Michael J. Pugia

Bikunin (Urinary Trypsin Inhibitor): Structure, Biological Relevance, And Measurement

Inflammatory processes, such as phagocytosis, coagulation, and vascular dilation, promote the release of serine proteases by neutrophils, macrophages, mast cells, lymphocytes, and the epithelial or endothelial cells. These proteases further facilitate the release of inflammatory cytokines and growth factors as well as take part in signal-cell proliferation through protease-activated receptors (PARs). Controlling the action of this cascade is necessary to prevent further damage to the normal tissues. One of the main anti-inflammatory response mediators is bikunin (Bik) that is responsible for inhibiting the activity of many serine proteases such as trypsin, thrombin, chymotrypsin, kallikrein, plasmin, elastase, cathepsin, Factors IXa, Xa, XIa, and XlIa. During the acute-phase response, Bik is released into plasma from proinhibitors primarily due to increased elastase activity. Bik is a glycoprotein, also referred to as urinary trypsin inhibitor, which in plasma inhibits the trypsin family of serine proteases by binding to either of the two Kunitz-binding domains. Bik also accumulates in urine. In conditions such as infection, cancer, tissue injury during surgery, kidney disease, vascular disease, coagulation, and diabetes, the concentrations of Bik in plasma and urine are increased. Several trypsin inhibitory assays for urine and immunoassays for both blood and urine have been described for measuring Bik. In addition to presenting the synthesis, structure, and pathophysiology of Bik, we will summarize various diagnostic approaches for measuring Bik. Analysis of Bik may provide a rapid approach in assessing various conditions involving the inflammatory processes.

An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI) ☆

Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC-MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC-MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC-MS/MS. The assay is performed on 350μL of plasma, and has a linear range from 0.08 to 5.3ng/L/s in the reflector mode, and 0.04 to 5.3ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC-MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R(2) value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics.

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch® vs. our microfluidic filter method revealed moderate correlation (R2 = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

The utility of circulating LHCGR as a predictor of Down's syndrome in early pregnancy.

BackgroundPrevious studies showed that soluble LHCGR/hCG-sLHCGR concentrations in serum or plasma combined with PAPP-A and free βhCG significantly increased the sensitivity of Down’s syndrome screen at early pregnancy without altering the false positive rate. The goal of the present study was to further examine the role of sLHCGR forms as combinatorial markers and to investigate whether sLHCGR could serve as an independent biomarker for Down’s syndrome in first trimester pregnancy screens.MethodsThe PAPP-A, free βhCG, and hCG-sLHCGR concentrations together with nuchal translucency (NT) were measured in 40 Down’s and 300 control pregnancies. The sLHCGR concentration was analysed in 40 Down’s and 206 control pregnancies.ResultsThe hCG-LHCGR in combination with PAPP-A and free βhCG increased the detection rate (DR) by 35% without altering the false positive rate (FPR). The sLHCGR: hCG-sLHCGR ratio alone detected 80% of Down’s pregnancies in first trimester screening, with a false positive rate of 0.5%.ConclusionsWhile measurement of sLHCGR forms in combination with PAPP-A and free βhCG significantly increases the detection rate of Down’s syndrome at first trimester, the ratio of sLHCGR: hCG-sLHCGR acts as an independent marker with a detection rate that is significantly higher than the existing biochemical markers individually for prenatal first trimester screening of Down’s syndrome.

Adiponectin Receptor-1 C-Terminal Fragment (CTF) in Plasma: Putative Biomarker for Diabetes

IntroductionPolypeptide fragments from cell surface receptors when found in plasma may be indicators of receptor regulation in disease conditions. It is known that subjects with diabetes have significantly lower plasma concentrations of adiponectin, a hormone released by adipose tissue, compared with nondiabetic controls. This hormone interacts with cell surface receptors in muscle (AdipoR1) and liver (AdipoR2).MethodsWe analyzed the relative distribution of specific fragments of AdipoR1 in healthy and diabetic individuals using an immunoaffinity mass spectrometry approach. We used antibodies raised against AdipoR1 immobilized on pre-activated protein chip surfaces to determine the molecular weights of bound polypeptide fragments using immunomass spectrometry (immuno-MS).ResultsInitially, immuno-MS analyses using a polyclonal antibody revealed two peaks (m/z 3,902 and 7,812) in plasma from normal, healthy individuals (n = 5) that were not present in the plasma of diabetics (n = 5). To confirm the detection of these fragments, a monoclonal antibody was developed against the last 25 amino acids of the AdipoR1 C-terminal fragment (CTF). Using the immuno-MS method, the monoclonal antibody detected the AdipoR1 CTF (m/z 3475) in all healthy controls (n = 10), but did not detect these fragments in the diabetic patients (n = 10).DiscussionThese preliminary observations suggest that the plasma levels of this receptor fragment may serve as an indicator of diabetic condition.

Enrichment and Detection of Circulating Tumor Cells and Other Rare Cell Populations by Microfluidic Filtration

The current standard methods for isolating circulating tumor cells (CTCs) from blood involve EPCAM-based immunomagnetic approaches. A major disadvantage of these strategies is that CTCs with low EPCAM expression will be missed. Isolation by size using filter membranes circumvents the reliance on this cell surface marker, and can facilitate the capture not only of EPCAM-negative CTCs but other rare cells as well. These cells that are trapped on the filter membrane can be characterized by immunocytochemistry (ICC) , enumerated and profiled to elucidate their clinical significance. In this chapter, we discuss advances in filtration systems to capture rare cells as well as downstream ICC methods to detect and identify these cells. We highlight our recent clinical study demonstrating the feasibility of using a novel method consisting of automated microfluidic filtration and sequential ICC for detection and enumeration of CTCs, as well as circulating mesenchymal cells (CMCs), circulating endothelial cells (CECs), and putative circulating stem cells (CSCs). We hypothesize that simultaneous analysis of circulating rare cells in blood of cancer patients may lead to a better understanding of disease progression and development of resistance to therapy.

Forms of Circulating Luteinizing Hormone Human Chorionic Gonadotropin Receptor for the Prediction of Early and Late Preeclampsia in the First Trimester of Pregnancy

Objective: To explore the value of circulating luteinizing human chorionic gonadotropin receptor (LHCGR) forms for the prediction of preeclampsia (PE) in the first trimester of pregnancy. Methods: Case-control study, based on a cohort of 5,759 pregnancies, including 20 early PE, 20 late PE, and 300 controls. We recorded/measured maternal characteristics, mean arterial pressure (MAP), uterine artery (UtA) Doppler, placental growth factor (PlGF), soluble Fms-like tyrosine kinase-1 (sFtl-1), and LHCGR forms (hCG-LHCGR and soluble LHCGR), and their independent predictive values were analyzed by logistic regression. Results: For early PE, the model included black ethnicity, chronic hypertension, previous PE, MAP, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, achieving detection rates (DR) of 83% at 10% of false-positive rates (FPR) [AUC: 0.961 (95% CI: 0.921-1)]. For late PE, the model included body mass index, previous PE, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, with DR of 75% at 10% of FPR [AUC: 0.923 (95% CI: 0.871-0.976)]. In both early and late PE, LHCGR forms improved DR by 6-15%. Conclusions: LHCGR forms improved the prediction for early and late PE. These results should be confirmed in larger prospective studies.

Automated Microfluidic Filtration and Immunocytochemistry Detection System for Capture and Enumeration of Circulating Tumor Cells and Other Rare Cell Populations in Blood

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.

Tumor Cell Detection by Mass Spectrometry Using Signal Ion Emission Reactive Release Amplification.

A method is presented for the detection of circulating tumor cells (CTC) using mass spectrometry (MS), through reporter-ion amplification. Particles functionalized with short-chain peptides are bound to cells through antibody-antigen interactions. Selective release and MS detection of peptides is shown to detect as few as 690 cells isolated from a 10 mL blood sample. Here we present proof-of-concept results that pave the way for further investigations.

Cardiovascular and Metabolic Syndrome Impact on Uristatin

Urinary trypsin inhibitors (uTi) were modestly elevated in cardiovascular disease patients (CVD), coronary artery disease (CAD) or metabolic syndrome patients (MetSyn). Urinary and plasma immunoassay values for Uristatin (Uri) and Bikunin (Bik) were within their normal ranges for 94 % of CVD patients and 97 % of MetSyn patients. Urinary and plasma Bik and Uri values were also only slightly elevate with metabolic syndrome risk factors such as diabetes (DM), lipidemia (LIP), hypertension (HTN), and obesity (OBS). Bik and Uri were typically elevated in CVAD patients with clinically significant events such as acute myocardial infarction (AMI) or congestive heart failure (CHF). Here urinary trypsin inhibitors (uTi) are indicative of a white blood cell (WBC) response, as acute events also elevated granulocytes in blood. Granulocytes values significantly correlated (P < .005) with the degree of coronary artery stenosis and coronary angiography. Generally patients with cardiovascular and metabolic conditions and without renal disease or infection had uTi values below the acute phase level (<12 mg/L).