Homogeneous Biosensing Based on Magnetic Particle Labels

The growing availability of biomarker panels for molecular diagnostics is leading to an increasing need for fast and sensitive biosensing technologies that are applicable to point-of-care testing. In that regard, homogeneous measurement principles are especially relevant as they usually do not require extensive sample preparation procedures, thus reducing the total analysis time and maximizing ease-of-use. In this review, we focus on homogeneous biosensors for the in vitro detection of biomarkers. Within this broad range of biosensors, we concentrate on methods that apply magnetic particle labels. The advantage of such methods lies in the added possibility to manipulate the particle labels by applied magnetic fields, which can be exploited, for example, to decrease incubation times or to enhance the signal-to-noise-ratio of the measurement signal by applying frequency-selective detection. In our review, we discriminate the corresponding methods based on the nature of the acquired measurement signal, which can either be based on magnetic or optical detection. The underlying measurement principles of the different techniques are discussed, and biosensing examples for all techniques are reported, thereby demonstrating the broad applicability of homogeneous in vitro biosensing based on magnetic particle label actuation

An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner

Novel Biomarkers of Gastrointestinal Cancer

Gastrointestinal (GI) cancer is a major cause of morbidity and mortality in the world. Since early diagnosis and optimal treatment selection are crucial to improving the prognosis of these diseases, the discovery of useful biomarkers has the potential to greatly reduce their burden. Recent technical and mechanical developments have allowed for the detection of tiny differences in various factors modified in physical conditions, which could contribute to the discovery of novel biomarkers for some diseases.In this Special Issue, we aim to focus on novel biomarkers for GI cancers, including esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer and biliary cancer. In addition, any samples (tissue, blood, urine and feces) are useful as biomarker sources, although body-fluid-based biomarkers are promising as diagnostic biomarkers due to their noninvasiveness. This Special Issue aims to collect novel insights clarifying the current situation and future perspective in this field

Study of the thrombogenicity induced by the cytotoxic treatment of malignant disease

Cancer and its treatment are frequently complicated by the development of venous thromboembolism (VTE). Interestingly, VTE incidence rates vary according to tumour type and the chemotherapy regimen administered. Yet, the precise mechanisms responsible for the increase in VTE in cancer patients remain unclear. Tissue factor (TF), the primary cellular initiator of the coagulation cascade, is over-expressed in many solid malignancies, particularly pancreatic cancer, and the number of circulating TF+ microparticles (MP) are increased in cancer patients. It is hypothesised that the increased risk of VTE in cancer patients generated by cytotoxic treatment may be partly attributed to the apoptotic process, involving the exposure of procoagulant phosphatidylserine (PS) on tumour cells or other chemotherapy damaged cells and increased release of TF+ MP into the circulation. The aim of this study was to explore how coagulation can be initiated in cancer cells and be potentiated by chemotherapy, with respects to the specific expression of TF and PS, and generation of MP.Flow cytometry was used to evaluate expression of cell surface TF and level of apoptosis in untreated/doxorubicin (Dox)-treated cancer cell lines, and number of MP in platelet-free plasma (PFP) from untreated pancreatic cancer patients, myeloproliferative disorder (MPD) patients, and multiple myeloma (MM) patients before, during, and after chemotherapy. The cell proliferation of untreated/Dox-treated cancer cells was assessed by an MTS assay. Procoagulant activity (PCA) of untreated/Dox-treated cancer cells and their isolated cell-free supernatants containing MP, and also MM and MPD patient PFP was measured using a prothrombin time assay. Enzyme-linked immunosorbent assay quantified levels of TF in unfiltered/0.1 μm filtered cell-free supernatants isolated from cancer cell lines, and serum levels of soluble cell adhesion molecules CD106 and CD54 from MM patients.Pancreatic (AsPC-1, CFPAC-1, MIA-PaCa-2), ovarian (A270, ES2, SKOV-3), colorectal (CaCo-2, LoVo), breast (MCF-7, MDA-MB-231, T47D), and haematological (JJN3, U937) cancer cells were found to support coagulation in a cell number-dependent manner, defined by a logarithmic relationship that was consistent across all cell lines. Furthermore, single cell clotting time (CT) was determined for each cancer cell line from the slope and y axis intercept of a logarithmically transformed data plot. A near linear relationship was observed between TF expression and single cell CT where a higher expression of TF results in a proportionally faster CT (P = 0.01). In addition, tumour cell-derived MP were shown to be procoagulant and the majority of procoagulant potential could be removed by passing isolated cell-free supernatants through a 0.1 μm filter. A dose-dependent CT was observed with AsPC-1, CFPAC-1, ES2, SKOV-3, LoVo, and MDA-MB-231 cell-free supernatants.The cytotoxic chemotherapeutic agent Dox was found to decrease the number of viable ovarian (ES2), breast (MDA-MB-231, T47D), and haematological (MM.1S, U937) cancer cells in a time- and dose-dependent manner, and cell death was shown to be induced by apoptosis and subsequently necrosis at higher drug concentrations. Cell surface expression of PS was found to increase following Dox treatment, while TF was not upregulated. Furthermore, Dox was shown to dose-dependently increase PCA in all cancer cells examined, although the effect of cell-free supernatants on PCA was less consistent; ES2, MDA-MB-231, and U937 cell-free supernatants isolated from Dox-treated cells demonstrated increased PCA at 0.01 and 0.1 μM concentrations. The Dox-induced increase in PCA of cancer cells and cell-free supernatants were found to correlate with tumour cell viability (r = 0.79 to 0.97, P < 0.01).Numbers of TF+ MP were significantly higher in 35 untreated pancreatic cancer patients in comparison with 15 MM patients prior to chemotherapy (P < 0.0005). Furthermore, numbers of endothelial cell-derived MP (EMP), monocyte-derived MP, plasma cell-derived MP, and PS+ MP, were significantly higher in 6 MPD patients in comparison with 15 MM patients prior to chemotherapy (P < 0.005), but not levels of platelet-derived MP (PMP) or TF+ MP. Markers of endothelial dysfunction, including EMP and soluble cell adhesion molecules CD106 and CD54, were elevated after thalidomide (Thal)- or lenalidomide (Len)-based therapies in MM patients. Furthermore, PCA was significantly increased in MM patients after treatment (P = 0.007), and also levels of PMP, plasma cell-derived MP, and PS+ MP (P < 0.05), but not monocyte-derived MP (P = 0.33) and TF+ MP (P = 0.41).In summary, this study shows that across a range of tumour sites a consistent relationship is seen between procoagulant potential and both cell number and TF cell surface expression. Dox can increase PCA of cancer cells through reduced cell viability that leads to PS exposure. Importantly, this Dox-induced procoagulant mechanism was not found to involve an upregulation of surface TF antigen on ovarian, breast, or haematological cancer cells. In MM patients treated with Thal- or Len-based therapies increased PCA was observed, which may be mediated by endothelial dysfunction and increased generation of MP

Novel pharmacological strategies for antagonizing anti-apoptosis protein function in malignancy.

Historically, cytotoxic therapies have provided the greatest advances in the treatment of malignant disease. Although some cancers are curable, many are not. Chemotherapeutic drugs rely upon the induction of a phylogenically old, cell suicide programme termed apoptosis for their efficacy. Apoptotic sensitivity is associated with curability, whereas, intrinsic resistance plays a major role in limiting therapeutic effectiveness. Mitochondria, the centres for aerobic respiration in the cell also play a pivotal role in regulating apoptosis. The anti-death proteins Bcl-2 and Bc1-XL localize to the outer mitochondrial membrane, and are expressed at high levels in many resistant malignacies compared with normal tissues. These proteins contribute to resistance by blocking apoptosis, and therefore represent valid targets for the development of novel inhibitory strategies. This thesis presents two strategies with therapeutic potential for antagonizing the anti-death action of Bcl-2 and Bc1-XL in haematological and epithelial malignancies. The first, involves the suppression of Bcl-2 and Bc1-XL gene expression by antisense oligodeoxynucleotides in vitro and in vivo. Mathematical models of antisense pharmacology are presented. The second, targets the mitochondrial megachannel that is intimately involved in apoptosis, and is regulated by binding to Bcl-2 and Bc1-XL In order to quantitatively measure the putative apoptosis sensitizing efficacy of these approaches at single cell resolution, stochastic models are described, enabling robust estimation of the distribution of tolerances and latency preceding apoptosis. The peripheral benzodiazepine receptor interacts with the mitochondrial megachannel. Evidence is provided, that Bcl-2 resistant apoptosis sensitization is mediated in vitro, in vivo, and ex vivo, by the ligand PK11195, through a mechanism involving direct megachannel regulation. This occurs not via the peripheral benzodiazepine receptor as previously thought, but through de novo generation of reactive oxygen species. Investigations of PK11195 pharmacodynamics, and molecular structural studies using proton nuclear magnetic resonance spectroscopy support a novel mechanism of action

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease