Surface Adjustment of Reverse Phase Protein Arrays using Positive Control Spots

Reverse phase protein arrays (RPPA) measure the relative expression levels of a protein in many samples simultaneously. Observed signal from these arrays is a combination of true signal, additive background, and multiplicative spatial effects. Background subtraction alone is not sufficient to remove all nonbiological trends from the data. We developed a surface adjustment that uses information from positive control spots to correct for spatial trends on the array beyond additive background. This method uses a generalized additive model to estimate a smoothed surface from positive controls. When positive controls are printed in a dilution series, a nested surface adjustment performs an intensity-based correction. When applicable, surface adjustment is able to remove spatial trends and increase within slide replicate agreement better than background subtraction alone as demonstrated on two sets of arrays. This work demonstrates the importance of including positive control spots on the array

Chemokine-Releasing Nanoparticles for Manipulation of Lymph Node Microenvironment

Chemokines (CKs) secreted by the host cells into surrounding tissue establish concentration gradients directing the migration of leukocytes. We propose an in vivo CK gradient remodeling approach based on sustained release of CKs by the crosslinked poly(N-isopropylacrylamide) hydrogel open meshwork nano-particles (NPs) containing internal crosslinked dye affinity baits for a reversible CK binding and release. The sustained release is based on a new principle of affinity off-rate tuning. The NPs with Cibacron Blue F3G-A and Reactive Blue-4 baits demonstrated a low-micromolar affinity binding to IL-8, MIP-2, and MCP-1 with a half-life of several hours at 37\ub0C. The capacity of NPs loaded with IL-8 and MIP-1\u3b1 to increase neutrophil recruitment to lymph nodes (LNs) was tested in mice after footpad injection. Fluorescently-labeled NPs used as tracers indicated the delivery into the sub-capsular compartment of draining LNs. The animals administered the CK-loaded NPs demonstrated a widening of the sub-capsular space and a strong lymph node influx of leukocytes, while mice injected with control NPs without CKs or bolus doses of soluble CKs alone showed only a marginal neutrophil response. This technology provides a new means therapeutically direct or restore immune cell traffic, and can also be employed for simultaneous therapy delivery

Application of analyte harvesting nanoparticle technology to the measurement of urinary HGH in healthy individuals

Urine represents a valuable biofluid for noninvasive measurement of Human Growth Hormone (HGH) secretion. Unfortunately, currently available commercial HGH immunoassays do not achieve the sensitivity needed for urinary HGH measurement in the low picogram per milliliter range, the expected normal concentration range of HGH in urine. A nanotechnology based sample preprocessing step was used to extract and concentrate HGH in urine so that urinary HGH could be measured with a clinical grade standard immunoassay designed for serum (Immulite 1000, Siemens). We applied the nanoparticle enhanced immunoassay to evaluate the baseline value of urinary HGH in a population of healthy young adults (age 18-30, N=33, median 21, M: F=39%:61%, with no reported medical therapies). Nanoparticle sample preprocessing effectively improved the lower limit of detection of the Immulite HGH assay by more than 50 fold, shifting the linear range of the assay to encompass the expected value of urinary HGH. The full process between run and within run CV% was 7.9 and 9.0%, respectively. On 33 healthy volunteers, the 95% reference values for hGH in spot urine normalized to specific gravity were 0.64 - 16.85 pg/mL (0.05-5.82 ng/g creatinine). Nanoparticle preprocessing constitutes a reliable means of measuring urinary HGH with a clinical grade immunoassay, now establishing a normal baseline value for HGH in urine. Nanoparticles can be used to study the kinetics of HGH excretion in urine, and the factors that influence urinary HGH secretion and HGH isoform proportions

Expression of matrix metalloproteinase-9 in nasopharyngeal carcinoma and association with Epstein-Barr virus infection

Objective: To evaluate the expression of matrix metalloproteinase-9 (MMP9) in nasopharyngeal carcinoma and the association between MMP9 and Epstein-Barr virus infection. Methods: The MMP9 expression was studied by immunohistochemical analysis; and Epstein-Barr virus encoded small nuclear mRNA-1 (EBER-1) produced by in situ hybridization were examined in 41 nasopharyngeal carcinoma sections, and the relation between them, and the associations of MMP9 with clinical features were statistically analyzed. Results: Positive expression rate of MMP9 was 73.17%. The expression of MMP9 showed significant positive correlation with the expression of EBER-1 (γ=0.483, P=0.001). There was significant association of MMP9 expression with lymph nodes metastasis and clinical stage (P<0.001), non-significant association with age, gender, pathological classification and T classification. Conclusions: The highly pronounced expression of MMP9 is associated with cervical lymph nodes metastasis. Epstein-Barr virus can enhance NPC metastasis by up-regulating the expression of MMP9

Modeling of protein signaling networks in clinical proteomics

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomicend points in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states.One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor micro environment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination,clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens