Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories.

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Clustering algorithms for anti-money laundering using graph theory and social network analysis

HEMOLIA (a project under European community’s 7th framework programme) is a new generation Anti-Money Laundering (AML) intelligent multi-agent alert and investigation system which in addition to the traditional financial data makes extensive use of modern society’s huge telecom data source, thereby opening up a new dimension of capabilities to all Money Laundering fighters (FIUs, LEAs) and Financial Institutes (Banks, Insurance Companies, etc.). This Master-Thesis project is done at AIA, one of the partners for the HEMOLIA project in Barcelona. The objective of this thesis is to find the clusters in a network drawn by using the financial data. An extensive literature survey has been carried out and several standard algorithms related to networks have been studied and implemented. The clustering problem is a NP-hard problem and several algorithms like K-Means and Hierarchical clustering are being implemented for studying several problems relating to sociology, evolution, anthropology etc. However, these algorithms have certain drawbacks which make them very difficult to implement. The thesis suggests (a) a possible improvement to the K-Means algorithm, (b) a novel approach to the clustering problem using the Genetic Algorithms and (c) a new algorithm for finding the cluster of a node using the Genetic Algorithm.

In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas.

PURPOSE: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. EXPERIMENTAL DESIGN: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. RESULTS: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. CONCLUSIONS: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Predictive value of anti-neutrophil cygtoplasmic antibodies (ANCA) in small vessel vasculitis

Résumé Valeur prédictive des anticorps dirigés contre le cytoplasme des neutrophiles (ANCA) dans les vasculites des vaisseaux de petit calibre But du travail : Les vasculites sont des pathologies le plus souvent sévères et parfois létales ; elles nécessitent une reconnaissance et un traitement précoces. Il est donc utile de pouvoir disposer de marqueurs diagnostiques, et éventuellement de marqueurs qui puissent prédire l'activité de la maladie. Les « antineutrophil cytoplasm antibodies » (ANCA) constituent une famille d'autoanticorps dirigés contre des antigènes du cytoplasme des neutrophiles, cellules clés du processus inflammatoire au cours des vasculites. De nombreuses études ont tenté de préciser l'utilité des ANCA dans le diagnostic et le suivi des vasculites avec des résultats contradictoires. Le but de ce travail a été de passer en revue l'évolution clinique des patients suivis dans notre service pour une vasculite à ANCA et évaluer la valeur prédictive des ANCA comme marqueur de récidive. Méthode: Les dossiers médicaux de 36 patients, suivis à notre consultation ambulatoire d'immunologie et allergie du CHUV pour une vasculite à ANCA entre janvier 1990 et décembre 2001, ont été analysés de manière rétrospective afin d'établir une base de données. Les données démographiques, le type de vasculite (granulomatose de Wegener ou polyangéite microscopique) et ses caractéristiques (organes touchés), les traitements reçus, les dosages des ANCA (par immunofluorescence et par ELISA avec détermination des anti-PR3 et/ou anti-MPO), et l'évolution clinique (récidive/rémission) ont été considérés. La valeur pronostique des ANCA dans notre population a été calculée utilisant les valeurs prédictives positive et négative, la likelihood et les odds ratios. La valeur statistique a été examinée par les tests Chi-square test ou Fisher's exact test (valeur significative définie comme p <0.05) à l'aide du programme GraphPad Instat software version 3, San Diego, CA. Résultats : Vingt-trois patients atteints d'une maladie de Wegener et treize d'une polyangéite microscopique ont été suivis pour une durée médiane de cinq ans (entre 1 mois et 16 ans). La plupart des patients ont été traités avec des corticostéroïdes associés à du cyclophosphamide. Une rémission a été obtenue chez 21 patients (91 %) atteints d'une maladie de Wegener, mais 74% ont présenté par la suite une récidive. Tous les patients atteints d'une polyangéite microscopique sont entrés en rémission et 33% ont par la suite récidivé. Une élévation persistante (définie comme supérieure à 6 mois) des ANCA ne s'est pas révélée associée à un risque statistiquement significatif de récidive (p=0.14). En revanche, une élévation soudaine du taux des ANCA s'est démontrée prédictive d'une récidive (table 3). Conclusion : Durant le suivi de certaines vasculites, comme la granulomatose de Wegener et la polyangéite microscopique une élévation des ANCA doit faire redouter une exacerbation de la maladie et, par conséquent, justifie une surveillance accrue.

Off-label uses of anti-TNF therapy in three frequent disorders: Behçet's disease, sarcoidosis, and noninfectious uveitis.

Tumoral necrosis factor α plays a central role in both the inflammatory response and that of the immune system. Thus, its blockade with the so-called anti-TNF agents (infliximab, etanercept, adalimumab, certolizumab pegol, and golimumab) has turned into the most important tool in the management of a variety of disorders, such as rheumatoid arthritis, spondyloarthropatties, inflammatory bowel disease, and psoriasis. Nonetheless, theoretically, some other autoimmune disorders may benefit from these agents. Our aim is to review these off-label uses of anti-TNF blockers in three common conditions: Behçet's disease, sarcoidosis, and noninfectious uveitis. Due to the insufficient number of adequate clinical trials and consequently to their lower prevalence compared to other immune disorders, this review is mainly based on case reports and case series.

Multicenter evaluation of the elecsys® anti-HCV II assay for the diagnosis of hepatitis C virus infection.

Routine screening of patients at risk of hepatitis C virus (HCV) infection has become a priority given recent improvements in therapeutic options and the asymptomatic nature of most chronic infections. The aim of this study was to evaluate the performance of the Elecsys® Anti-HCV II assay, a new qualitative antibody immunoassay, compared with currently available assays, and assess its suitability for routine diagnostic testing. The sensitivity of the Elecsys® Anti-HCV II, ARCHITECT® Anti-HCV, AxSYM® HCV 3.0, PRISM® HCV, Vitros® ECi Anti-HCV, Elecsys® Anti-HCV, and ADVIA Centaur® HCV assays was compared using commercially available seroconversion panels and samples from patients known to be HCV positive and infected with HCV genotypes 1-6. Specificity was investigated using samples from blood donors, unselected hospitalized patients, and patients with potential cross-reacting factors or from high-risk groups. The Elecsys® Anti-HCV II assay detected more positive bleeds than the comparator assays, was more sensitive in recognizing early HCV infection, and correctly identified all 765 samples known to be HCV positive, regardless of genotype. The overall specificity of the Elecsys(®) Anti-HCV II assay was 99.84% (n = 6,850) using blood donor samples, 99.66% (n = 3,922) using samples from unselected hospitalized patients, and 99.66% (n = 2,397) using samples from patients with potentially cross-reacting factors or from high-risk groups. The specificity of the Elecsys® Anti-HCV II assay was superior or equal to the comparator assays. In conclusion, the Elecsys® Anti-HCV II assay is a sensitive and specific assay suitable for routine use in the reliable detection of anti-HCV antibodies. J. Med. Virol. 85:1362-1368, 2013. © 2013 Wiley Periodicals, Inc.

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy

Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era

Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks.

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.

TRAF1/C5 but not PTPRC variants are potential predictors of rheumatoid arthritis response to anti-tumor necrosis factor therapy.

BACKGROUND The aim of our work was to replicate, in a Southern European population, the association reported in Northern populations between PTPRC locus and response to anti-tumor necrosis factor (anti-TNF) treatment in rheumatoid arthritis (RA). We also looked at associations between five RA risk alleles and treatment response. METHODS We evaluated associations between anti-TNF treatment responses assessed by DAS28 change and by EULAR response at six months in 383 Portuguese patients. Univariate and multivariate linear and logistic regression analyses were performed. In a second step to confirm our findings, we pooled our population with 265 Spanish patients. RESULTS No association was found between PTPRC rs10919563 allele and anti-TNF treatment response, neither in Portuguese modeling for several clinical variables nor in the overall population combining Portuguese and Spanish patients. The minor allele for RA susceptibility, rs3761847 SNP in TRAF1/C5 region, was associated with a poor response in linear and logistic univariate and multivariate regression analyses. No association was observed with the other allellic variants. Results were confirmed in the pooled analysis. CONCLUSION This study did not replicate the association between PTPRC and the response to anti-TNF treatment in our Southern European population. We found that TRAF1/C5 risk RA variants potentially influence anti-TNF treatment response.

Allergo-immunologie. 2 : Les réactions allergiques aux anti-inflammatoires non stéroïdiens [Allergic reactions to nonsteroidal anti-inflammatory drugs]

Allergy to nonsteroidal antiinflammatory drugs (NSAIDs) is a very common affliction, especially among patients with asthma and chronic urticaria. These reactions are most often of a non-immunological nature but related to pharmacologic intolerance and linked to arachidonic acid metabolism and leukotriene release. Therefore, crossed reactions implying all non-selective and semi-selective NSAIDs constitute the rule, especially during respiratory reactions to NSAIDs and for patients with chronic urticaria. In isolated acute urticaria, crossed reactions are difficult to predict so caution is necessary. Tolerance induction is possible, especially when aspirin has to be administered in small doses as antiplatelet agent. Finally, acetaminophen and selective NSAIDs as celecoxib are well tolerated by most of these patients. L'allergie aux anti-inflammatoires non stéroïdiens (AINS) est très fréquente, en particulier chez les asthmatiques ou dans l'urticaire chronique. Il s'agit en général de réactions non immunologiques, mais dues à une intolérance pharmacologique liée au métabolisme de l'acide arachidonique et à la formation de leucotriènes. Ainsi, les réactions croisées impliquant tous les AINS non sélectifs et semi-sélectifs sont la règle, surtout lors de réactions respiratoires aux AINS et dans l'urticaire chronique. Lors d'urticaire aiguë isolée, les réactions croisées sont difficiles à prédire, ainsi la prudence s'impose. Une induction de tolérance est possible, en particulier lorsque l'aspirine est nécessaire à dose faible, comme antiagrégant plaquettaire. Enfin, le paracétamol et les AINS sélectifs sont supportés par la grande majorité de ces patients.

Que faire des nouveaux anti-épileptiques [Clinical utilization of new anti-epileptic agents].

Since the beginning of the 1990's, a dozen of new anti-epileptic drugs have been on the market or will be soon. This article reviews the daily clinical utilisation of new anti-epileptic drugs. It considers, without being complete, the current opinions and tendencies. The new anti-epileptic substances are generally as efficient as conventional medications. However, they are better tolerated and are more easily used in combination with conventional anti-epileptic drugs. Polytherapy is certainly the form of treatment, which is used in the most cases of resistant epilepsies. The surgical treatment can be used in only a very limited number of cases. The objective of treatment is the complete control of seizures, with minimum secondary effects. Though this objective is rarely reached, the NAE significantly improves the quality of life of patients suffering from severe epilepsy. The utilisation of NAE is not without risk. Increase in the frequency and severity of seizures may occur; we should remember that severe adverse effects appeared in the post-marketing period of the use of Vigabatrine and Felbamate. Therefore, we must remain vigilant in the clinical use of the anti-epileptic drugs.

Biological treatments in Behçet's disease: beyond anti-TNF therapy.

Behçet's disease (BD) is universally recognized as a multisystemic inflammatory disease of unknown etiology with chronic course and unpredictable exacerbations: its clinical spectrum varies from pure vasculitic manifestations with thrombotic complications to protean inflammatory involvement of multiple organs and tissues. Treatment has been revolutionized by the progressed knowledge in the pathogenetic mechanisms of BD, involving dysfunction and oversecretion of multiple proinflammatory molecules, chiefly tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, and IL-6. However, although biological treatment with anti-TNF-α agents has been largely demonstrated to be effective in BD, not all patients are definite responders, and this beneficial response might drop off over time. Therefore, additional therapies for a subset of refractory patients with BD are inevitably needed. Different agents targeting various cytokines and their receptors or cell surface molecules have been studied: the IL-1 receptor has been targeted by anakinra, the IL-1 by canakinumab and gevokizumab, the IL-6 receptor by tocilizumab, the IL12/23 receptor by ustekinumab, and the B-lymphocyte antigen CD-20 by rituximab. The aim of this review is to summarize all current experiences and the most recent evidence regarding these novel approaches with biological drugs other than TNF-α blockers in BD, providing a valuable addition to the actually available therapeutic armamentarium.