Application of Modified Shell Vial Culture Procedure for Arbovirus Detection

The isolation of arboviruses from patient's low titer sera can be difficult. Here we compared the detection efficiency of Dengue (DEN), Yellow Fever (YF), Saint Louis Encephalitis (SLE), West Nile (WN), Ilheus (ILH), Group C (GC), Oropouche (ORO), Mayaro (MAY) and Venezuela Encephalitis Equine (VEE) viruses using a Modified Shell Vial Culture (MSVC) protocol to a Standard Cell Culture (SCC) protocol. First the MSVC and SCC protocols were compared using five dilutions for each of the following stock viruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN, ILH, GC, ORO, MAY and VEE. Next, patients' original sera from which viruses (DEN-1, DEN-2, DEN-3, YF, GC, ORO, MAY and VEE) had been previously isolated were compare by the two methods using five sera dilutions. In addition, seven sera that were positive for DEN-3 by RT-PCR and negative by SCC were processed by MSVC. The MSVC protocol was consistently 1-2 logs higher virus dilution more sensitive for virus detection than the SCC protocol for all stock Flaviviruses tested (DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN and ILH). MSVC was equal to or one log more sensitive for virus detection than SCC for the stock Bunyaviruses (GC and ORO). For the stock Alphavirus MAY, MSVC was equally or one log more sensitive for virus detection than SCC, while for VEE SCC was equally or one log more sensitive for virus detection than MSVC. MSVC was consistently one to two sera dilutions more sensitive than SCC for the detection of Flaviviruses from patients' sera. Both methods were approximately equally sensitive for the detection of Bunyaviruses from patients' sera and equal or one dilution less sensitive for the detection of Alphaviruses from patients' sera. Additionally, MSVC detected DEN virus in five of seven DEN-3 RT-PCR positive, SCC negative patients' sera

Efficacy and safety of autologous platelet rich plasma for the treatment of vascular ulcers in primary care: Phase III study

Background: Vascular ulcers are commonly seen in daily practice at all levels of care and have great impact at personal, professional and social levels with a high cost in terms of human and material resources. Given that the application of autologous platelet rich plasma has been shown to decrease healing times in various different studies in the hospital setting, we considered that it would be interesting to assess the efficacy and feasibility of this treatment in primary care. The objectives of this study are to assess the potential efficacy and safety of autologous platelet rich plasma for the treatment of venous ulcers compared to the conventional treatment (moist wound care) in primary care patients with chronic venous insufficiency (C, clinical class, E, aetiology, A, anatomy and P, pathophysiology classification C6). Design: We will conduct a phase III, open-label, parallel-group, multicentre, randomized study. The subjects will be 150 patients aged between 40 and 100 years of age with an at least 2-month history of a vascular venous ulcer assigned to ten primary care centres. For the treatment with autologous platelet rich plasma, all the following tasks will be performed in the primary care setting: blood collection, centrifugation, separation of platelet rich plasma, activation of coagulation adding calcium chloride and application of the PRP topically after gelification. The control group will receive standard moist wound care. The outcome variables to be measured at baseline, and at weeks 5 and 9 later include: reduction in the ulcer area, Chronic Venous Insufficiency Quality of Life Questionnaire score, and percentage of patients who require wound care only once a week. Discussion: The results of this study will be useful to improve the protocol for using platelet rich plasma in chronic vascular ulcers and to favour wider use of this treatment in primary care.This study can be undertaken thanks to the financial support of the Spanish Carlos III Health Institute. We are grateful for funding from the Department of Health and Consumer Affairs of the Government of the Basque Country, the Basque Health Service (Osakidetza) for the pilot support and the Ezkerraldea Enkarterri health region

Bordetella Adenylate Cyclase Toxin Mobilizes Its β2 Integrin Receptor into Lipid Rafts to Accomplish Translocation across Target Cell Membrane in Two Steps

Bordetella adenylate cyclase toxin (CyaA) binds the αMβ2 integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin ‘translocation intermediate’, which can be ‘locked’ in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the ‘intermediate’ permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca2+ influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the ‘translocation intermediate’ promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol

Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that primarily affects the lining of the synovial joints and is associated with progressive disability, premature death, and socioeconomic burdens. A better understanding of how the pathological mechanisms drive the deterioration of RA progress in individuals is urgently required in order to develop therapies that will effectively treat patients at each stage of the disease progress. Here we dissect the etiology and pathology at specific stages: (i) triggering, (ii) maturation, (iii) targeting, and (iv) fulminant stage, concomitant with hyperplastic synovium, cartilage damage, bone erosion, and systemic consequences. Modern pharmacologic therapies (including conventional, biological, and novel potential small molecule disease-modifying anti-rheumatic drugs) remain the mainstay of RA treatment and there has been significant progress toward achieving disease remission without joint deformity. Despite this, a significant proportion of RA patients do not effectively respond to the current therapies and thus new drugs are urgently required. This review discusses recent advances of our  understanding of RA pathogenesis, disease modifying drugs, and provides perspectives on next generation therapeutics for RA

A short history of the 5-HT2C receptor: from the choroid plexus to depression, obesity and addiction treatment

This paper is a personal account on the discovery and characterization of the 5-HT2C receptor (first known as the 5- HT1C receptor) over 30 years ago and how it translated into a number of unsuspected features for a G protein-coupled receptor (GPCR) and a diversity of clinical applications. The 5-HT2C receptor is one of the most intriguing members of the GPCR superfamily. Initially referred to as 5-HT1CR, the 5-HT2CR was discovered while studying the pharmacological features and the distribution of [3H]mesulergine-labelled sites, primarily in the brain using radioligand binding and slice autoradiography. Mesulergine (SDZ CU-085), was, at the time, best defined as a ligand with serotonergic and dopaminergic properties. Autoradiographic studies showed remarkably strong [3H]mesulergine-labelling to the rat choroid plexus. [3H]mesulergine-labelled sites had pharmacological properties different from, at the time, known or purported 5-HT receptors. In spite of similarities with 5-HT2 binding, the new binding site was called 5-HT1C because of its very high affinity for 5-HT itself. Within the following 10 years, the 5-HT1CR (later named 5- HT2C) was extensively characterised pharmacologically, anatomically and functionally: it was one of the first 5-HT receptors to be sequenced and cloned. The 5-HT2CR is a GPCR, with a very complex gene structure. It constitutes a rarity in theGPCR family: many 5-HT2CR variants exist, especially in humans, due to RNA editing, in addition to a few 5-HT2CR splice variants. Intense research led to therapeutically active 5-HT2C receptor ligands, both antagonists (or inverse agonists) and agonists: keeping in mind that a number of antidepressants and antipsychotics are 5- HT2CR antagonists/inverse agonists. Agomelatine, a 5-HT2CR antagonist is registered for the treatment of major depression. The agonist Lorcaserin is registered for the treatment of aspects of obesity and has further potential in addiction, especially nicotine/ smoking. There is good evidence that the 5-HT2CR is involved in spinal cord injury-induced spasms of the lower limbs, which can be treated with 5-HT2CR antagonists/inverse agonists such as cyproheptadine or SB206553. The 5-HT2CR may play a role in schizophrenia and epilepsy. Vabicaserin, a 5-HT2CR agonist has been in development for the treatment of schizophrenia and obesity, but was stopped. As is common, there is potential for further indications for 5-HT2CR ligands, as suggested by a number of preclinical and/or genome-wide association studies (GWAS) on depression, suicide, sexual dysfunction, addictions and obesity. The 5-HT2CR is clearly affected by a number of established antidepressants/antipsychotics and may be one of the culprits in antipsychotic-induced weight gain