Comparison between one and two-dimensional liquid chromatographic approaches for the determination of plasmatic stroke biomarkers by isotope dilution and tandem mass spectrometry.

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

Peripheral, Interictal Serum S100B Levels are Not Increased in Chronic Migraine Patients.

BACKGROUND: The trigemino-vascular system (TVS) plays a key role in migraine pathophysiology. Glial cells are abundant in the TVS system and mainly in the trigeminal ganglion. S100B protein is a calcium-binding protein, found in the cytoplasm of glial cells in the central nervous system, which is released in response to inflammatory stimuli. Previous works analyzing S100B in migraineurs have offered contradictory results. OBJECTIVE: In this case-control study, we analyzed serum levels of S100B as a possible biomarker of the glial TVS activation in chronic migraine (CM). PATIENTS AND METHODS: The study group consisted of patients attending our clinic with CM and, as control groups, patients with episodic migraine (EM), cluster headache (CH) outside of a bout and healthy volunteers (HV) with no headache history. S100B levels were determined interictally in peripheral blood samples by ELISA. RESULTS: We assessed serum samples from 43 patients with CM, 19 with EM, 29 HV (mostly women), and 22 with (CH). S100B levels in CM (mean 22.9 ± 9.8 pg/mL) were not different (P = .727) when compared to EM patients (21.2 ± 9.3 pg/mL), difference of 1.7 (95% CI -5.7 to 8.9), CH patients (22.4 ± 7.8 pg/mL), difference of 0.5 (-5.7 to 6.7), and HV (20.6 ± 8.3 pg/mL), difference of 2.3 (-3.7 to 8.3). CONCLUSION: In contrast to other neuropeptides such as calcitonin gene-related-peptide and vasoactive intestinal peptide, which are increased in CM, interictal serum S100B levels are not elevated in these patients. According to our results, S100B levels do not seem to be a useful peripheral biomarker of the glial TVS activation in CM.

¿Existe relación entre las lesiones de la sustancia blanca asociadas a migraña y el foramen oval permeable? Análisis de una serie de pacientes con migraña crónica / Is there a relationship between white matter lesions associated with migraine and patent foramen ovale? Analysis of a series of patients with chronic migraine

INTRODUCCIÓN: Las lesiones de la sustancia blanca son más prevalentes en los pacientes migrañosos que en la población general, especialmente en los que tienen una alta frecuencia de ataques. El foramen oval permeable se ha descrito como posible nexo de unión entre la migraña y las lesiones de la sustancia blanca. OBJETIVO: Determinar la existencia de una posible relación entre el foramen oval permeable y las lesiones de la sustancia blanca en una serie de pacientes con migraña crónica. PACIENTES Y MÉTODOS: Estudio observacional, unicéntrico, de casos y controles. Se seleccionó a 89 mujeres con migraña crónica. La persistencia y las características del foramen oval permeable se evaluaron mediante un estudio Doppler transcraneal. El foramen oval permeable se clasificó como pequeño, moderado o masivo. Se consideraron permanentes los detectados en reposo, y latentes, el resto. El protocolo de resonancia magnética incluyó imágenes sagitales potenciadas en T1, axiales potenciadas en FLAIR-T2 y secuencia combinada de densidad protónica y T2-FSE. Las lesiones de la sustancia blanca se clasificaron como profundas, periventriculares o ambas. RESULTADOS: La prevalencia de foramen oval permeable (53,6% frente a 48,5%; p = 0,80) y la proporción de foramen oval permeable masivo y permanente fueron similares entre los pacientes con y sin lesiones de la sustancia blanca. Tampoco se encontraron diferencias en la prevalencia (55,6% frente a 52,6%; p = 1,00) o las características del foramen oval permeable en función de la distribución de las lesiones de la sustancia blanca. CONCLUSIÓN: Los resultados no sugieren la intervención del foramen oval permeable en la fisiopatología de las lesiones de la sustancia blanca observadas en pacientes migrañosos

INTRODUCTION. White matter lesions are more prevalent in migraine patients than in the general population, especially those with a high frequency of attacks. A patent foramen ovale has been described as a possible link between migraine and white matter lesions. AIM. To determine the existence of a possible relationship between a patent foramen ovale and white matter lesions in a series of patients with chronic migraine. PATIENTS AND METHODS. Observational, single-centre, case-control study. Eighty-nine women with chronic migraine were selected. The persistence and characteristics of the patent foramen ovale were assessed by means of a transcranial Doppler study. The patent foramen ovale was classified as small, moderate or massive. Those detected at rest were considered permanent, and the others were classified as latent. The MRI protocol included T1-enhanced sagittal images, FLAIR-T2-enhanced axial images, and a proton density and T2-FSE combined sequence. The white matter lesions were classified as deep, periventricular or both. RESULTS. The prevalence of patent foramen ovale (53.6% versus 48.5%; p = 0.80) and the proportion of massive, permanent patent foramen ovale were similar among patients with and without white matter lesions. Neither was there any difference in the prevalence (55.6% versus 52.6%; p = 1.00) or the characteristics of the patent foramen ovale as a function of the distribution of white matter lesions. CONCLUSION. The results do not suggest that a patent foramen ovale intervenes in the pathophysiology of the white matter lesions observed in patients with migraine

Characterization of Plasma-Derived Extracellular Vesicles Isolated by Different Methods: A Comparison Study.

Extracellular vesicles (EV) are small membrane structures released by cells that act as potent mediators of intercellular communication. The study of EV biology is important, not only to strengthen our knowledge of their physiological roles, but also to better understand their involvement in several diseases. In the field of biomedicine they have been studied as a novel source of biomarkers and drug delivery vehicles. The most commonly used method for EV enrichment in crude pellet involves serial centrifugation and ultracentrifugation. Recently, different protocols and techniques have been developed to isolate EV that imply less time and greater purification. Here we carry out a comparative analysis of three methods to enrich EV from plasma of healthy controls: ultracentrifugation, ExoQuickTM precipitation solution (System Biosciences), and Total Exosome Isolation kit (Invitrogen). Our results show that commercial precipitation reagents are more efficient and enable higher EV enrichment factors compared with traditional ultracentrifugation, although subsequent imaging analysis is not possible with some of them. We hope that this work will contribute to the current research on isolation techniques to assist the progress of clinical applications with diagnostic or therapeutic objectives.

Neuropeptides as a Marker for Chronic Headache.

PURPOSE OF REVIEW: The purpose of this review is to revise current evidence on trigemino-vascular system (TVS) neuropeptides as potential biomarkers for chronic primary headaches, mainly for chronic migraine (CM). RECENT FINDINGS: Within sensory neuropeptides, released by an activated trigeminal nerve, calcitonin gene-related peptide (CGRP) levels seem to be a good biomarker of acute migraine and somewhat sensitive and specific for CM. CGRP, however, is not increased in 20-30% of CM patients, which suggests that CGRP is not the only neuropeptide involved in migraine pain generation and maintenance. Data for other sensory neuropeptides are inconsistent (neurokinin, substance P) or absent (amylin and cholecystokinin-8). Among parasympathetic neuropeptides, vasoactive intestinal polypeptide (VIP) is increased interictally in CM, and in at least some migraine cases ictally, pituitary adenylate cyclase-activating peptide (PACAP) has been shown to be increased ictally in jugular blood, but interictal, peripheral data do not indicate such an increase, and there are no data for other parasympathetic peptides. Finally, S100B, as a potential marker of glial TVS activation, has been studied with inconsistent results in migraine patients. Current data on TVS neuropeptides as potential migraine biomarkers must be taken with caution, even for the promising case of CGRP. We do not know with certainty whether increased levels are the reflection of TVS activation, the reliability and homogeneity of the different laboratory tests, or what is the influence on these measurements of the short half-life of many of these peptides or of preventive treatments. One further limitation would be whether the described increases in levels of some neuropeptides such as CGRP are specific for migraine versus other headaches.

Calcitonin gene-related peptide in peripheral blood as a biomarker for migraine.

PURPOSE OF REVIEW: There is no available biomarker for any of the primary headaches, including migraine. As demonstrated in jugular blood, during a migraine attack, trigeminal activation releases several neuropeptides, very especially calcitonin gene-related peptide (CGRP), which gives rise to the typical throbbing migraine pain. Here, we review the current evidence for measurement of peripheral CGRP levels as a potential biomarker for trigemino-vascular activation in migraine. RECENT FINDINGS: Several studies, including a limited number of migraine patients, have shown increased peripheral CGRP levels during migraine attacks. The maximum increase in plasma CGRP levels was reached within 2 h of the onset of the attacks and can be reverted by triptans. In addition, CGRP levels measured in peripheral blood outside migraine attacks and in the absence of symptomatic medication have been shown to be increased in chronic migraine patients. Increased CGRP levels were able to predict the response to onabotulinumtoxinA treatment and were reduced 1 month after onabotulinumtypeA therapy. SUMMARY: Although CGRP data must be confirmed and expanded in future studies and specificity of CGRP levels should be studied in entities able to resemble migraine, it seems that peripheral CGRP levels are a good biomarker of acute migraine and somewhat specific and sensitive interictally in chronic migraine.

Receiver operating characteristic curve generalization for non-monotone relationships.

The receiver operating characteristic curve is a popular graphical method frequently used in order to study the diagnostic capacity of continuous markers. It represents in a plot true-positive rates against the false-positive ones. Both the practical and theoretical aspects of the receiver operating characteristic curve have been extensively studied. Conventionally, it is assumed that the considered marker has a monotone relationship with the studied characteristic; i.e., the upper (lower) values of the (bio)marker are associated with a higher probability of a positive result. However, there exist real situations where both the lower and the upper values of the marker are associated with higher probability of a positive result. We propose a receiver operating characteristic curve generalization, [Formula: see text], useful in this context. All pairs of possible cut-off points, one for the lower and another one for the upper marker values, are taken into account and the best of them are selected. The natural empirical estimator for the [Formula: see text] curve is considered and its uniform consistency and asymptotic distribution are derived. Finally, two real-world applications are studied.

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.

OBJECTIVE: 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. METHODS: Women older than 17 and diagnosed as CM were recruited. Healthy women with no headache history and women with episodic migraine (EM) served as control groups. Total PACAP and vasoactive intestinal peptide (VIP) levels were determined in blood samples obtained from the right antecubital vein by ELISA outside a migraine attack and having taken no symptomatic medication the day before. RESULTS: We assessed serum samples from 86 women with CM, 32 healthy women, and 35 women with EM. There were no differences in PACAP levels in CM patients (109.8 ± 43.8, 97.4 [32.5-253.1] pg/mL), controls (108.7 ± 43.0, 98.7 [50.7-197.3] pg/mL), or EM patients (98.8 ± 34.3, 94.2 [52.0-190.7] pg/mL). VIP levels were significantly increased (P = .027) in CM as compared to control healthy women (136.0 ± 111.5 pg/mL; 103.1 [20.5-534.0] pg/mL vs 88.6 ± 61.0 pg/mL; 66.0 [21.1-256.1]) and EM patients (103.0 ± 56.7 pg/mL; 103.5 [15.2-263.0] pg/mL). In the range of this study variables such as age, CM duration, the presence of aura, analgesic overuse, depression, fibromyalgia, vascular risk factors, history of triptan consumption or kind of preventative treatment did not significantly influence PACAP or VIP levels. CONCLUSION: In contrast to VIP, interictal PACAP level measured in peripheral blood does not seem to be a biomarker reflecting parasympathetic activation in CM.

Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation.

Prevalence of cranial autonomic parasympathetic symptoms in chronic migraine: Usefulness of a new scale.

BACKGROUND: Cranial autonomic symptoms (CAS) seem to appear in around half of migraine patients. OBJECTIVE: Our aim was to analyse the prevalence and profile of CAS, mainly of cranial autonomic parasympathetic symptoms (CAPS), in a series of patients with chronic migraine (CM) according the new criteria for autonomic symptoms in the current IHS classification. PATIENTS AND METHODS: We recruited consecutive CM patients attending our headache clinic. Five CPAS were surveyed: lacrimation, conjunctival injection, eyelid oedema, ear fullness and nasal congestion. They were graded as 0 (absent), 1 (present and mild) and 2 (present and conspicuous); therefore the score in this CAPS scale ranges from 0 to 10 points. As a cranial autonomic sympathetic symptom (CSAS), we also asked about the presence of ptosis. RESULTS: We interviewed 100 CM patients. Their mean age was 45 years (18-63 years); 93 were females. Eighteen had no CAPS, while 82 reported at least one CAPS. There were only six patients with scores higher than 5, the mean and median CAPS being 2.1 and 2, respectively. Prevalence of CAPS was lacrimation (49%), conjunctival injection (44%), eyelid oedema (39%), ear fullness (30%) and nasal congestion (20%). Ptosis was reported by 42. CONCLUSION: These results, by using for the first time an easy quantitative scale, confirm that (mild) CAPS are not the exception but the rule in CM patients. The score in this CAPS scale could be of help as a further endpoint in clinical trials or to be correlated with potential biomarkers of parasympathetic activation in primary headaches.

Patología arterial en la migraña: disfunción endotelial y cambios estructurales en la vasculatura cerebral y sistémica / Arterial pathology in migraine: endothelial dysfunction and structural changes in the brain and systemic vasculature

Introducción. La fisiopatología subyacente a la asociación entre migraña y otras enfermedades vasculares sistémicas no aterotrombóticas no se conoce con certeza. La disfunción endotelial se ha propuesto como nexo común. A su vez, la disfunción endotelial se considera como precursora de cambios estructurales en las paredes arteriales. Objetivo. Revisar el conocimiento actual acerca de las alteraciones funcionales (disfunción endotelial) y estructurales (rigidez arterial y cambios ateroescleróticos) del lecho arterial asociadas a la migraña. Desarrollo. Estudios de marcadores biológicos de disfunción endotelial en sangre periférica, vasorreactividad sistémica y cerebral, cálculo de índices de rigidez arterial y visualización directa de cambios macroscópicos en la pared arterial han mostrado diferencias entre pacientes con y sin migraña, así como entre los distintos subtipos de migraña. Conclusiones. La disfunción endotelial, como precursora de cambios estructurales a nivel arterial, se postula como sustrato de la patología vascular asociada a la migraña. La alteración de marcadores biológicos es sugestiva de disfunción endotelial en los pacientes con migraña; sin embargo, la correlación con estudios de vasorreactividad no permite establecer conclusiones definitivas. Los datos disponibles no permiten concluir que la migraña se asocie con alteraciones macroscó- picas fuera del lecho arterial cerebral (AU)

Introduction. The pathophysiology underlying the association between migraine and other non-atherosclerotic vascular diseases is largely unknown. Endothelial dysfunction has been proposed as a common link. Besides, endothelial dysfunction is considered as a predictor of structural changes in the arterial walls. Aim. To review the current knowledge about the functional (endothelial dysfunction) and structural (arterial stiffness and atherosclerotic diseases) arterial properties associated with migraine. Development. Studies of biological markers of endothelial dysfunction in peripheral blood, systemic and cerebral vasoreactivity, arterial stiffness indexes and direct visualization of macroscopic changes in the arterial wall have shown differences between patients with and without migraine, as well as between the different migraine subtypes. Conclusions. Endothelial dysfunction, as a predictor of structural changes in arteries, has been proposed as an early marker for vascular pathology associated with migraine. In migraine patients there is an increase of biomarkers of endothelial dysfunction, but the correlation with vasoreactivity studies does not allow definite conclusions. Available data do not allow to conclude that migraine is associated with macroscopic alterations outside the cerebral arterial bed (AU)

[Arterial pathology in migraine: endothelial dysfunction and structural changes in the brain and systemic vasculature]. / Patologia arterial en la migraña: disfuncion endotelial y cambios estructurales en la vasculatura cerebral y sistemica.

INTRODUCTION: The pathophysiology underlying the association between migraine and other non-atherosclerotic vascular diseases is largely unknown. Endothelial dysfunction has been proposed as a common link. Besides, endothelial dysfunction is considered as a predictor of structural changes in the arterial walls. AIM: To review the current knowledge about the functional (endothelial dysfunction) and structural (arterial stiffness and atherosclerotic diseases) arterial properties associated with migraine. DEVELOPMENT: Studies of biological markers of endothelial dysfunction in peripheral blood, systemic and cerebral vasoreactivity, arterial stiffness indexes and direct visualization of macroscopic changes in the arterial wall have shown differences between patients with and without migraine, as well as between the different migraine subtypes. CONCLUSIONS: Endothelial dysfunction, as a predictor of structural changes in arteries, has been proposed as an early marker for vascular pathology associated with migraine. In migraine patients there is an increase of biomarkers of endothelial dysfunction, but the correlation with vasoreactivity studies does not allow definite conclusions. Available data do not allow to conclude that migraine is associated with macroscopic alterations outside the cerebral arterial bed.

TITLE: Patologia arterial en la migraña: disfuncion endotelial y cambios estructurales en la vasculatura cerebral y sistemica.Introduccion. La fisiopatologia subyacente a la asociacion entre migraña y otras enfermedades vasculares sistemicas no aterotromboticas no se conoce con certeza. La disfuncion endotelial se ha propuesto como nexo comun. A su vez, la disfuncion endotelial se considera como precursora de cambios estructurales en las paredes arteriales. Objetivo. Revisar el conocimiento actual acerca de las alteraciones funcionales (disfuncion endotelial) y estructurales (rigidez arterial y cambios ateroescleroticos) del lecho arterial asociadas a la migraña. Desarrollo. Estudios de marcadores biologicos de disfuncion endotelial en sangre periferica, vasorreactividad sistemica y cerebral, calculo de indices de rigidez arterial y visualizacion directa de cambios macroscopicos en la pared arterial han mostrado diferencias entre pacientes con y sin migraña, asi como entre los distintos subtipos de migraña. Conclusiones. La disfuncion endotelial, como precursora de cambios estructurales a nivel arterial, se postula como sustrato de la patologia vascular asociada a la migraña. La alteracion de marcadores biologicos es sugestiva de disfuncion endotelial en los pacientes con migraña; sin embargo, la correlacion con estudios de vasorreactividad no permite establecer conclusiones definitivas. Los datos disponibles no permiten concluir que la migraña se asocie con alteraciones macroscopicas fuera del lecho arterial cerebral.

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.

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

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.

Long-term experience with onabotulinumtoxinA in the treatment of chronic migraine: What happens after one year?

BACKGROUND: OnabotulinumtoxinA (onabotA) has shown its efficacy over placebo in chronic migraine (CM), but clinical trials lasted only up to one year. OBJECTIVE: The objective of this article is to analyse our experience with onabotA treatment of CM, paying special attention to what happens after one year. PATIENTS AND METHODS: We reviewed the charts of patients with CM on onabotA. Patients were injected quarterly during the first year but the fifth appointment was delayed to the fourth month to explore the need for further injections. RESULTS: We treated 132 CM patients (mean age 47 years; 119 women). A total of 108 (81.8%) showed response during the first year. Adverse events, always transient and mild-moderate, were seen in 19 (14.4%) patients during the first year; two showed frontotemporal muscle atrophy after being treated for more than five years. The mean number of treatments was 7.7 (limits 2-29). Among those 108 patients with treatment longer than one year, 49 (45.4%) worsened prior to the next treatment, which obliged us to return to quarterly injections and injections were stopped in 14: in 10 (9.3%) due to a lack of response and in four due to the disappearance of attacks. In responders, after an average of two years of treatment, consumption of any acute medication was reduced by 53% (62.5% in triptan overusers) and emergency visits decreased 61%. CONCLUSIONS: Our results confirm the long-term response to onabotA in three-quarters of CM patients. After one year, lack of response occurs in about one out of 10 patients and injections can be delayed, but not stopped, to four months in around 40% of patients. Except for local muscle atrophy in two cases treated more than five years, adverse events are comparable to those already described in short-term clinical trials.

Increased VIP levels in peripheral blood outside migraine attacks as a potential biomarker of cranial parasympathetic activation in chronic migraine.

AIM: The aim of this article is to determine vasoactive intestinal peptide (VIP) levels outside migraine attacks in peripheral blood as a potential biomarker for chronic migraine (CM). METHODS: Women older than 17 and diagnosed as CM were recruited. Matched healthy women with no headache history and women with episodic migraine (EM) served as control groups, together with a series of patients with episodic cluster headache in a pain-free period. VIP levels were determined in blood samples obtained from the right antecubital vein by ELISA outside a migraine attack, the patients having taken no symptomatic medication the day before. For ethical reasons, preventives were not stopped. RESULTS: We assessed plasma samples from 119 women with CM, 33 healthy women, 51 matched women with EM and 18 patients (16 males) with cluster headache matched for age. VIP levels were significantly increased in CM (165.1 pg/ml) as compared to control healthy women (88.5 pg/ml) and episodic cluster headache patients (101.1 pg/ml). VIP levels in EM (134.9 pg/ml) were significantly higher compared to controls and numerically lower than those of CM. Thresholds of 71.8 and 164.5 pg/ml optimized the sensitivity and specificity to differentiate CM from healthy controls and EM, respectively. Variables such as age, CM duration, the presence of aura, analgesic overuse, depression, fibromyalgia, vascular risk factors, history of triptan consumption or kind of preventive treatment did not significantly influence VIP levels. CONCLUSION: Increased interictal VIP level measured in peripheral blood could be a biomarker helping in CM diagnosis, though it does not clearly differentiate between EM and CM.

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

BACKGROUND: Cell-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. RESULTS: Platelet-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. CONCLUSIONS: Plasma 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.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.