10 research outputs found

    Caffeine Inhibits EGF-Stimulated Trophoblast Cell Motility through the Inhibition of mTORC2 and Akt.

    Get PDF
    Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo

    KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation

    Get PDF
    The cell junction–associated receptor CAR coordinates the dynamics between EGFR signaling and the cytoskeleton during cell proliferation.</jats:p

    Immunogenicity of protein therapeutics: The key causes, consequences and challenges

    No full text
    The immunogenicity of protein therapeutics has so far proven to be difficult to predict in patients, with many biologics inducing undesirable immune responses directed towards the therapeutic resulting in reduced efficacy, anaphylaxis and occasionally life threatening autoimmunity. The most common effect of administrating an immunogenic protein therapeutic is the development of a high affinity anti-therapeutic antibody response. Furthermore, it is clear from clinical studies that protein therapeutics derived from endogenous human proteins are capable of stimulating undesirable immune responses in patients, and as a consequence, the prediction and reduction of immunogenicity has been the focus of intense research. This review will outline the principle causes of the immunogenicity in protein therapeutics, and describe the development of pre-clinical models that can be used to aid in the prediction of the immunogenic potential of novel protein therapeutics prior to administration in man

    Conversion activity of brain derived PrP<sup>C</sup> in the CAA seeded with infected brain homogenates.

    No full text
    <p>(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS<sup>−</sup> without PK treatment), and protease resistant (DPBS<sup>+</sup> with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrP<sup>C</sup> expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).</p

    Conversion activity of brain derived PrP<sup>C</sup> in the CAA seeded with infected brain homogenates is sensitive to ionic strength and inhibited by the specific depletion of heparan sulphate.

    No full text
    <p>(A) The CAA was performed using IBH diluted in UBH prepared from WT and KO mice in Tris-HCl pH 7.4 and the indicated concentrations of NaCl. ** Indicates a significant reduction in conversion activity relative to 125mM NaCl. B) The CAA was performed using IBH diluted in UBH prepared from WT mice in 125mM NaCl/Tris-HCl pH 7.4 after treatment with Heparinase I (H), Heparinase III (HS), Chondroitinase ABC (Ch), their corresponding buffers (underlined) or without treatment (Con). Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Quantification (A, B) is based on at least three experiments, mean and SEM are shown. **p&lt;0.01, ***p&lt;0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism). C) The amount of sGAG purified from UBH treated with Heparinase I (H), Heparinase III (HS) and Chondrotinase ABC (Ch) or untreated (Con) was determined by Blyscan analysis and normalised to the amount of sGAG recovered from buffer controls (not shown). D) The absorbance (254nm) of sGAG eluted from a Q-Sepharose HiTrap anion exchange column in increasing concentrations of NaCl (0–1M). GAGs were purified from control (□), Heparinase I treated (⋄) and Heparinase III treated (○) or Chondroitinase ABC treated (+) brain homogenates. Quantification (C, D) is based on an analysis performed in duplicate.</p

    Conversion activity of wild-type and mutant PrP<sup>C</sup> expressed in RK-13 cells in the CAA following chlorate treatment to modify the sulphation of GAG.

    No full text
    <p>The CAA was performed using lysates prepared from RK-13 cells expressing WT (101P) and mutant (101L) moPrP. A) Quantification of conversion activity of 101P and 101L moPrP left untreated (−) or treated with 30mM chlorate (+) or UBH (Brain). B) Western blot analysis of PrP<sup>C</sup> expression in 101P and 101L moPrP left untreated (−) or treated with 30mM chlorate (+). Equivalent protein loaded in each lane, blots probed with beta-tubulin. CAA performed using (C) 101P-moPrP and (D) 101L-moPrP cells left untreated (−) or treated with 30mM chlorate (+). Conversion activity was determined as the fold increase in immunoreactive signal relative to puroRK reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hour at 37°C). Blots developed with 03R19. Molecular weight (kDa) is shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p&lt;0.05 two-tailed t-test of indicated pairs. In (C) and (D) CAA performed using KO and WT mouse brain homogenates (with quantitation shown as brain in A) and cell lysate derived from puroRK (N), 101P (P) and 101L (L) moPrP expressing cell lines. Truncated fragment (←) was not a consistently observed in either wildtype or mutant cell lines and was not included in analysis.</p

    Binding of wild-type and mutant PrP<sup>C</sup> expressed in RK-13 to sGAG.

    No full text
    <p>A) The ability of wildtype (101P) and mutant (101L) moPrP expressed in RK-13 cells to bind heparin sepharose beads in the presence of increasing concentrations of NaCl (50, 100, 125, 300, 1000mM), was determined by western blot analysis. N shows neat input. B) PrP<sup>C</sup> bound to heparin sepharose in the presence of 50mM NaCl was treated with PNGaseF before western blot analysis. Full length (F) and truncated (T) PrP species are shown. C) The percentage of 101P (dotted line) and 101L (solid line) moPrP bound to heparin sepharose in increasing concentrations of NaCl was determined relative to binding in 50mM NaCl. Blots developed with 03R19. Molecular weight (kDa) is shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. Binding differed significantly by Two-way ANOVA (p&lt;0.001).</p

    Conversion activity in the CAA following Benzonase treatment of UBH or IBH.

    No full text
    <p>A) The CAA was performed in the presence of the indicated concentrations of MgCl<sub>2</sub> and B) performed following pre-treatment as indicated. Conversion activity was determined as the fold increase in immunoreactive signal of treated samples relative to their equivalent KO reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Quantification is based on at least three experiments, mean and SEM are shown. **p&lt;0.001 or *p&lt;0.05 using one-way analysis of variance (ANOVA) with Dunnet's test for multiple comparisons against the indicated control (GraphPad, Prism).</p

    Systematic approach to selecting licensed drugs for repurposing in the treatment of progressive multiple sclerosis.

    Get PDF
    OBJECTIVE: To establish a rigorous, expert-led, evidence-based approach to the evaluation of licensed drugs for repurposing and testing in clinical trials of people with progressive multiple sclerosis (MS). METHODS: We long-listed licensed drugs with evidence of human safety, blood-brain barrier penetrance and demonstrable efficacy in at least one animal model, or mechanistic target, agreed by a panel of experts and people with MS to be relevant to the pathogenesis of progression. We systematically reviewed the preclinical and clinical literature for each compound, condensed this into a database of summary documents and short-listed drugs by scoring each one of them. Drugs were evaluated for immediate use in a clinical trial, and our selection was scrutinised by a final independent expert review. RESULTS: From a short list of 55 treatments, we recommended four treatments for immediate testing in progressive MS: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. We also prioritised clemastine, lamotrigine, oxcarbazepine, nimodipine and flunarizine. CONCLUSIONS: We report a standardised approach for the identification of candidate drugs for repurposing in the treatment of progressive MS.JB received expense payments from Novartis for speaking as patient representative during Siponimod licensing. AJC receives funding from the MRC and MS Society UK. DF is funded by the Wellcome and BBSRC, and has a project with Sangamo. A.G. de la Fuente has been supported by the ECTRIMS postdoctoral fellowship during this period. GG declares current research funding from Merck KGa (CLAD-B study), Roche (ORATORIO-HAND study) and Takeda (SIZOMUS Study). DM received funding previously from Biogen, MedDay and SanofiGenzyme. BN received funding from the Cambridge Centre for Myelin Repair, funded by MS Society UK. SP declares current funding from Italian and US Multiple Sclerosis Societies. LP has been supported by a senior research fellowship FISM - Fondazione Italiana Sclerosi Multipla - cod. 2017/B/5 and financed or co financed with the ‘5 per mille' public funding, by the Isaac Newton Trust RG 97440 and the Addenbrooke’s Charitable Trust RG 97519. KS declares current funding from Fondation Leducq, Multiple Sclerosis Society, Rosetrees Trust. A. Wilkins received a research grant from Sanofy (2018). A. Williams declares funding from MS Society UK, Roche, MRC, Lifearc
    corecore