Advances in microfabrication allow for highly sensitive calorimeters with dramatically reduced volume, decreased response time and increased energy resolution. These calorimeters hold the potential for designs of ELISA platforms competitive with fluorescent and chemiluminescent technologies. We have developed a new assay platform using conventional ELISA reagents to produce a thermal signal quantifiable using calorimetry. Our optimized micromachined calorimeters have nL reaction volumes and a minimum detectable power of 375 pW/Hz1/2. We demonstrate rapid quantification in a model system of trastuzumab, a humanized monoclonal antibody used in the treatment of HER2 overexpressing breast cancers, in human serum using a HER2 peptide mimetic. Trastuzumab concentration and reaction time constant correlated well (R2 = 0.954) and can be used to determine trastuzumab concentrations. The limit of detection for the ThermometricELISA (TELISA) was 10 μg/ml trastuzumab in human serum. TELISA allows for a simple readout, reduction in assay time, sample and reagent volumes and has the potential to become a point of care multiplexed platform technology.

Point of care (POC) diagnostics represents one of the fastest growing health care technology segments. Developments in microfabrication have led to the development of highly-sensitive nanocalorimeters ideal for directly measuring heat generated in POC biosensors. Here we present a novel nano-calorimeter-based biosensor design with differential sensing to eliminate common mode noise and capillary microfluidic channels for sample delivery to the thermoelectric sensor. The calorimeter has a resolution of 1.4 ± 0.2 nJ/(Hz)1/2 utilizing a 27 junction bismuth/titanium thermopile, with a total Seebeck coefficient of 2160 μV/K. Sample is wicked to the calorimeter through a capillary channel making it suitable for monitoring blood obtained through a finger prick (<1 μL sample required). We demonstrate device performance in a model assay using catalase, achieving a threshold for hydrogen peroxide quantification of 50 μM. The potential for our device as a POC blood test for metabolic diseases is shown through the quantification of phenylalanine (Phe) in serum, an unmet necessary service in the management of Phenylketonuria (PKU). Pegylated phenylalanine ammonia-lyase (PEG-PAL) was utilized to react with Phe, but reliable detection was limited to <5 mM due to low enzymatic activity. The POC biosensor concept can be multiplexed and adapted to a large number of metabolic diseases utilizing different immobilized enzymes.

Background— Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16α-hydroxyestrone (16αOHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16αOHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. Methods and Results— MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16αOHE 1.25 μg/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator–activated receptor-γ and CD36 in those mice exposed to 16αOHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti–miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti–miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti–miR-29 treatment. Conclusions— 16αOHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.

Xinping Chen, PhD; Megha Talati, PhD; Joshua P. Fessel, MD, PhD; Anna R. Hemnes, MD; Santhi Gladson, MS; Jaketa French, BS; Sheila Shay, BS; Aaron Trammel, MD; John A. Phillips, MD, PhD; Rizwan Hamid, MD, PhD; Joy D. Cogan, PhD; Elliott P. Dawson, MS; Kristie E. Womble, BS; Lora K. Hedges, BS; Elizabeth G. Martinez, DO; Lisa A. Wheeler, BS; James E. Loyd, MD; Susan J. Majka, PhD; James West, PhD; Eric D. Austin, MD, MSCI

e11567 Background: By itself, native trastuzumab is considered non-immunogenic. However, unfolded/denatured antibodies can elicit an anti-antibody response. We developed an anti-trastuzumab antibody serum test indicative of shorter disease free survival in a small cohort of patients with HER2 positive breast cancer treated with trastuzumab ( Pohlmann PR, M.T., Blum DL. Trastuzumab-reactive antibodies (TR-abs) in serum and trastuzumab (Tzb) benefit prediction in patients with HER2-overexpressing breast cancer. J Clin Oncol 30, Abstract 77 (2012). Aim: To further develop/characterize the multiplex ELISA array prior to prospective validation. Methods: Trastuzumab F(ab’)2and synthetic peptide fragments representing components of trastuzumab, or a HER2 synthetic peptide mimetic, were used. Trastuzumab-treated, HER2 positive breast cancer patient plasma/serum samples (n=34; 22 adjuvant; 12 metastatic) obtained from tissue repositories were analyzed by the multiplex ELISA array. Results: For IgG, 26% (9 of 34) of samples from trastuzumab-treated HER2 positive breast cancer patients had detectable antibodies that reacted with components of trastuzumab in the assay, with an absorbency reading greater than 1.0 OD. Assay performance has been improved by optimizing reaction plate preparation, antigen binding, patient dilution, HRP-labeled secondary antibody source and dilution, and colorimetric signal generation and assay performance temperature (range ~20°C-25°C). Assay linearity was established over the range of 1:50 to 1:400 dilution for serum from a sample yielding >3.4 OD using immobilized trastuzumab F(ab’)2 as antigen and for peptides from 1:50 up to 1:2700 with linear correlation coefficients of R2=0.99. Serum or plasma from same blood draw gave equivalent results. Inter-day and intra-day coefficient of variation were less than 5.0 % for the panel of analytes. Conclusions: We have been able to reproduce the original findings in a different cohort of trastuzumab treated HER2 positive breast cancer patients using an improved multiplexed ELISA array. The assay was found to be reliable and robust and is ready for further clinical development and validation.

CYP4A11 arachidonic acid monooxygenase oxidizes endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid, a renal vasoconstrictor and natriuretic. Cyp4a deficiency causes hypertension in male mice, and a loss-of-function variant (T8590C) of CYP4A11 is associated with hypertension in white individuals. Hypertension and hypertensive renal disease are more common among black than white individuals, but the relationship between genetic variation at CYP4A11 and hypertension in black individuals is not known. This study tested the hypothesis that the CYP4A11 T8590C polymorphism is associated with higher BP or clinical outcomes in 732 black Americans with hypertensive renal disease participating in the African American Study of Kidney Disease (AASK). Men with the 8590CC genotype had significantly higher systolic BP (CC 156.5 +/- 22.6 versus 148.4 +/- 24.3 mmHg in CT and TT combined; P = 0.04) and pulse pressure (P = 0.04) at baseline; this association was not observed among women. In addition, this genotype was associated with higher systolic and diastolic BP at 36-mo follow-up among those randomly assigned to the lower BP arm of the AASK. Among all participants (or men but not women) with proteinuria, the 8590CC genotype was associated with an increased cumulative incidence of ESRD or death, controlling for randomization and clinical characteristics. In summary, the CYP4A11 8590CC genotype is associated with increased BP in black men with hypertensive nephrosclerosis and is associated with adverse clinical outcomes in those with baseline proteinuria. These data support a role for renal monooxygenases and 20-hydroxyeicosatetraenoic acid in the regulation of BP and renal function in men.

Genetic variants in the beta(2)-adrenergic receptor (ADRB2) coding block have been associated with different parameters of asthma severity, but there is no consensus on which variants are most important. Our objective was to determine whether the genetic variants in the 5'- or 3'-flanking regions of ADRB2 impact the response to therapy. DNA was obtained initially from 72 adults hospitalized for an asthma exacerbation. We sequenced a 5,000 bp region of the ADRB2 gene that spanned the flanking regions and identified 31 single nucleotide polymorphisms (SNPs). Nonresponders to asthma therapy were defined as patients whose forced expiratory volume in 1 second (FEV(1)) worsened by >10% at 24 hours after admission. We then evaluated the relationship between the 19 common SNPs and response to asthma-specific therapy during acute disease exacerbations. Our results showed a significant association between nonresponders and a haplotype of five promoter SNPs in a nearly complete linkage disequilibrium. An analysis of the promoter and coding block polymorphisms in an extended cohort of 99 patients confirmed that promoter haplotype was the genetic component most strongly associated with asthmatic nonresponders, which was statistically significant among whites (p < 0.05). An identification of this promoter haplotype may provide an alternate explanation for the variation in the asthma responses observed with ADRB2 coding block polymorphisms.

AIMS Vascular α2B adrenergic receptor (ADRA2B) mediate vasoconstriction and contribute to peripheral regulation of vascular tone. In vitro, ADRA2B undergoes agonist-induced desensitization and a common genetic variation, 301–303 deletion, was associated with loss of desensitization. We previously characterized variation in ADRA2B and observed 24 variant sites. This study examines the hypothesis that the 301–303del or other variants alter vascular desensitization in response to the selective ADRA2 agonist, dexmedetomidine (D) in vivo. METHODS Venoconstriction in response to D (0.01–1000 ng/min) was measured in the dorsal hand vein using a linear variable differential transformer in 40 healthy subjects (age 18–45 yrs). On a separate day, the dose that had caused > 50% constriction in the dose-response study was infused for 180 min and response measured. The area under the curve for response, normalized to the initial response (nAUC) was calculated. 5812 bp of contiguous sequence of ADRA2B were examined by bidirectional sequencing, and relationship of ADRA2B variants to nAUC determined. RESULTS Six variants (1 promoter, 3 coding, 2 3′ UTR) were found with an allele frequency> 5%. nAUC was 0.93 ± 0.22 (mean±SD). 301–303del or other common variant did not contribute to variability in nAUC.[Ins/Ins (n=22): 0.89 ± 0.23, Ins/del (n=11): 0.98 ±0.24, del/del (n=7): 0.98 ±0.16; pANOVA= 0.47]. CONCLUSION Common variants in ADRA2B do not contribute substantially to ADRA2B vascular desensitization in vivo. Clinical Pharmacology & Therapeutics (2005) 79, P31–P31; doi: 10.1016/j.clpt.2005.12.112