future multibiomarker test development will be enhanced by advancements in the number of proteins that can be analyzed, analytical sensitivity and throughput, and sample volume requirements, all of which depend on the further advancement of microfluidics, detection technologies and affinity-based reagents.

Development of a type 2 diabetes risk model from a panel of serum biomarkers from the Inter99 cohort.

OBJECTIVE: The purpose of this study was to develop a model for assessing the 5-year risk of developing type 2 diabetes from a panel of 64 circulating candidate biomarkers. RESEARCH DESIGN AND METHODS: Subjects were selected from the Inter99 cohort, a longitudinal population-based study of approximately 6,600 Danes in a nested case-control design with the primary outcome of 5-year conversion to type 2 diabetes. Nondiabetic subjects, aged >or=39 years, with BMI >or=25 kg/m(2) at baseline were selected. Baseline fasting serum samples from 160 individuals who developed type 2 diabetes and from 472 who did not were tested. An ultrasensitive immunoassay was used to measure of 58 candidate biomarkers in multiple diabetes-associated pathways, along with six routine clinical variables. Statistical learning methods and permutation testing were used to select the most informative biomarkers. Risk model performance was estimated using a validated bootstrap bias-correction procedure. RESULTS: A model using six biomarkers (adiponectin, C-reactive protein, ferritin, interleukin-2 receptor A, glucose, and insulin) was developed for assessing an individual's 5-year risk of developing type 2 diabetes. This model has a bootstrap-estimated area under the curve of 0.76, which is greater than that for A1C, fasting plasma glucose, fasting serum insulin, BMI, sex-adjusted waist circumference, a model using fasting glucose and insulin, and a noninvasive clinical model. CONCLUSIONS: A model incorporating six circulating biomarkers provides an objective and quantitative estimate of the 5-year risk of developing type 2 diabetes, performs better than single risk indicators and a noninvasive clinical model, and provides better stratification than fasting plasma glucose alone.

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide.

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.