New advances in genomics and epigenetics in antiphospholipid syndrome.

APS patients exhibit a wide clinical heterogeneity in terms of the disease's origin and progression. This diversity can be attributed to consistent aPL profiles and other genetic and acquired risk factors. Therefore, understanding the pathophysiology of APS requires the identification of specific molecular signatures that can explain the pro-atherosclerotic, pro-thrombotic and inflammatory states observed in this autoimmune disorder. In recent years, significant progress has been made in uncovering gene profiles and understanding the intricate epigenetic mechanisms and microRNA changes that regulate their expression. These advancements have highlighted the crucial role played by these regulators in influencing various clinical aspects of APS. This review delves into the recent advancements in genomic and epigenetic approaches used to uncover the mechanisms contributing to vascular and obstetric involvement in APS. Furthermore, we discuss the implementation of novel bioinformatics tools that facilitate the investigation of these mechanisms and pave the way for personalized medicine in APS.

Splicing machinery is profoundly altered in systemic lupus erythematosus and antiphospholipid syndrome and directly linked to key clinical features.

OBJECTIVES: To characterize the splicing machinery (SM) of leukocytes from primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome with lupus (APS + SLE) patients, and to assess its clinical involvement. METHODS: Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS + SLE) and 50 HD were purified, and 45 selected SM components were evaluated by qPCR-microfluidic array. Relationship with clinical features and underlying regulatory mechanisms were assessed. RESULTS: APS, SLE and APS + SLE leukocytes displayed significant and specific alterations in SM-components (SMC), associated with clinical features [autoimmune profiles, disease activity, lupus nephritis (LN), and CV-risk markers]. A remarkable relationship among dysregulated SMC in monocytes and the presence of LN in SLE was highlighted, revealing a novel pathological mechanism, which was further explored. Immunohistology analysis of renal biopsies highlighted the pathological role of the myeloid compartment in LN. Transcriptomic analysis of monocytes from SLE-LN(+) vs SLE-LN(-) identified 271 genes differentially expressed, mainly involved in inflammation and IFN-signaling. Levels of IFN-related genes correlated with those of SMC in SLE-LN(+). These results were validated in two external SLE-LN(+) datasets of whole-blood and kidney biopsies. In vitro, SLE-LN(+)-serum promoted a concomitant dysregulation of both, the IFN signature and several SMC, further reversed by JAKinibs treatment. Interestingly, IFNs, key inflammatory cytokines in SLE pathology, also altered SMC. Lastly, the over/down-expression of selected SMC in SLE-monocytes reduced the release of inflammatory cytokines and their adhesion capacity. CONCLUSION: Overall, we have identified, for the first time, a specific alteration of SMC in leukocytes from APS, SLE and APS + SLE patients that would be responsible for the development of distinctive clinical profiles.

Voriconazole-induced periostitis.

Voriconazole is a fluorinated drug from the triazole group that is widely used in the prophylaxis and treatment of fungal infections in immunosuppressed patients. Chronic use of this medication can generate, as an adverse effect, a multifocal, asymmetric, diffuse and nodular periosteal reaction, associated with severe and disabling skeletal pain and elevated alkaline phosphatase and serum fluoride. Radiography is the imaging technique of choice for periostitis diagnosis. In general, clinical manifestations and radiographic findings disappear, when the drug is discontinued. We report the clinical case of a 44 year-old woman diagnosed with acute myeloid leukemia, who developed an invasive fungal infection treated with voriconazole after a stem cell transplant. Nine months after starting antifungal treatment, she manifested symptoms and radiological signs compatible with periostitis. Due to clinical suspicion, we decided to suspend voriconazole, with consequent resolution of clinical manifestations and radiological findings.

Granular flow through an aperture: Influence of obstacles near the outlet.

We study how the presence of obstacles in a confined system of monodisperse disks affects their discharge through an aperture. The disks are driven by a horizontal conveyor belt that moves at constant velocity. The mean packing fraction at the outlet decreases as the distance between the obstacles and the aperture decreases. The obstacles organize the dynamics of the stagnant zones in two characteristic behaviors that differ mainly in the magnitude of the fluctuations of the fraction of stagnant disks in the system. It is shown that the effective aperture is reduced by the presence of obstacles.

Accuracy of different diagnostic tests for early, delayed and late prosthetic joint infection.

BACKGROUND: A combination of laboratory, histopathological and microbiological tests for diagnosis of prosthetic joint infection (PJI) have been strongly recommended. This study aims to characterize the accuracy of individual or group tests, such as culture of sonicate fluid, synovial fluid and peri-implant tissue, C-reactive protein (CRP) and histopathology for detection of early, delayed and late PJI. METHODS: A prospective study of patients undergoing hip or knee arthroplasty from February 2009 to February 2014 was performed in a Spanish tertiary health care hospital. The diagnostic accuracy of the different methods was evaluated constructing receiver-operating-characteristic (ROC) curve areas. RESULTS: One hundred thirty consecutive patients were included: 18 (13.8%) early PJI, 35 (27%) delayed PJI and 77 (59.2%) late PJI. For individual parameters, the area under the ROC curve for peri-implant tissue culture was larger for early (0.917) than for delayed (0.829) and late PJI (0.778), p = 0.033. There was a significantly larger difference for ROC area in the synovial fluid culture for delayed (0.803) than for early (0.781) and late infections (0.679), p = 0.039. The comparison of the areas under the ROC curves for the two microbiological tests showed that sonicate fluid was significantly different from peri-implant tissue in delayed (0.951 vs 0.829, p = 0.005) and late PJI (0.901 vs 0.778, p = 0.000). The conjunction of preoperative parameters, synovial fluid culture and CRP, improved the accuracy for late PJI (p = 0.01). The conjunction of histopathology and sonicate fluid culture increased the area under ROC curve of sonication in early (0.917 vs 1.000); p = 0.06 and late cases (0.901 vs 0.999); p < 0.001. CONCLUSION: For early PJI, sonicate fluid and peri-implant tissue cultures achieve the same best sensitivity. For delayed and late PJI, sonicate fluid culture is the most sensitive individual diagnostic method. By combining histopathology and peri-implant tissue, all early, 97% of delayed and 94.8% of late cases are diagnosed. The conjunction of histopathology and sonicate fluid culture yields a sensitivity of 100% for all types of infection.

Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

'Atherothrombosis-associated microRNAs in Antiphospholipid syndrome and Systemic Lupus Erythematosus patients'.

MicroRNAs markedly affect the immune system, and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs' expression was linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes, and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression, and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

Immunotherapy in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a disorder characterized by the association of arterial or venous thrombosis and/or pregnancy morbidity with the presence of antiphospholipid antibodies (anticardiolipin antibodies, lupus anticoagulant antibodies, and/or anti-ß2-glycoprotein I antibodies). Thrombosis is the major manifestation in patients with aPLs, but the spectrum of symptoms and signs associated with aPLs has broadened considerably, and other manifestations, such as thrombocytopenia, non-thrombotic neurological syndromes, psychiatric manifestations, livedo reticularis, skin ulcers, hemolytic anemia, pulmonary hypertension, cardiac valve abnormality, and atherosclerosis, have also been related to the presence of those antibodies. Several studies have contributed to uncovering the basis of antiphospholipid antibody pathogenicity, including the targeted cellular components, affected systems, involved receptors, intracellular pathways used, and the effector molecules that are altered in the process. Therapy for thrombosis traditionally has been based on long-term oral anticoagulation; however, bleeding complications and recurrence despite high-intensity anticoagulation can occur. The currently accepted first-line treatment for obstetric APS (OAPS) is low-dose aspirin plus prophylactic unfractionated or low-molecular-weight heparin (LMWH). However, in approximately 20% of OAPS cases, the final endpoint, i.e. a live birth, cannot be achieved. Based on all the data obtained in different research studies, new potential therapeutic approaches have been proposed, including the use of new oral anticoagulants, statins, hydroxychloroquine, coenzyme Q10, B-cell depletion, platelet and TF inhibitors, peptide therapy or complement inhibition among others. Current best practice in use of these treatments is discussed.

Suspension flow: do particles act as mixers?

Recently, Roht et al. [J. Contam. Hydrol., 2013, 145, 10-16] observed that the presence of suspended non-Brownian macroscopic particles decreased the dispersivity of a passive solute, for a pressure-driven flow in a narrow parallel-plate channel at low Reynolds numbers. This result contradicts the idea that the streamline distortion caused by the random diffusive motion of the particles increases the dispersion and mixing of the solute. Therefore, to estimate the influence of this motion on the dispersivity of the solute, and investigate the origin of the reported decrease, we experimentally studied the probability density function (pdf) of the particle velocities, and spatio-temporal correlations, in the same experimental configuration. We observed that, as the mean suspension velocity exceeds a critical value, the pdf of the streamwise velocity of the particles markedly changes from a symmetric distribution to an asymmetric one strongly skewed to high velocities and with a peak of the most probable velocity close to the maximum velocity. The latter observations and the analysis of the suspension microstructure indicate that the observed decrease in the dispersivity of the solute is due to particle migration to the mid-plane of the channel, and consequent flattening of the velocity profile. Moreover, we estimated the contribution of particle diffusive motion to the solute dispersivity to be three orders of magnitude smaller than the reported decrease, and thus negligible. Solute dispersion is then much more affected by how particles modify the flow velocity profile across the channel than by their random diffusive motion.

Dispersive liquid-liquid microextraction for metals enrichment: a useful strategy for improving sensitivity of laser-induced breakdown spectroscopy in liquid samples analysis.

A rapid and efficient Dispersive Liquid-Liquid Microextraction (DLLME) followed by Laser-Induced Breakdown Spectroscopy detection (LIBS) was evaluated for simultaneous determination of Cr, Cu, Mn, Ni and Zn in water samples. Metals in the samples were extracted with tetrachloromethane as pyrrolidinedithiocarbamate (APDC) complexes, using vortex agitation to achieve dispersion of the extractant solvent. Several DLLME experimental factors affecting extraction efficiency were optimized with a multivariate approach. Under optimum DLLME conditions, DLLME-LIBS method was found to be of about 4.0-5.5 times more sensitive than LIBS, achieving limits of detection of about 3.7-5.6 times lower. To assess accuracy of the proposed DLLME-LIBS procedure, a certified reference material of estuarine water was analyzed.

Granular flow through an aperture: influence of the packing fraction.

For the last 50 years, the flow of a granular material through an aperture has been intensely studied in gravity-driven vertical systems (e.g., silos and hoppers). Nevertheless, in many industrial applications, grains are horizontally transported at constant velocity, lying on conveyor belts or floating on the surface of flowing liquids. Unlike fluid flows, that are controlled by the pressure, granular flow is not sensitive to the local pressure but rather to the local velocity of the grains at the outlet. We can also expect the flow rate to depend on the local density of the grains. Indeed, vertical systems are packed in dense configurations by gravity, but, in contrast, in horizontal systems the density can take a large range of values, potentially very small, which may significantly alter the flow rate. In the present article, we study, for different initial packing fractions, the discharge through an orifice of monodisperse grains driven at constant velocity by a horizontal conveyor belt. We report how, during the discharge, the packing fraction is modified by the presence of the outlet, and we analyze how changes in the packing fraction induce variations in the flow rate. We observe that variations of packing fraction do not affect the velocity of the grains at the outlet, and, therefore, we establish that flow-rate variations are directly related to changes in the packing fraction.

Flow of a thin liquid film coating a horizontal stationary cylinder.

An experimental and theoretical study of the flow of liquid films around a stationary horizontal cylinder is reported. The film presents two different behaviors: The flow is stable in the upper zone (up to ∼150° with the vertical) and Rayleigh-Taylor-like instabilities appear in the lower zone. For the stable region, film thickness evolution could be described by numerically integrating an evolution equation obtained using a lubrication approximation. For the unstable region, a linear stability analysis allows us to determine the maximum growth wavelength for the Rayleigh-Taylor instability. Approximate analytical solutions were obtained for generatrices at an angle with the vertical θ=0 (stable region) and θ=π (where the instability appears).

Pressure independence of granular flow through an aperture.

We experimentally demonstrate that the flow rate of granular material through an aperture is controlled by the exit velocity imposed on the particles and not by the pressure at the base, contrary to what is often assumed in previous work. This result is achieved by studying the discharge process of a dense packing of monosized disks through an orifice. The flow is driven by a conveyor belt. This two-dimensional horizontal setup allows us to independently control the velocity at which the disks escape the horizontal silo and the pressure in the vicinity of the aperture. The flow rate is found to be proportional to the belt velocity, independent of the amount of disks in the container and, thus, independent of the pressure in the outlet region. In addition, this specific configuration makes it possible to get information on the system dynamics from a single image of the disks that rest on the conveyor belt after the discharge.

Proteomics in antiphospholipid syndrome: a review.

The presence of antiphospholipid antibodies (aPL) has been closely related to the development of thrombosis and complications in pregnancy. However, not all patients with aPL will develop those clinical features. The exact pathogenic mechanisms leading to thrombosis and/or pregnancy morbidity are poorly understood. Currently, biomarkers which enable one to predict the prognosis of patients with positive aPL are not readily available. Current advances in genomics and proteomics provide the opportunity to discover novel biomarkers based on changes in concentration levels or post-translational modifications of proteins and peptides. These techniques are now being applied in various areas of medicine with very promising results. This review covers recent studies that have used this approach for a better understanding of the pathogenic mechanisms involved in the development of thrombosis in patients with antiphospholipid syndrome (APS). Although, there are very few qualified biomarkers that have arisen as a result of efforts in proteomics, it is expected that these techniques will deliver biomarkers that might ultimately identify different subgroups of APS patients with various prognoses that might have implications with respect to management and prognosis.

Genomics and proteomics: a new approach for assessing thrombotic risk in autoimmune diseases.

Several systemic autoimmune conditions, including rheumatoid arthritis, systemic lupus erythematosus and antiphospholipid syndrome, are characterised by enhanced atherosclerosis and, consequently, higher cardiovascular morbidity and mortality rates. The association of these diseases with atherosclerosis suggests a common pathogenic mechanism. Genomic and proteomic studies performed on atherosclerotic plaques have further confirmed the presence of a gene and protein profile similar to that observed in autoimmune diseases with cardiovascular risks. Human sera and body fluids have been analysed and have resulted in the identification of auto-antibodies that can be used as diagnostic markers in specific autoimmune diseases, and proteomic fingerprints of blood cells, tissues and body fluids have resulted in the identification of individual proteins or patterns of protein expression that are deregulated. The information provided by these proteomic studies is of diagnostic and therapeutic potential. In this review, we discuss new approaches available for assessing thrombotic risk in autoimmune diseases, focusing in the genomic and proteomic methods now available to deep into the origin of the mechanisms associated with vascular involvement in systemic autoimmune diseases. The increasing data available suggests that when treating patients with these autoimmune disorders, paying attention to the increased risk of cardiovascular disease is essential.

Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome.

BACKGROUND: One of the described mechanisms leading to thrombosis in antiphospholipid syndrome (APS) is overexpression of tissue factor (TF) in the monocytes and endothelial cells of patients with antiphospholipid antibodies (aPL). Vascular endothelial growth factor (VEGF) may stimulate monocyte TF expression through its receptor, the tyrosine kinase Flt-1. OBJECTIVES: This study aimed to analyze the following in monocytes of 55 primary APS patients: VEGF and Flt-1 expression levels, their potential regulation by aPL, and the association of VEGF and Flt-1 expression with the increased TF expression found in APS patients. RESULTS: Purified monocytes from APS patients showed higher levels of VEGF and Flt-1 than healthy donors, which further correlated with immunoglobulin G (IgG) anticardiolipin titers and TF expression rank. Moreover, monocyte VEGF and Flt-1 levels were significantly higher in patients with than in patients without previous thrombosis. In vitro, IgG from APS patients increased monocyte VEGF and Flt-1 expression in a dose-dependent manner. VEGF and Flt-1 expression was significantly inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; this suggests the involvement of this kinase in the aPL-induced VEGF and Flt-1 upregulation. CONCLUSIONS: Our data show, for the first time in vivo, that monocytes from primary APS patients have an increased expression of VEGF and Flt-1. Furthermore, in vitro results indicated that this cytokine is produced by monocytes when treated with aPL, and that the p38 MAPK signaling pathway plays an important role. Thus, VEGF might act as a regulatory factor in aPL-mediated monocyte activation and TF expression, thereby contributing to the proinflammatory-prothrombotic phenotype of APS patients.

Rearrangements in a two-dimensional packing of disks.

Several aspects of the dynamics of a granular two-dimensional (2D) packing of disks slowly tilted until the system loses stability and an avalanche takes place are discussed. The evolution of the system, constructed with monodisperse disks placed on a thin cell, is studied by image analysis. As in the 3D case (packing of spheres), the system undergoes several rearrangements of different magnitude before the avalanche takes place. For thick systems, not only are small rearrangements detected but also displacements of large clusters of disks are observed in the bulk and on the free surface of the packing. In particular, characteristic angles and the avalanche mass were determined for samples of different heights. On thick systems, velocity fields of large rearrangements are presented and changes in the internal structure of the packing produced by these rearrangements are analyzed. It is found that the main effects of rearrangements is to increase the disorder of the system. Also, as the disorder of the system increases its stability threshold decreases.

Antiphospholipid syndrome and tissue factor: a thrombotic couple.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). Among the thrombogenic mechanisms proposed, it has been suggested that aPL can stimulate tissue factor (TF) expression by endothelial cells (ECs) and monocytes. Moreover, our in vivo studies have shown that APS patients (particularly those with thrombosis) have increased monocyte TF expression. Yet, the molecular mechanism(s) by which aPL induce TF expression has not been completely underscored. In a recent study, we have demonstrated that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK-1/ERK proteins, and the p38 MAP kinase-depenent nuclear translocation and activation of NFkappaB/Rel proteins. Understanding the intracellular mechanism(s) of aPL-mediated monocyte activation may help to establish new therapeutic approaches, such as selective inhibition of MAP kinases, to reverse the prothrombotic state in APS. Furthermore, the contribution of TF to a protrombotic state in the APS provides a renewed focus on antithrombotic therapies in current use, including the oral anticoagulation and, more recently, the use of statins, which have been proven to be effective in the inhibition of EC and monocyte TF-expression.

Instabilities in slowly driven granular packing.

In this work, a digital imaging technique is used to study the superficial fluctuations observed when a granular packing is slowly driven to the threshold of instability. The experimental results show the presence of three types of events. Small superficial rearrangements of grains are observed during all the experiments. They present a power-law behavior although the system is not in a critical state as predicted by self-organized criticality models. In thick granular piles, large rearrangements are detected at regular angular intervals. They are related to the threshold of instability of the contact network that relaxes to stable configurations producing internal rearrangements of the grains. Finally, an avalanche is triggered when the superficial beads that are set in motion acquire enough momentum to destabilize grains from layers below.

Simulations in the mathematical modeling of the spread of the Hantavirus.

The range of validity of a recently proposed deterministic (mean field) model of the spread of the Hantavirus infection is studied with the help of Monte Carlo simulations for the evolution of mice populations. The simulation is found to reproduce earlier results on the average but to display additional behavior stemming from discreteness in mice number and from fluctuations of the finite size system. It is shown that mice diffusion affects those additional features of the simulation in a physically understandable manner, higher diffusion constants leading to greater agreement with the mean field results.