Engineering topics

Zangar, Richard C

Publications and source records attributed to Zangar, Richard C.

11 records

DOE OSTI2013

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts, but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures. - Highlights: ► Obesity modulates inflammatory markers in BAL fluid after LPS exposure. ► Obese animals have a unique transcriptional signature in lung after LPS exposure. ► Obesity elevates inflammatory stress and reduces antioxidant capacity in the lung. ► Toxicant-specific biomarkers predict exposure independent of systemic inflammation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Multiplexed Cancer Biomarker Detection Using Quartz-based Photonic Crystal Surfaces

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20–99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1–41 pg/mL), resulting in a three-log range of quantitative detection.

Huang, Cheng-Sheng↗

DOE OSTI2011

Plasma Biomarkers for Detecting Hodgkin's Lymphoma in HIV Patients

The lifespan of AIDS patients has increased as a result of aggressive antiretroviral therapy, and the incidences of the AIDS-defining cancers, Hodgkin's lymphoma and Kaposi sarcoma, are declining, Still, the increased longevity of AIDS patients is now associated with increased incidence of other cancers, including Hodgkin's lymphoma (HL). In order to determine if we could identify biomarkers for the early detection of HL, we undertook an accurate mass and elution time tag proteomics analysis of individual plasma samples from AIDS patients without HL (n=14) and with HL (n=22). This analysis identified 33 proteins, included C-reactive protein and three serum amyloid proteins, that were statistically (p<0.05) altered by at least 1.5-fold between the two groups. At least three of these proteins have previously been reported to be altered in the blood of HL patients. Ingenuity Pathway Analysis software identified 'inflammatory response' and 'cancer' as the top two, biological functions commonly associated with these proteins. The clear association of these proteins with cancer and inflammation suggests that they are truly associated with HL and that they would be useful in the detection of this disease.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Reducing heterophilic antibody interference in immunoassays using single chain antibodies

Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2011

Reactive Oxygen Species Alter Autocrine and Paracrine Signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endo-plasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A customELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-{kappa}B pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Development of a Fibrinogen-Specific Sandwich Enzyme-Linked Immunosorbent Assay Microarray Assay for Distinguishing Between Blood Plasma and Serum Samples

We have developed a fibrinogen-specific sandwich ELISA microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies, 49D2, HPA001900, and F8512, were evaluated in conjunction with 1D6 as detection antibody, and the data show that 49D2 and, to a lesser extent, F8512 successfully identify previously unknown plasma and serum samples based upon a ~28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high throughput manner prior to proteomics analyses.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2011

Plasma Biomarker Profiles Differ Depending on Breast Cancer Subtype but RANTES is Consistently Increased

Background: Current biomarkers for breast cancer have little potential for detection. We determined if breast cancer subtypes influence circulating protein biomarkers. Methods: A sandwich-ELISA microarray platform was used to evaluate 23 candidate biomarkers in plasma samples that were obtained from subjects with either benign breast disease or invasive breast cancer. All plasma samples were collected at the time of biopsy, after a referral due to a suspicious screen (e.g., mammography). Cancer samples were evaluated based on breast cancer subtypes, as defined by the HER2 and estrogen receptor statuses. Results: Ten proteins were statistically altered in at least one breast cancer subtype, including four epidermal growth factor receptor ligands, two matrix metalloproteases, two cytokines, and two angiogenic factors. Only one cytokine, RANTES, was significantly increased (P<0.01 for each analysis) in all four subtypes, with areas under receiver operating characteristic curves (AUC) that ranged from 0.76 to 0.82, depending on cancer subtype. The best AUC values were observed for analyses that combined data from multiple biomarkers, with values ranging from 0.70 to 0.99, depending on the cancer subtype. Although the results for RANTES are consistent with previous publications, the multi-assay results need to be validated in independent sample sets. Conclusions: Different breast cancer subtypes produce distinct biomarker profiles, and circulating protein biomarkers have potential to differentiate between true and false positive screens for breast cancer. Impact: Subtype-specific biomarker panels may be useful for detecting breast cancer or as an adjunct assay to improve the accuracy of current screening methods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Comparative Proteomics and Pulmonary Toxicity of Instilled Single Walled Carbon Nanotubes, Crocidolite Asbestos and Ultrafine Carbon Black in Mice

Reflecting their exceptional potential to advance a range of biomedical, aeronautic, and other industrial products, carbon nanotube (CNT) production, and the potential for human exposure to aerosolized CNT’s, is increasing. CNT’s have toxicologically significant structural and chemical similarities to asbestos, and have repeatedly been shown to cause pulmonary inflammation, granuloma formation and fibrosis after inhalation/instillation/aspiration exposure in rodents, a pattern of effects similar to those observed following exposure to asbestos. To determine the degree to which responses to SWCNT and asbestos are similar or different, the pulmonary response of C57BL/6 mice to repeated exposure to SWCNT, crocidolite asbestos and ultrafine carbon black (UFCB) were compared using high-throughput global HPLC-FTICR-MS proteomics, histopathology and BAL cytokine analyses. Mice were exposed to material suspensions (40 μg/mouse) twice a week, for 3 weeks by pharyngeal aspiration. Histologically, the incidence and severity of inflammatory and fibrotic responses were greatest in mice treated with SWCNT. SWCNT treatment affected the greatest changes in abundance of identified lung tissue proteins. The trend in number of proteins affected (SWCNT (376)>asbestos (231)>UFCB (184)) followed the potency of these materials in 3 biochemical assays of inflammation (cytokines). SWCNT treatment uniquely affected the abundance of 109 proteins, but these proteins largely represent cellular processes affected by asbestos treatment as well, further evidence of broad similarity in the tissue-level response to asbestos and SWCNT. Two high sensitivity markers of inflammation, one (S100a9) observed in humans exposed to asbestos, were found and may be promising biomarkers of human response to SWCNT exposure.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Analysis of High-Throughput ELISA Microarray Data

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

Protein Secretion in Human Mammary Epithelial Cells following HER1 Receptor Activation: Influence of HER2 and HER3 Expression

Background: Secretion of proteins by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a very limited understanding of the cellular regulatory processes that regulate protein secretion. Method: In this study, we utilize an ELISA microarray platform to evaluate the effects of epidermal growth factor receptor (HER) expression on protein secretion in human epithelial mammary cells (HMEC). These secreted proteins included several HER1 ligands, interleukins 1α and 18, RANTES, vascular endothelial and platelet derived growth factors, matrix metalloproteases 1, 2 and 9, and the extracellular portion of the HER1 and HER2 proteins. Result: We utilized HMEC lines that were engineered to express different levels of HER1, HER2 and HER3. We determined the effects of these receptors on the secretion of a variety of growth factors, cytokines, and proteases. Conclusion: Overall, this study suggests that HER overexpression orchestrate broad affects on the tumor microenvironment by altering the secretion of a diverse group of biologically active proteins.

59 BASIC BIOLOGICAL SCIENCES↗