Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings

Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus

BACKGROUND: Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity. METHODS AND FINDINGS: We performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity. CONCLUSIONS: These data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus

Circulating protein biomarkers of pharmacodynamic activity of sunitinib in patients with metastatic renal cell carcinoma: modulation of VEGF and VEGF-related proteins

<p>Abstract</p> <p>Background</p> <p>Sunitinib malate (SUTENT^®) is an oral, multitargeted tyrosine kinase inhibitor, approved multinationally for the treatment of advanced RCC and of imatinib-resistant or – intolerant GIST. The purpose of this study was to explore potential biomarkers of sunitinib pharmacological activity via serial assessment of plasma levels of four soluble proteins from patients in a phase II study of advanced RCC: VEGF, soluble VEGFR-2 (sVEGFR-2), placenta growth factor (PlGF), and a novel soluble variant of VEGFR-3 (sVEGFR-3).</p> <p>Methods</p> <p>Sunitinib was administered at 50 mg/day on a 4/2 schedule (4 weeks on treatment, 2 weeks off treatment) to 63 patients with metastatic RCC after failure of first-line cytokine therapy. Predose plasma samples were collected on days 1 and 28 of each cycle and analyzed via ELISA.</p> <p>Results</p> <p>At the end of cycle 1, VEGF and PlGF levels increased >3-fold (relative to baseline) in 24/54 (44%) and 22/55 (40%) cases, respectively (P < 0.001). sVEGFR-2 levels decreased ≥ 30% in 50/55 (91%) cases and ≥ 20% in all cases (P < 0.001) during cycle 1, while sVEGFR-3 levels were decreased ≥ 30% in 48 of 55 cases (87%), and ≥ 20% in all but 2 cases. These levels tended to return to near-baseline after 2 weeks off treatment, indicating that these effects were dependent on drug exposure. Overall, significantly larger changes in VEGF, sVEGFR-2, and sVEGFR-3 levels were observed in patients exhibiting objective tumor response compared with those exhibiting stable disease or disease progression (P < 0.05 for each analyte; analysis not done for PlGF).</p> <p>Conclusion</p> <p>Sunitinib treatment in advanced RCC patients leads to modulation of plasma levels of circulating proteins involved in VEGF signaling, including soluble forms of two VEGF receptors. This panel of proteins may be of value as biomarkers of the pharmacological and clinical activity of sunitinib in RCC, and of angiogenic processes in cancer and other diseases.</p

Modulation of innate immunity during endotoxemia and gram-negative infection

Lower respiratory tract infection is the fourth most common cause of death in the world. Pneumonia can result in sepsis, which is a major cause or morbidity and mortality worldwide. The research described in this thesis focuses on the innate immune response during gram-negative (bacterial) infection, particularly pneumonia and sepsis. For this we made use of established human (endotoxemia) and mouse models (bacterial pneumonia and sepsis). We first studied the role of mesenchymal stem cells (MSCs) as potential new sepsis therapy. We used adipose-derived mesenchymal stem cells (ASCs) in a randomized placebo controlled endotoxemia trial. We found that ASC infusion was well tolerated and resulted in mixed proinflammatory and anti-inflammatory responses. In addition, ASC infusion resulted in a transient systemic procoagulant effect. In a murine model of Klebsiella pneumoniae derived pneumosepsis, we show that treatment with human ASCs resulted in immune modulatory effects in the lung, associated with less bacterial outgrowth and reduced lung inflammation. We furthermore found that ASCs express tissue factor dependent procoagulant activity. To gain more knowledge on the pathophysiology we studied two important mediatiors of innate immunity, MMP-8 and caspase-11. Using MMP-8 and caspase-11 knock-out mice respectively, we were able to show their effects in a murine model of pneumosepsis. We show a limited role for MMP-8 in the host response. Caspase-11 deficiency was associated with less fibrin formation and more bacterial outgrowth in the lungs

Modulation of innate immunity during endotoxemia and gram-negative infection

Lower respiratory tract infection is the fourth most common cause of death in the world. Pneumonia can result in sepsis, which is a major cause or morbidity and mortality worldwide. The research described in this thesis focuses on the innate immune response during gram-negative (bacterial) infection, particularly pneumonia and sepsis. For this we made use of established human (endotoxemia) and mouse models (bacterial pneumonia and sepsis). We first studied the role of mesenchymal stem cells (MSCs) as potential new sepsis therapy. We used adipose-derived mesenchymal stem cells (ASCs) in a randomized placebo controlled endotoxemia trial. We found that ASC infusion was well tolerated and resulted in mixed proinflammatory and anti-inflammatory responses. In addition, ASC infusion resulted in a transient systemic procoagulant effect. In a murine model of Klebsiella pneumoniae derived pneumosepsis, we show that treatment with human ASCs resulted in immune modulatory effects in the lung, associated with less bacterial outgrowth and reduced lung inflammation. We furthermore found that ASCs express tissue factor dependent procoagulant activity. To gain more knowledge on the pathophysiology we studied two important mediatiors of innate immunity, MMP-8 and caspase-11. Using MMP-8 and caspase-11 knock-out mice respectively, we were able to show their effects in a murine model of pneumosepsis. We show a limited role for MMP-8 in the host response. Caspase-11 deficiency was associated with less fibrin formation and more bacterial outgrowth in the lungs