Sphingosine 1-phosphate receptor signaling.

The sphingosine 1-phosphate (S1P) receptor signaling system is a productive model system. A hydrophobic zwitterionic lysophospholipid ligand with difficult physical properties interacts with five high-affinity G protein-coupled receptors to generate multiple downstream signals. These signals modulate homeostasis and pathology on a steep agonist concentration-response curve. Ligand presence is essential for vascular development and endothelial integrity, while acute increases in ligand concentrations result in cardiac death. Understanding this integrated biochemical system has exemplified the impact of both genetics and chemistry. Developing specific tools with defined biochemical properties for the reversible modulation of signals in real time has been essential to complement insights gained from genetic approaches that may be irreversible and compensated. Despite its knife-edge between life and death, this system, based in part on receptor subtype-selectivity and in part on differential attenuation of deleterious signals, now appears to be on the cusp of meaningful therapy for multiple sclerosis.

Non-Linear Regression Modelling to Estimate the Global Warming Potential of a Newspaper.

Technological innovations are not enough by themselves to achieve social and environmental sustainability in companies. Sustainable development aims to determine the environmental impact of a product and the hidden price of products and services through the concept of radical transparency. This means that companies should show and disclose the impact on the environment of any good or service. This way, the consumer can choose in a transparent manner, not only for the price. The use of the eco-label as a European eco-label, which bases its criteria on life cycle assessment, could provide an indicator of corporate social responsibility for a given product. However, it does not give a full guarantee that the product was obtained in a sustainable manner. The aim of this work is to provide a way of calculating the value of the environmental impacts of an industrial product, under different operating conditions, so that each company can provide detailed information on the impacts of its products, information that can form part of its "green product sheet". As a case study, the daily production of a newspaper, printed by coldset, has been chosen. Each process involved in production was configured with raw material and energy consumption information from production plants, manufacturer data and existing databases. Four non-linear regression models have been trained to estimate the impact of a newspaper's circulation from five input variables (pages, grammage, height, paper type, and print run) with 5508 data samples each. These non-linear regression models were trained using the Levenberg-Marquardt nonlinear least squares algorithm. The mean absolute percentage errors (MAPE) obtained by all the non-linear regression models tested were less than 5%. Through the proposed correlations, it is possible to obtain a score that reports on the impact of the product for different operating conditions and several types of raw materials. Ecolabelling can be further developed by incorporating a scoring system for the impact caused by the product or process, using a standardised impact methodology.

Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure.

Efficient treatments against Acanthamoeba Keratitis (AK), remains until the moment, as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. In this study, two antiglaucoma eye drops were tested for their activity against Acanthamoeba. Moreover, this study was based on previous data which gave us evidence of a possible link between the absences of Acanthamoeba at the ocular surface in patients treated with beta blockers for high eye pressure both containing timolol as active principle. The amoebicidal activity of the tested eye drops was evaluated against four strains of Acanthamoeba using Alamar blue method. For the most active drug the cysticidal activity against A. castellanii Neff cysts and further experiments studying changes in chromatin condensation levels, in the permeability of the plasmatic membrane, the mitochondrial membrane potential and the ATP levels in the treated amoebic strains were done. Even though both eye drops were active against the different tested strains of Acanthamoeba, statistical analysis revealed that one of them (Timolol Sandoz) was the most effective one against all the tested strains presenting IC50s ranging from 0.529% ± 0.206 for the CLC 16 strain to 3.962% ± 0.150 for the type strain Acanthamoeba castellanii Neff. Timolol Sandoz 0.50% seems to induce amoebic cell death by damaging the amoebae at the mitochondrial level. Considering its effect, Timolol Sandoz 0.50% could be used in the case of contact lens wearers and patients with glaucoma.

Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

Incremental cost of implementing residual insecticide treatment with delthametrine on top of intensive routine Aedes aegypti control.

OBJECTIVE: Information on the cost of implementing residual insecticide treatment (RIT) for Aedes control is scarce. We evaluated the incremental cost on top of intensive conventional routine activities of the Aedes control programme (ACP) in the city of Santiago de Cuba, Cuba. METHODS: We conducted the cost analysis study in 2011-2012, from the perspective of the ACP. Data sources were bookkeeping records, activity registers of the Provincial ACP Centre and the accounts of an RIT implementation study in 21 clusters of on average four house blocks comprising 5180 premises. RESULTS: The annual cost of the routine ACP activities was 19.66 US$ per household. RIT applications in rounds at 4-month intervals covering, on average, 97.2% and using 8.5 g of delthametrine annually per household, cost 3.06 US$ per household per year. Delthametrine comprised 66.5% of this cost; the additional cost for deploying RIT comprised 15.6% of the total ACP routine cost and 27% of the cost related to routine adult stage Aedes control. CONCLUSIONS: The incremental cost of implementing RIT is high. It should be weighed against the incremental effect on the burden caused by the array of pathogens transmitted by Aedes. The cost could be reduced if the insecticide became cheaper, by limiting the number of yearly applications or by targeting transmission hot spots.

The organization of the sphingosine 1-phosphate signaling system.

The understanding of the role of the sphingosine 1-phosphate signaling system in immunology and host defense has deepened exponentially over the past 12 years since the discovery that lymphocyte egress was reversibly modulated by sphingosine 1-phosphate receptors, and with the development of fingolimod, a prodrug of a nonselective S1P receptor agonist, for therapeutic use in the treatment of relapsing, remitting multiple sclerosis. Innovative genetic and chemical approaches, together with structural biology, now provide a more detailed molecular understanding of a regulated lysophospholipid ligand with a variety of autocrine, paracrine, and systemic effects in physiology and pathology, based upon selective interactions with a high affinity and selective evolutionary cluster of G-protein-coupled receptors.

Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is a G protein-coupled receptor that is critical for proper lymphocyte development and recirculation. Agonists to S1P(1) are currently in use clinically for the treatment of multiple sclerosis, and these drugs may act on both S1P(1) expressed on lymphocytes and S1P(1) expressed within the central nervous system. Agonists to S1P(1) and deficiency in S1P(1) both cause lymphocyte sequestration in the lymph nodes. In the present study, we show that S1P(1) antagonism induces lymphocyte sequestration in the lymph nodes similar to that observed with S1P(1) agonists while upregulating S1P(1) on lymphocytes and endothelial cells. Additionally, we show that S1P(1) antagonism reverses experimental autoimmune encephalomyelitis in mice without acting on S1P(1) expressed within the central nervous system, demonstrating that lymphocyte sequestration via S1P(1) antagonism is sufficient to alleviate autoimmune pathology.

Actions of a picomolar short-acting S1P1 agonist in S1P1-eGFP knock-in mice.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo.

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.

S1P(1) receptor modulation with cyclical recovery from lymphopenia ameliorates mouse model of multiple sclerosis.

Multiple sclerosis (MS) therapies modulate T-cell autoimmunity in the central nervous system (CNS) but may exacerbate latent infections. Fingolimod, a nonselective sphingosine-1-phosphate (S1P) receptor agonist that induces sustained lymphopenia and accumulates in the CNS, represents a new treatment modality for MS. We hypothesized that sustained lymphopenia would not be required for efficacy and that a selective, CNS-penetrant, peripherally short-acting, S1P(1) agonist would show full efficacy in a mouse MS model. Using daily treatment with 10 mg/kg 2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino)ethanol (CYM-5442) at the onset of clinical signs in myelin oligodendrocyte glycoprotein MOG(35-55)- induced experimental allergic encephalomyelitis (EAE), we assessed clinical scores, CNS cellular infiltration, demyelination, and gliosis for 12 days with CYM-5442, vehicle, or fingolimod. CYM-5442 levels in CNS and plasma were determined at experiment termination, and blood lymphopenia was measured 3 and 24 h after the last injection. Plasma levels of cytokines were assayed at the end of the protocol. Changes in S1P(1)-enhanced green fluorescent protein expression on neurons and astrocytes during active EAE and upon CYM-5442 treatment were quantified with flow cytometry and Western blotting by using native-locus enhanced green fluorescent protein-tagged S1P(1) mice. S1P(1) agonism alone reduced pathological features as did fingolimod (maximally lymphopenic throughout), despite full reversal of lymphopenia within each dosing interval. CYM-5442 levels in CNS but not in plasma were sustained. Neuronal and astrocytic S1P(1) expression in EAE was suppressed by CYM-5442 treatment, relative to vehicle, and levels of key cytokines, such as interleukin 17A, were also significantly reduced in drug-treated mice. S1P(1)-selective agonists that induce reversible lymphopenia while persisting in the CNS may be effective MS treatments.

Umbilical KeyPort bilateral laparoscopic orchiectomy in patient with complete androgen insensitivity syndrome.

MAIN FINDINGS: A 22-year-old woman with complete androgen insensitivity syndrome (CAIS) presenting with primary amenorrhea and normal female external genitalia was referred for laparoscopic gonadectomy. She had been diagnosed several years earlier but was reluctant to undergo surgery. Case HYPOTHESIS: Diagnosis of this X-linked recessive inherited syndrome characterizes by disturbance of virilization in males with an AR mutation, XY karyotipe, female genitalia and severely undescended testis with risk of malignization. The optimal time to orchidectomy is not settled; neither the real risk of malignancy in these patients. Early surgery impacts development of a complete female phenotype, with enlargement of the breasts. Based on modern diagnostic imaging using DCE-MRI and surgical technology with single port laparoscopic access we hypothesize that the optimum time for gonadectomy is not at the time of diagnosis, but once feminization has completed. PROMISING FUTURE IMPLICATIONS: An umbilical laparoendoscopic single-site access for bilateral gonadectomy appears to be the first choice approach as leaves no visible incision and diminishes the psychological impact of surgery in a patient with CAIS absolutely reassured as female. KeyPort, a single port access with duo-rotate instruments developed by Richard Wolf facilitates this surgery and allows excellent cosmetic results.

Modulating tone: the overture of S1P receptor immunotherapeutics.

SUMMARY: Modulation of complex functions within the immune system has proven to be surprisingly sensitive to alterations in the lysophospholipid sphingosine 1-phosphate (S1P) receptor-ligand rheostat. This has become increasingly evident from both chemical and genetic manipulation of the S1P system, with pharmacological effects upon lymphoid cells, dendritic cell function, as well as vascular interfaces. The integrated immune system, perhaps as a result of its relatively recent evolutionary ontogeny, has selected for a number of critical control points regulated by five distinct high affinity G-protein-coupled receptor subtypes with a shared ligand, with receptors distributed on lymphocytes, dendritic cells, and endothelium. All of these cellular components of the axis are capable of modulating immune responses in vivo, with the impact on the immune response being very different from classical immunosuppressants, by virtue of selective spatial and temporal sparing of humoral and myeloid elements of host defense. Pharmacological subversion of the S1P rheostat is proving to be clinically efficacious in multiple sclerosis, and both the scope and limitations of therapeutic modulation of the S1P axis in immunotherapy are becoming clearer as understanding of the integrated chemical physiology of the S1P system emerges.

Gastric lymphoma at high risk of perforation successfully managed with chemotherapy alone.

We report the case of a previously healthy 49-year-old woman who presented with upper gastrointestinal bleeding, which was found at laparotomy to be due to high-grade B cell gastric lymphoma. CT scans showed that this was partially adherent to the spleen, with erosion of the gastric wall and suggested impending perforation. Given the risk of perforation, further surgical intervention (gastrectomy and splenectomy) was considered; however, after multidisciplinary team discussion, we chose to offer chemotherapy and careful inpatient observation instead.Our patient made a full recovery with no perforation.The message from our experience and literature review is that medical management may lead to a more favourable outcome in gastric lymphoma than surgery, despite radiological appearances suggesting impending perforation. This approach avoids the risk of the lymphoma progressing at other anatomical sites secondary to delays in giving chemotherapy. If this approach is followed, the patient must be carefully monitored.

Autoimmune pancytopenia occurring late after simultaneous pancreas and kidney transplantation.

A 35-year-old woman presented with a widespread petechial rash and pancytopenia. She underwent simultaneous pancreas and kidney transplantation for type 1 diabetes 8 years previously followed by a renal transplant 1 year prior to presentation, and was taking tacrolimus as long-term immunosuppression. The full blood count showed haemoglobin 97 g/L, platelet count 2×109/L and neutrophil count 0.22×109/L. Peripheral blood film examination confirmed genuine thrombocytopenia in the absence of any haemolytic or malignant features. Serological testing identified autoantibodies against all three blood lineages, consistent with a diagnosis of autoimmune pancytopenia. Treatment with steroids, intravenous immunoglobulins, romiplostim and mycophenolate mofetil achieved only fleeting remissions. Blood counts eventually normalised following the administration of rituximab and a change from tacrolimus to ciclosporin immunosuppression. Cytopenias are a well-recognised complication of post-transplantation care but we believe this to be the first reported case of autoimmune pancytopenia following solid organ transplantation. In this case report, we discuss the approach to investigation of haematological abnormalities post-transplant and the rationale for, and outcome of, the management of this rare case.

In vitro evaluation of commercial foam Belcils® on Acanthamoeba spp.

Interest in periocular (eyelid and eyelashes margins) hygiene has attracted attention recently and a growing number of commercials eye cleanser and shampoos have been marketed. In the present study, a particular eye cleanser foam, Belcils® has been tested against trophozoites and cysts on the facultative pathogen Acanthamoeba. Viability was tested by the alamarBlue™ method and the foam was tested for the induction of programmed cell death in order to explore its mode of action. We found that a 1% solution of the foam eliminated both trophozoite and cyst stage of Acanthamoeba spp. After 90 min of incubation, Belcils® induced, DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We conclude that the foam destroys the cells by the induction of an apoptosis-like process. The current eye cleanser could be used as part of AK therapy protocol and as prevention from AK infections for contact lens users and post-ocular trauma patients.

In Vitro Activity of Statins against Naegleria fowleri.

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, and also global warming, as these amoebae thrive in warm water bodies, seem to be the key factors. Until present, no fully effective drugs have been developed to treat PAM, and the current options are amphotericin B and miltefosine, which present side effects such as liver and kidney toxicity. Statins are able to inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme for the synthesis of ergosterol of the cell membrane of these amoebae. Therefore, the in vitro activity of a group of statins was tested in this study against two types of strains of Naegleria fowleri. The obtained results showed that fluvastatin was the most effective statin tested in this study and was able to eliminate these amoebae at concentrations of 0.179 ± 0.078 to 1.682 ± 0.775 µM depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen.