Optical Biosensors for the Detection of Rheumatoid Arthritis (RA) Biomarkers: A Comprehensive Review.

A comprehensive review of optical biosensors for the detection of biomarkers associated with rheumatoid arthritis (RA) is presented here, including microRNAs (miRNAs), C-reactive protein (CRP), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA), interleukin-6 (IL-6) and histidine, which are biomarkers that enable RA detection and/or monitoring. An overview of the different optical biosensors (based on fluorescence, plasmon resonances, interferometry, surface-enhanced Raman spectroscopy (SERS) among other optical techniques) used to detect these biomarkers is given, describing their performance and main characteristics (limit of detection (LOD) and dynamic range), as well as the connection between the respective biomarker and rheumatoid arthritis. It has been observed that the relationship between the corresponding biomarker and rheumatoid arthritis tends to be obviated most of the time when explaining the mechanism of the optical biosensor, which forces the researcher to look for further information about the biomarker. This review work attempts to establish a clear association between optical sensors and rheumatoid arthritis biomarkers as well as to be an easy-to-use tool for the researchers working in this field.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

CXCR7 impact on CXCL12 biology and disease.

It is known that the chemokine receptor CXCR7 (RDC1) can be engaged by both chemokines CXCL12 (SDF-1) and CXCL11 (I-TAC), but the exact expression pattern and function of CXCR7 is controversial. CXCR7 expression seems to be enhanced during pathological inflammation and tumor development, and emerging data suggest this receptor is an attractive therapeutic target for autoimmune diseases and cancer. CXCR7/CXCR4 heterodimerization, ß-arrestin-mediated signaling, and modulation of CXCL12 responsiveness by CXCR7 suggest that the monogamous CXCR4/CXCL12 signaling axis is an oversimplified model that needs to be revisited. Consequently, research into CXCR7 biology is of great interest and further studies are warranted. This review summarizes recent findings about the CXCR7 receptor and analyses its impact on understanding the roles of CXCL12 biology in health and disease.

The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression.

Monocytes are versatile cells that can express different functional programs in response to microenvironmental signals. We show that primary blood monocytes secrete the CXCL12 chemokine, and express the CXCR4 and CXCR7 receptors, leading to an autocrine/paracrine loop that contribute to shape monocyte differentiation to a distinct type of macrophages, with an enhanced expression of CD4, CD14, and CD163, or dendritic cells, with a reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The in vivo relevance of CXCL12 production by mononuclear phagocytes was studied in metastatic melanoma tissues by a thoroughly immunofluorescence phenotyping of CXCL12(high) expressing cells, which were CD45(+), coexpressed the macrophage antigens CD68, CD163, and CD209 and constituted the 60%-90% of tumor-associated macrophages. Microarray analysis of primary monocytes revealed that the vascular endothelial growth factor and the angiogenic chemokine CCL1 mRNA levels were up-regulated in response to CXCL12, leading to enhanced expression of both proteins. In addition, we found that CXCL12 autocrine/paracrine signaling down-regulates the expression of the transcription factor RUNX3 and contributes to maintain the long-term CD4 and CD14 expression in monocytes/macrophages. Together, these results suggest that autocrine CXCL12 production modulates differentiation of monocytes toward a distinct program with proangiogenic and immunosuppressive functions.

Moesin orchestrates cortical polarity of melanoma tumour cells to initiate 3D invasion.

Tumour cell dissemination through corporal fluids (blood, lymph and body cavity fluids) is a distinctive feature of the metastatic process. Tumour cell transition from fluid to adhesive conditions involves an early polarization event and major rearrangements of the submembrane cytoskeleton that remain poorly understood. As regulation of cortical actin-membrane binding might be important in this process, we investigated the role of ezrin and moesin, which are key crosslinking proteins of the ERM (ezrin, radixin, moesin) family. We used short interfering RNA (siRNA) to show that moesin is crucial for invasion by melanoma cells in 3D matrices and in early lung colonization. Using live imaging, we show that following initial adhesion to the endothelium or 3D matrices, moesin is redistributed away from the region of adhesion, thereby generating a polarized cortex: a stable cortical actin dome enriched in moesin and an invasive membrane domain full of blebs. Using Lifeact-GFP, a 17-amino-acid peptide that binds F-actin, we show the initial symmetry breaking of cortical actin cytoskeleton during early attachment of round cells. We also demonstrated that ezrin and moesin are differentially distributed during initial invasion of 3D matrices, and, specifically, that moesin controls adhesion-dependent activation of Rho and subsequent myosin II contractility. Our results reveal that polarized moesin plays a role in orienting Rho activation, myosin II contractility, and cortical actin stability, which is crucial for driving directional vertical migration instead of superficial spreading on the fluid-to-solid tissue interface. We propose that this mechanism of cortical polarization could sustain extravasation of fluid-borne tumour cells during the process of metastasis.

Rho/ROCK and myosin II control the polarized distribution of endocytic clathrin structures at the uropod of moving T lymphocytes.

We have examined the spatio-temporal dynamics of clathrin-mediated endocytosis (CME) during T lymphocyte polarization and migration. Near the plasma membrane, we detected heterogeneous arrangements of GFP-clathrin that were clustered predominantly at the uropod; some diffraction limited spots ( approximately 200 nm) and a major population of larger clathrin structures (CSs) (300-800 nm). Membrane CSs fully co-localized with the endocytic adaptor complex AP-2, which was also polarized towards the rear membrane. During the direct incorporation of the endocytic cargo transferrin, large and relatively stable clathrin/AP-2 structures at the uropod membrane transiently co-localized with spots of transferrin, which suggests that they are endocytic competent platforms. The highly polarized distribution of membrane CSs towards the uropod and their endocytic ability support the existence of a preferential region of endocytosis located at or near the rear pole of T lymphocytes. Inactivation of Rho by dominant negative RhoA or C3 exoenzyme, and inhibition of Rho-kinase (ROCK) with Y-27632, or myosin II with blebbistatin, all resulted in suppression of CS polarization, which indicates that the posterior distribution of CSs relies on Rho/ROCK signaling and myosin II contractility. In addition, blocking CME with dominant negative mutants or by clathrin RNA interference, results in a remarkable inhibition of both basal and CXCL12-promoted migration, which suggests that CME is required for successful T-cell migration. We hypothesize that enhanced endocytic rates at the cell rear could provide a mechanism to remove leftover surface to accommodate cell retraction, and/or to spatially resolve signaling for guided cell migration.

Synaptic clusters of MHC class II molecules induced on DCs by adhesion molecule-mediated initial T-cell scanning.

Initial adhesive contacts between T lymphocytes and dendritic cells (DCs) facilitate recognition of peptide-MHC complexes by the TCR. In this report, we studied the dynamic behavior of adhesion and Ag receptors on DCs during initial contacts with T-cells. Adhesion molecules LFA-1- and ICAM-1,3-GFP as well as MHC class II-GFP molecules were very rapidly concentrated at the DC contact area. Binding of ICAM-3, and ICAM-1 to a lesser extent, to LFA-1 expressed by mature but not immature DC, induced MHC-II clustering into the immune synapse. Also, ICAM-3 binding to DC induced the activation of the Vav1-Rac1 axis, a regulatory pathway involved in actin cytoskeleton reorganization, which was essential for MHC-II clustering on DCs. Our results support a model in which ICAM-mediated MHC-II clustering on DC constitutes a priming mechanism to enhance antigen presentation to T-cells.

Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1.

Integrin LFA-1 is a receptor that is able to transmit multiple intracellular signals in leukocytes. Herein we show that LFA-1 induces a potent and transient increase in the activity of the small GTPase Rac-1 in T cells. Maximal Rac-1 activity peaked 10-15 min after LFA-1 stimulation and rapidly declined to basal levels at longer times. We have identified Vav, a guanine nucleotide exchange factor for Rac-1, and PI3K/Akt, as regulators of the activation and inactivation phases of the activity of Rac-1, respectively, in the context of LFA-1 signaling based on the following experimental evidence: (i) LFA-1 induced activation of Vav and PI3K/Akt with kinetics consistent with a regulatory role for these molecules on Rac-1, (ii) overexpression of a constitutively active Vav mutant induces activation of Rac independently of LFA-1 stimulation whereas overexpression of a dominant-negative Vav mutant blocks LFA-1-mediated Rac activation, (iii) pharmacological inhibition of PI3K/Akt prevented the fall in the activity of Rac-1 after its initial activation but had no effect on Vav activity, and (iv) overexpression of a dominant-negative or a constitutively active Akt-1 induced or inhibited, respectively, Rac-1 activity. Finally, we show that T cells with a sustained Rac activity have impaired capacity to elongate onto ICAM-1. These results demonstrate that down-regulation of the activity of this GTPase is a requirement for the regulation of T cell morphology and motility and highlight the importance of temporal regulation of the signaling triggered from this integrin.

LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca(2)(+)-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells.

Lymphocyte function-associated antigen (LFA-1) is a member of the beta2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca(2)(+) mobilizing agents A23187, thapsigargin, and ionomycin induce an increase in adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) and activation and redistribution of the proline-rich tyrosine kinase-2 (PYK2) to the microtubule-organizing center (MTOC) in T-lymphoblasts. These effects are similar to those observed upon direct induction of activation of LFA-1 with the stimulatory mAb KIM-127. Most importantly, Ca(2)(+) mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca(2)(+)-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca(2)(+) chelator EGTA, which depletes the intracellular Ca(2)(+), inhibited the effects of mAb KIM-127 on cell morphology and PYK2 activation. This inhibition with EGTA was not reversed by cross-linking integrin LFA-1 with specific antibodies, indicating that Ca(2)(+) exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca(2)(+) chelators affect the organization of the microtubular cytoskeleton and the localization of PYK2 to the MTOC area, suggesting that these agents could inhibit the activation of PYK2 by interfering with the microtubular network of T cells. Taken together, our results demonstrate for the first time an important role for the integrin LFA-1 and the microtubular cytoskeleton in the Ca(2)(+)-mediated activation of PYK2 in T-lymphocytes.