scholarly journals Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2956
Author(s):  
Suresh Veeramani ◽  
George J. Weiner

Molecular complexes, such as ligand–receptor complexes, are vital for both health and disease and can be shed into the circulation in soluble form. Relatively little is known about the biology of soluble ligand–receptor complexes. The functional importance of such complexes and their potential use as clinical biomarkers in diagnosis and therapy remains underappreciated. Most traditional technologies used to study ligand–receptor complexes measure the individual levels of soluble ligands or receptors rather than the complexes themselves. The fraction of receptors occupied by ligand, and the potential clinical relevance of such information, has been largely overlooked. Here, we review the biological significance of soluble ligand–receptor complexes with a specific focus on their potential as biomarkers of cancer and other inflammatory diseases. In addition, we discuss a novel RNA aptamer-based technology, designated ligand–receptor complex-binding aptamers (LIRECAP), that can provide precise measurement of the fraction of a soluble receptor occupied by its ligand. The potential applicability of the LIRECAP technology as a biomarker discovery platform is also described.

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3568
Author(s):  
Hina Mir ◽  
Neeraj Kapur ◽  
Dominique N. Gales ◽  
Praveen K. Sharma ◽  
Gabriela Oprea-Ilies ◽  
...  

Precise mechanisms underlying breast cancer (BrCa) metastasis are undefined, which becomes a challenge for effective treatments. Chemokine signaling instigates the trafficking of cancer cells in addition to leukocytes. This study aimed to ascertain the clinical and biological significance of the CXCR6/CXCL16 signaling axis in the pathobiology of BrCa. Our data show a higher expression of CXCR6 in BrCa cell lines and tissues. Stage-III BrCa tissues express significantly higher CXCR6 compared to stage-II tissues. The ligand, CXCL16, could remain tethered to the cell surface, and, after proteolytic shedding of the ectodomain, the N-terminal fragment is released, converting it to its oncogenic, soluble form. Like CXCR6, N-terminal CXCL16 and ADAM-10 were significantly higher in stage-III than stage-II, but no significant difference was observed in the C-terminal fragment of CXCL16. Further, stimulation of the CXCR6/CXCL16 axis activated Src, FAK, ERK1/2, and PI3K signaling pathways, as per antibody microarray analysis, which also underlie CXCL16-induced F-actin polymerization. The CXCR6/CXCL16 axis induces cytoskeleton rearrangement facilitating migration and invasion and supports BrCa cell survival by activating the PI3K/Akt pathway. This study highlights the significance of the CXCR6/CXCL16 axis and ADAM10 as potential therapeutic targets for advanced-stage BrCa.


2008 ◽  
Vol 13 (6) ◽  
pp. 515-526 ◽  
Author(s):  
Weidong Du ◽  
Xueling Ma ◽  
E. Marion Schneider

The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH 2 groups to N-hydroxysuccinimide (NHS)—activated surfaces and investigated the potential application of tumor necrosis factor—α (TNFα) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFα antigen and TNFα antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients ( r) in TNFα and TNFα antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFα-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFα, TNFα antibody, and TNFα-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFα, 30.9% (13/42) for TNFα antibody, 28.6% (12/42) for TNFα—receptor 1 complex, and 26.1% (11/42) for TNFα—receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients. ( Journal of Biomolecular Screening 2008:515-526)


2001 ◽  
Vol 100 (5) ◽  
pp. 567-575 ◽  
Author(s):  
Funmi M. BELGORE ◽  
Andrew D. BLANN ◽  
Gregory Y. LIP

Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. VEGF interacts with the endothelium via two membrane-spanning receptors, fms-like tyrosine kinase (Flt)-1 and kinase domain receptor. A soluble form of Flt-1 (sFlt-1) was isolated from endothelial cell media; however, its biological significance is still unknown, with limited data on plasma sFlt-1 levels in disease states. We have developed two new ELISAs for detecting free and VEGF-complexed sFlt-1, which were tested in accordance with standard validation and assessment methodologies employed in commercial settings. The intra-and inter-assay coefficients of variation are < 5% and 10% respectively, and results are highly reproducible. Applying these ELISAs in a clinical setting, we measured levels of VEGF, free and complexed sFlt-1 in citrated plasma from 40 patients with cardiovascular disease and 40 healthy controls. Median (interquartile range) plasma levels of VEGF in patients were significantly greater than controls [403 pg/ml (158–925 pg/ml) versus 113 pg/ml (33–231 pg/ml), P ⩽ 0.05]. Free sFlt-1 was significantly lower in patients compared with controls [8 ng/ml (2–22 ng/ml) versus 21 ng/ml (10–73 ng/ml), P ⩽ 0.05]. There was no significant difference in the levels of complexed sFlt-1 between the two groups. Plasma levels of VEGF-complexed sFlt-1 are minimal, despite the presence of excess free sFlt-1. Thus unbound plasma VEGF detected by ELISA may represent the majority of circulating VEGF, and justifies the measurement of plasma VEGF as an indicator of circulating VEGF levels. Furthermore, these results suggest that circulating sFlt-1 may serve as a selective inhibitor of VEGF activity, and that this regulatory mechanism may be altered by pathological conditions.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4012-4012 ◽  
Author(s):  
Zaiba Shamim ◽  
Stephanie Thiant ◽  
Sylvie Faucher ◽  
Bachra Choufi ◽  
Lars Ryder ◽  
...  

Abstract Abstract 4012 Background: Interleukin-7 (IL-7) is a cytokine essential for T cell development in the thymus and maintenance of peripheral T cells. IL-7 binds to cellular IL-7 receptors (IL-7Ra-common g chain heterodimer), in competition with a soluble form of the receptor, shed by the cells (sIL-7Ra). We have identified single nucleotide polymorphisms in the exons of the gene encoding IL-7Ra (+510T/C rs1494558, +1237 G/C rs1494555, 2087 C/T rs6897932), and previous results by us and by others indicate that IL-7R SNPs are associated with aGvHD and mortality after SCT. Moreover, the biological significance of +2087 C/T SNP has been suggested by the finding of elevated serum levels of sIL-7Ra in healthy individuals with 2087CC (Haplotype 4), also associated with increased alternative splicing of exon 6 and a higher frequency of recent thymic emigrants. We hypothesized that sIL-7Ra levels during SCT are influenced by genetic polymorphism and may play a role in immune reconstitution after SCT. Aims: 1) To investigate sIL-7Ra levels during SCT along with IL-7Ra genotypes and 2) to evaluate associations between sIL-7Ra levels and immune reconstitution and outcome in SCT. Patients and Methods: 122 patients undergoing SCT for haematological malignancies after either myeloablative (n= 52) or reduced intensity conditioning (n=70) were included. Donors were either matched siblings (n=68) or matched unrelated donors (n=54), and the patient age at the time of transplant was 50 years (6–67) (median (range)). sIL-7Ra levels were measured in plasma by a quantitative bead capture assay. IL-7Ra SNPs were determined by a SSP-PCR system and IL-7 was tested by ELISA (R&D) (n=77). Results: sIL-7Ra levels decreased during the course of transplantation from 113 ng/ml (32–558) at day −15 to 48 ng/ml (32–195) at day +14 (p=0.0001), and reached baseline levels again at day +60. sIL-7Ra levels were not associated with the intensity of the conditioning regimen. This pattern appeared to be inversely mirrored by the IL-7 levels, which increased from baseline values at day –14 of 2.4 pg/ml (0.3–17.6) to 11.3 pg/ml (2.0–30.2) (p<0.0001) at day +14, followed by a gradual decline down to baseline values at day +60. sIL-7Ra levels at day +90 were reduced in patients transplanted with donors carrying IL-7Ra 2087T allele, in line with our previous findings in healthy individuals (105 ng/ml (42–274) vs. 152 ng/ml (20–971), p=0.0015). In addition, post-transplant sIL-7Ra levels correlated with pre-transplant sIL-7Ra levels (r=0.39 p=0.0032), indicating that patient related factors in addition to the genotype of the donor lymphocytes may play a role in the regulation of sIL-7Ra levels post-transplant. sIL-7Ra appeared to be predictive of the rate of immune reconstitution by the finding that sIL-7Ra at day +14 correlated significantly with total lymphocyte counts post-transplant (day +90: r=0.28 (p=0.031) and day +180: r=0.55 (p<0.0001)). In contrast, IL-7Ra genotypes were not associated with immune lymphocyte counts post-transplant and early post-transplant IL-7 levels did not correlate significantly with lymphocyte counts at any stage. Outcomes: There was a trend towards an association between high sIL-7Ra levels and increased overall survival (p=0.057), but sIL-7Ra levels were unrelated to the occurrence of aGvHD. However, the IL-7/sIL-7Ra ratio at day +14 was significantly higher in patients with grade 2–4 aGvHD compared to grade 0–1 aGvHD (0.29 (0.04–0.73) vs 0.19 (0.01–0.8), p=0.033). Conclusion: The data of the present study indicates that sIL-7Ra levels after SCT are determined by the IL-7R 2087 genotype of the donor in addition to patient related factors. sIL-7Ra levels in the early phase post transplant is associated with the rate of lymphocyte recovery post-SCT. Thereby this study adds to the growing evidence suggesting the importance of the IL-7 axis in SCT. The study further suggests that monitoring sIL-7Ra levels post-transplant may help to guide the clinical use of IL-7. Disclosures: No relevant conflicts of interest to declare.


2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15583-e15583
Author(s):  
Juan-Sebstian Saldivar ◽  
Jason Harris ◽  
Sejal Desai ◽  
Erin Ayash ◽  
Prateek Tandon ◽  
...  

e15583 Background: While immunotherapy has become a pillar of cancer treatment, diagnostic biomarkers that consistently predict patient response to these therapies have remained elusive. There is an increasing need for the development of integrative, composite biomarkers that can model the complex biology driving response and/or resistance to immunotherapy more effectively than existing single-analyte approaches. However, the majority of current cancer diagnostic panels, with their focus on a small set of genes, provide limited ability to support these emerging advanced biomarkers. Methods: To address these limitations, we developed and validated NeXT Dx, a comprehensive enhanced exome and transcriptome based diagnostic platform designed to simultaneously characterize tumor and immune genomics from a single limited FFPE sample. To achieve higher accuracy and sensitivity for an exome scale diagnostic platform, we developed an augmented exome assay that improves uniformity of coverage across all ~20,000 genes, including boosted coverage of 248 clinically-relevant cancer genes. We validated this assay using genomic DNA and RNA extracted from tumor-derived cell-lines, constructs, clinical FFPE samples, and proficiency testing samples. The assay utilizes > = 25ng of co-extracted DNA and RNA which were sequenced using Illumina NovaSeq instruments at our CAP-accredited, CLIA-certified laboratory. Additional assay enhancements for HLA, immune repertoire, and oncoviruses were designed to further optimize the platform for immunotherapy biomarker discovery applications. Results: Validation of NeXT Dx demonstrated a performance of 99.5% sensitivity and 99.8% positive predictive value (PPV) for SNVs with > = 5% AF; 98.7% sensitivity and 97.4% PPV for indels with > = 10% AF; 97.2% sensitivity and 94.6% PPV for CNAs in samples with > = 30% tumor content; 94.9% sensitivity and 94.9% PPV for fusions; and a 2.1% error rate for MSI classification. TMB was calculated using gold-standard whole exome data from SNVs and indels. Typical median coverage depth was > 1,000X for 248 clinically-relevant genes, ~300X for the remaining (whole exome) footprint. Conclusions: With NeXT Dx, we demonstrate a exome/transcriptome scale diagnostic platform that can detect current clinical biomarkers with high sensitivity as well as support emerging, advanced biomarkers that integrate across both tumor and immune features.


1994 ◽  
Vol 179 (4) ◽  
pp. 1307-1316 ◽  
Author(s):  
R J Faull ◽  
N L Kovach ◽  
J M Harlan ◽  
M H Ginsberg

We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.


2003 ◽  
Vol 31 (6) ◽  
pp. 1462-1471 ◽  
Author(s):  
S.K. Dower ◽  
E.E. Qwarnstrom

We have been analysing the signalling systems that couple to receptors of the TIR (Toll/interleukin-1 receptor) family, which signal through a common cytoplasm region; the TIR domain. These systems are of both practical and fundamental biological significance, being central to the pathogenesis of chronic inflammatory diseases such as atherosclerosis, to host defence throughout the biological world, and are ancient in the context of life on earth, having originated more than 1 billion years ago: prior to the divergence of plants and animals. TIR domain receptors couple to at least two sets of well-characterized pathways: those leading to the activation of inhibitory κB kinase complexes/nuclear factor κB, and those leading to the activation of mitogen-activated protein kinase/AP-1/ATF-2 etc. We have been investigating these systems using a combination of expression screening methods to identify new components, and real-time green fluorescent protein-based techniques to observe execution of signalling programmes in real time. Our data reveal that there is a very large level of cell-to-cell variation in signal programme execution even in clonal populations and that at least one mechanism for dealing with this heterogeneity is the assembly of signal transduction components into large multiprotein complexes.


2008 ◽  
Vol 54 (1) ◽  
pp. 24-38 ◽  
Author(s):  
René R S Packard ◽  
Peter Libby

Abstract Recent investigations of atherosclerosis have focused on inflammation, providing new insight into mechanisms of disease. Inflammatory cytokines involved in vascular inflammation stimulate the generation of endothelial adhesion molecules, proteases, and other mediators, which may enter the circulation in soluble form. These primary cytokines also induce production of the messenger cytokine interleukin-6, which stimulates the liver to increase production of acute-phase reactants such as C-reactive protein. In addition, platelets and adipose tissue can generate inflammatory mediators relevant to atherothrombosis. Despite the irreplaceable utility of plasma lipid profiles in assessment of atherosclerotic risk, these profiles provide an incomplete picture. Indeed, many cardiovascular events occur in individuals with plasma cholesterol concentrations below the National Cholesterol Education Program thresholds of 200 mg/dL for total cholesterol and 130 mg/dL for low-density lipoprotein (LDL) cholesterol. The concept of the involvement of inflammation in atherosclerosis has spurred the discovery and adoption of inflammatory biomarkers for cardiovascular risk prediction. C-reactive protein is currently the best validated inflammatory biomarker; in addition, soluble CD40 ligand, adiponectin, interleukin 18, and matrix metalloproteinase 9 may provide additional information for cardiovascular risk stratification and prediction. This review retraces the biology of atherothrombosis and the evidence supporting the role of inflammatory biomarkers in predicting primary cardiovascular events in this biologic context.


2020 ◽  
Vol 11 ◽  
Author(s):  
Reema Bansal ◽  
Amod Gupta

The diseases affecting the retina or uvea (iris, ciliary body, or choroid) generate changes in the biochemical or protein composition of ocular fluids/tissues due to disruption of blood-retinal barrier. Ocular infections and inflammations are sight-threatening diseases associated with various infectious and non-infectious etiologies. Several etiological entities cause uveitis, a complex intraocular inflammatory disease. These causes of uveitis differ in different populations due to geographical, racial, and socioeconomic variations. While clinical appearance is sufficiently diagnostic in many diseases, some of the uveitic entities manifest nonspecific or atypical clinical presentation. Identification of biomarkers in such diseases is an important aid in their diagnostic armamentarium. Different diseases and their different severity states release varying concentrations of proteins, which can serve as biomarkers. Proteomics is a high throughput technology and a powerful screening tool for serum biomarkers in various diseases that identifies proteins by mass spectrometry and helps to improve the understanding of pathogenesis of a disease. Proteins determine the biological state of a cell. Once identified as biomarkers, they serve as future diagnostic and pharmaceutical targets. With a potential to redirect the diagnosis of idiopathic uveitis, ocular proteomics provide a new insight into the pathophysiology and therapeutics of various ocular inflammatory diseases. Tears, aqueous and vitreous humor represent potential repositories for proteomic biomarkers discovery in uveitis. With an extensive proteomics work done on animal models of uveitis, various types of human uveitis are being subjected to proteome analysis for biomarker discovery in different ocular fluids (vitreous, aqueous, or tears).


2020 ◽  
Vol 7 ◽  
Author(s):  
Jun Liao ◽  
Qian Fu ◽  
Wenfang Chen ◽  
Jun Li ◽  
Wenhui Zhang ◽  
...  

Previous studies have implicated the role of CD146 and its soluble form (sCD146) in the pathogenesis of inflammatory diseases. However, the association between CD146 and acute rejection in kidney transplant patients remains unexplored. In this study, fifty-six patients with biopsy-proved rejection or non-rejection and 11 stable allograft function patients were retrospectively analyzed. Soluble CD146 in plasma was detected in peripheral blood by enzyme linked immunosorbent assay (ELISA), and local CD146 expression in graft biopsy was detected by immunohistochemistry. We found that plasma soluble CD146 in acute rejection recipients was significantly higher than in stable patients without rejection, and the biopsy CD146 staining in the rejection group was higher than that of the non-rejection group. Multivariate analysis demonstrated soluble CD146 as an independent risk factor of acute rejection. The area under the receiver operating characteristic curve (AUC) of sCD146 for AR diagnosis was 0.895, and the optimal cut-off value was 75.64 ng/ml, with a sensitivity of 87.8% and a specificity of 80.8%, which was better than eGFR alone (P = 0.02496). Immunohistochemistry showed CD146 expression in glomeruli was positively correlated with the Banff-g score, and its expression in tubules also had a positive relationship with the Banff-t score. Therefore, soluble CD146 may be a potential biomarker of acute rejection. Increased CD146 expression in the endothelial or tubular epithelial cells may imply that endothelial/epithelial dysfunction is involved in the pathogenesis of immune injury.


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