Esther Serrano-Pertierra

CGRP and VIP Levels as Predictors of Efficacy of Onabotulinumtoxin Type A in Chronic Migraine

Onabotulinumtoxin type A (onabotA) has shown efficacy in chronic migraine (CM). Its precise mechanism of action, however, is unknown.

OnabotulinumtoxinA decreases interictal CGRP plasma levels in patients with chronic migraine.

Abstract OnabotulinumtoxinA (onabotA) has shown efficacy in chronic migraine (CM). Its mechanism of action, however, remains obscure. We have analysed whether treatment with onabotA is able to induce changes in interictal plasma calcitonin gene-related peptide (CGRP) concentrations, which have been shown to be increased in patients with CM. Calcitonin gene-related peptide levels were determined in samples obtained from the right antecubital vein using ELISA, outside a migraine attack and having taken no symptomatic medication in the previous 24 hours, in 83 patients with CM (average age 44 years; 94% females) before and 1 month after treatment with 155 to 195 U of onabotA. CGRP levels after onabotA treatment (median, 51.89 pg/mL; range, 199.4-10.2) were significantly lower as compared with CGRP levels obtained before onabotA treatment (median, 74.09 pg/mL; range, 241.0-11.4; P = 0.001). Pretreatment CGRP levels in responders (76.85 pg/mL) were significantly higher than those seen in nonresponders (50.45 pg/mL; P = 0.001). One month after treatment, the CGRP levels did not change in nonresponders (51.89 pg/mL; P not significant), but significantly decreased in responders (52.48 pg/mL; P = 0.003). A number of demographic factors, clinical features, and comorbidities were not different in responders as compared with those of nonresponders. These results confirm that interictal CGRP levels can be of help in predicting the response to onabotA and suggest that the mechanism of action of onabotA in CM is the reversal of sensitization as a result of the inhibition of CGRP release.

Microparticles in multiple sclerosis and clinically isolated syndrome: effect on endothelial barrier function.

BackgroundCell-derived microparticles are secreted in response to cell damage or dysfunction. Endothelial and platelet dysfunction are thought to contribute to the development of multiple sclerosis (MS). Our aim here is, first, to compare the presence of microparticles of endothelial and platelet origin in plasma from patients with different clinical forms of MS and with clinically isolated syndrome. Second, to investigate the effect of microparticles on endothelial barrier function.ResultsPlatelet-poor plasma from 95 patients (12 with clinically isolated syndrome, 51 relapsing-remitting, 23 secondary progressive, 9 primary progressive) and 49 healthy controls were analyzed for the presence of platelet-derived and endothelium-derived microparticles by flow cytometry. The plasma concentration of platelet-derived and endothelium-derived microparticles increased in all clinical forms of MS and in clinically isolated syndrome versus controls. The response of endothelial barriers to purified microparticles was measured by electric cell-substrate impedance sensing. Microparticles from relapsing-remitting MS patients induced, at equivalent concentrations, a stronger disruption of endothelial barriers than those from healthy donors or from patients with clinically isolated syndrome. MS microparticles acted synergistically with the inflammatory mediator thrombin to disrupt the endothelial barrier function.ConclusionsPlasma microparticles should be considered not only as markers of early stages of MS, but also as pathological factors with the potential to increase endothelial permeability and leukocyte infiltration.

NKG2D- and CD28-mediated costimulation regulate CD8 T cell chemotaxis through different mechanisms: the role of Cdc42/N-WASp

The activating receptor NKG2D is mainly expressed in NK cells and CD8+ T cells. NKG2D and CD28 recruit the p85 subunit of PI3K to propagate their signals through the YXXM signaling motif. The function of CD28 as a costimulatory molecule is well‐established in T cells. Ligation of NKG2D on T cells costimulates TCR signaling, although the intracellular signaling pathways triggered by the two receptors may not be identical. In this study, we analyzed the function of the NKG2D receptor in human CD8+ T cell chemotaxis toward a CXCL12 gradient. We found that costimulation of the TCR together with CD28 or NKG2D impairs cell migration, although the signaling pathways responsible for this effect differ. Whereas the Rho GTPase Rac1 is activated upon TCR and costimulation via CD28 and NKG2D, the activity of Cdc42 is increased only upon CD3/NKG2D activation. Moreover, knockdown of N‐WASp expression with siRNA rescues migration rates after NKG2D‐mediated costimulation but not after CD3/CD28 activation. CD28‐ and NKG2D‐mediated costimulation induces cofilin activation by dephosphorylation. Inhibition of N‐WASp by wiskostatin further decreases phosphorylation levels of cofilin, although this effect is especially severe upon CD3/NKG2D activation. Thus, our findings reveal new differences in the signaling pathways between CD28‐ and NKG2D‐mediated costimulation in the regulation of cell chemotaxis in human CD8+ T cells.

Point-of-care detection of extracellular vesicles: Sensitivity optimization and multiple-target detection

Extracellular vesicles (EVs) are membrane-bound nanovesicles delivered by different cellular lineages under physiological and pathological conditions. Although these vesicles have shown relevance as biomarkers for a number of diseases, their isolation and detection still has several technical drawbacks, mainly related with problems of sensitivity and time-consumed. Here, we reported a rapid and multiple-targeted lateral flow immunoassay (LFIA) system for the detection of EVs isolated from human plasma. A range of different labels (colloidal gold, carbon black and magnetic nanoparticles) was compared as detection probe in LFIA, being gold nanoparticles that showed better results. Using this platform, we demonstrated that improvements may be carried out by incorporating additional capture lines with different antibodies. The device exhibited a limit of detection (LOD) of 3.4×106EVs/µL when anti-CD81 and anti-CD9 were selected as capture antibodies in a multiple-targeted format, and anti-CD63 labeled with gold nanoparticles was used as detection probe. This LFIA, coupled to EVs isolation kits, could become a rapid and useful tool for the point-of-care detection of EVs, with a total analysis time of two hours.

No Change in Interictal PACAP Levels in Peripheral Blood in Women With Chronic Migraine

To determine total pituitary adenylate cyclase activating polypeptide (PACAP) in peripheral blood as a potential marker of the activation of the parasympathetic arm of the trigemino‐vascular system in chronic migraine (CM) in a case‐control study.

Increased natural killer cell chemotaxis to CXCL12 in patients with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease characterized by leukocyte infiltration into the central nervous system (CNS). Migration of lymphocyte subpopulations towards CXCL12 was analyzed coupled to six-color flow cytometry in untreated patients in the remitting phase, during relapse, in patients with clinically isolated syndrome (CIS), and in healthy volunteers. Significantly higher migration rates of natural killer cells (CD45+CD3-CD16/56+) were observed in patients in remission and CIS patients than in patients during relapse and in controls. Moreover, the frequency of CD3-CD16/56+CXCR4+ cells is higher in patients in remission and in CIS patients, but not during relapse.

Wiskott-Aldrich syndrome protein (WASp) and N-WASp are involved in the regulation of NK-cell migration upon NKG2D activation

NKG2D is a transmembrane receptor mainly expressed on CD8+ T cells and NK cells. Engagement of NKG2D with its ligands can trigger a cytotoxic response. It has been shown that tumor cells deliver soluble NKG2D ligands as a mechanism of immune evasion through the downregulation of surface‐expressed NKG2D. These ligands may be also secreted in microvesicles and regulate NK‐cell function, but the existence of alternative mechanisms has not been explored. In this study, we describe that NKG2D activation inhibits NK‐cell chemotaxis toward a CXCL12 gradient. Costimulation of the inhibitory receptor NKG2A rescues NK‐cell migration rates. Thus, the balance of NKG2D/NKG2A activation may determine the migratory ability of NK cells. Furthermore, our data indicated that NKG2D cross‐linking induces the activation of the Rho GTPases Rac1 and Cdc42, while RhoA activity is decreased. Pharmacological inhibition of the Cdc42 effectors Wiskott‐Aldrich syndrome protein (WASp)/N‐WASp, and the reduction of their levels using RNA interference partially abolished NKG2D‐mediated impairment of cell migration, suggesting a pivotal role of Cdc42 in the regulation of NK‐cell migration by NKG2D activation. Therefore, our results provide a new mechanism that may contribute to the immune response or evasion in tumors.

L-plastin is involved in NKG2D recruitment into lipid rafts and NKG2D-mediated NK cell migration

Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β‐OG‐selective solubility in the NKL cell line. We found that the NKG2D‐DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin‐bundling protein L‐plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L‐plastin participates in NKG2D‐mediated inhibition of NK cell chemotaxis.

Circulating extracellular vesicles as potential biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis: an exploratory pilot study

ABSTRACT Chronic Fatigue Syndrome (CFS), also known as Myalgic Encephalomyelitis (ME) is an acquired, complex and multisystem condition of unknown etiology, no established diagnostic lab tests and no universally FDA-approved drugs for treatment. CFS/ME is characterised by unexplicable disabling fatigue and is often also associated with numerous core symptoms. A growing body of evidence suggests that extracellular vesicles (EVs) play a role in cell-to-cell communication, and are involved in both physiological and pathological processes. To date, no data on EV biology in CFS/ME are as yet available. The aim of this study was to isolate and characterise blood-derived EVs in CFS/ME. Blood samples were collected from 10 Spanish CFS/ME patients and 5 matched healthy controls (HCs), and EVs were isolated from the serum using a polymer-based method. Their protein cargo, size distribution and concentration were measured by Western blot and nanoparticle tracking analysis. Furthermore, EVs were detected using a lateral flow immunoassay based on biomarkers CD9 and CD63. We found that the amount of EV-enriched fraction was significantly higher in CFS/ME subjects than in HCs (p = 0.007) and that EVs were significantly smaller in CFS/ME patients (p = 0.014). Circulating EVs could be an emerging tool for biomedical research in CFS/ME. These findings provide preliminary evidence that blood-derived EVs may distinguish CFS/ME patients from HCs. This will allow offer new opportunities and also may open a new door to identifying novel potential biomarkers and therapeutic approaches for the condition.