Structural and Functional Characterization of Fucoidan and Its Procoagulant Effect in An Ex Vivo Model of Factor VIII-Inhibited Guinea Pigs

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2262-2262
Author(s):  
Michael Dockal ◽  
Werner Hoellriegl ◽  
Zhenqing Zhang ◽  
Susanne Till ◽  
Sabine Knappe ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Sulfated Polysaccharides ◽  
In Vitro Tests ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Normal Ranges ◽  
Procoagulant Effect

Abstract Abstract 2262 Fucoidans are sulfated polysaccharides extracted and purified from brown seaweeds. Described as non-anticoagulant polysaccharides (NASPs), they have been shown to improve clotting in FVIII- and FIX-deficient plasma (Liu et al. Thromb Haemost 2006;95:68), making them good candidates for hemophilia treatment. NASP orally administered to hemophilia A dogs over several weeks resulted in correction to normal ranges in thromboelastography (TEG) parameters and improvement of the cuticle bleeding time (Prasad et al. Blood 2008;111:672). We screened extracts of eleven brown algae species to select the most active, high-quality, homogeneous fucoidan. In-depth structural analysis and assessment of the pro-/anticoagulant and TFPI-inhibiting activities of a selected set of fucoidan preparations was performed. The effect of the best fucoidan candidate was studied in a novel guinea pig model by whole blood TEG monitoring supported by calibrated automated thrombographic (CAT) analysis. Comparative structural studies of fucoidans included monosaccharide composition and elemental analysis, molecular weight and overall structural determination by NMR. 13C-NMR spectroscopy gave a comprehensive picture of fucoidan structure including fucose and alginate content and the relative heterogeneity of the preparation distinguishing between fucoidans of different algae species. Heterogeneity of the materials was highest in U. pinnatifida and L. japonica and lowest in F. vesiculosus (F.v.) samples. Procoagualant activity was shown by CAT and rotation thromboelastometry. Clotting parameters of FVIII-inhibited human blood or plasma were corrected to normal levels at 1 and 3 μg/mL depending on the fucoidan. In addition, the fucoidans were analyzed by BiaCore and a modified dilute prothrombin time assay to show binding to TFPI and inhibition of full-length TFPI activity. The screening process supported the identification and optimization of new fucoidan-specific analytical methods. Furthermore, a CAT protocol optimized for guinea pig plasma was used to show the procoagulant effect of the overall best fucoidan candidate. We spiked F.v. fucoidan into FVIII-inhibited guinea pig plasma between 1 and 100μg/mL and triggered thrombin generation with low amounts of tissue factor. The FVIII-inhibited guinea pig plasma was corrected to normal CAT parameters at ∼30μg/mL NASP. Based on our findings in the CAT assay, we designed a dosage pattern for studying the procoagulant effect using TEG in FVIII-inhibited Dunkin Hartley guinea pigs (n=10) after intravenous administration of 0.1 to 1.6mg/kg NASP. The primary endpoint assessed was R-time (time to clotting). FVIII-inhibited guinea pigs showed a median R-time of ≥120min (no clotting), while R-time was reduced in a non-linear dose-dependent manner after administration of NASP down to 69min (0.4mg/kg NASP). Thus, the results generated in in vitro tests, were confirmed in vivo. This new animal model of induced hemophilia will support the development of alternative hemophilia therapeutics. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Zhang:Baxter Healthcare Corporation: Employment. Till:Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Szabo:Baxter Healthcare Corporation: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

scholarly journals No Relationship Between Perceived Health Anomalies and Perceived Experimental Success in Retired Breeder Male Hartley Albino Guinea Pigs

2020 ◽  
Author(s):  
Chandra B. Bain ◽  
Julie M. Settlage ◽  
Grace A. Blair ◽  
Steven Poelzing
Keyword(s):  
Guinea Pig ◽  
Hair Loss ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Perceived Health ◽  
Electrophysiological Recordings ◽  
Electrophysiological Studies ◽  
Monthly Pattern ◽  
Time Of Year ◽  
Whole Heart

ABSTRACTGuinea pigs used in our laboratory for cardiac research sometimes exhibit physical abnormalities. These issues may abate or intensify during the time they are housed in our facility. After using a guinea pig for research, experimentalists note the apparent health of an animal based on visible features and/or abnormal electrophysiology of the heart. There was an existing anecdotal observation that the health of the Guinea Pigs, and subsequently the experimental success rate, had a seasonal variation; therefore we sought to determine if there is a time of year in which our guinea pigs are more likely to be perceived as unhealthy, and whether any determined monthly pattern correlates with an experimentalist’s ability to complete an experimental protocol. An electronic log was created to record the perceived health of the animal and the ability to complete the experiment successfully. Irregular symptoms included, but were not limited to, severe weight or hair loss and irregularities with the heart found post thoracotomy or during baseline electrophysiological recordings of whole-heart preparations. Animals that did not exhibit significant weight or hair loss, or other ailments were considered “healthy”. Overall, our results indicate that there are no monthly variations in perceived Hartley Albino guinea pig health or correlations with experimental completion rates, suggesting mild hair or weight loss that is common when shipping animals may not significantly affect the ability to conduct ex vivo whole-heart electrophysiological studies.

scholarly journals Visualising virulence factors: Trichophyton benhamiaes subtilisins demonstrated in a guinea pig skin ex vivo model

Mycoses ◽  
2020 ◽  
Vol 63 (9) ◽  
pp. 970-978 ◽  
Author(s):  
Christina‐Marie Baumbach ◽  
Jule Kristin Michler ◽  
Pietro Nenoff ◽  
Silke Uhrlaß ◽  
Wieland Schrödl
Keyword(s):  
Guinea Pig ◽  
Virulence Factors ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Pig Skin

scholarly journals Inhibition of Chondroitin-4-Sulfate-Specific Adhesion of Plasmodium falciparum-Infected Erythrocytes by Sulfated Polysaccharides

2005 ◽  
Vol 73 (7) ◽  
pp. 4288-4294 ◽  
Author(s):  
Katherine T. Andrews ◽  
Nicole Klatt ◽  
Yvonne Adams ◽  
Petra Mischnick ◽  
Reinhard Schwartz-Albiez
Keyword(s):  
Plasmodium Falciparum ◽  
Ex Vivo ◽  
Severe Disease ◽  
Placental Tissue ◽  
Peripheral Circulation ◽  
Sulfated Polysaccharides ◽  
Mature Stage ◽  
Dependent Manner ◽  
Inhibitory Compounds

ABSTRACT Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin 4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Soluble polysaccharides that release mature-stage parasitized erythrocytes into the peripheral circulation may help elucidate these interactions and have the potential to aid in developing therapeutic strategies. We have screened a panel of 11 sulfated polysaccharides for their capacities to inhibit adhesion of infected erythrocytes to CSA expressed on CHO-K1 cells and ex vivo human placental tissue. Two carrageenans and a cellulose sulfate (CS10) were able to inhibit adhesion to CSA and to cause already bound infected erythrocytes to de-adhere in a dose-dependent manner. CS10, like CSA and in contrast to all other compounds tested, remained bound to infected erythrocytes after washing and continued to inhibit binding. Both carrageenans and CS10 inhibited adhesion to placental tissue. Although highly sulfated dextran sulfate can inhibit CSA-mediated adhesion to CHO cells, this polysaccharide amplified adhesion to placental tissue severalfold, demonstrating the importance of evaluating inhibitory compounds in systems as close to in vivo as possible. Interestingly, and in contrast to all other compounds tested, which had a random distribution of sulfate groups, CS10 exhibited a clustered sulfate pattern along the polymer chain, similar to that of the undersulfated placental CSA preferred by placental-tissue-binding infected erythrocytes. Therefore, the specific antiadhesive capacity observed here seems to depend not only on the degree of charge and sulfation but also on a particular pattern of sulfation.

scholarly journals Phasic and Tonic Smooth Muscle Function of the Partially Obstructed Guinea Pig Intestine

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Jingbo Zhao ◽  
Donghua Liao ◽  
Jian Yang ◽  
Hans Gregersen
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Muscle Function ◽  
Circumferential Stress ◽  
Outer Diameter ◽  
Dependent Manner ◽  
Contraction Force ◽  
Jejunal Segment ◽  
Smooth Muscle Function

This study was to generate phasic and tonic stress-strain curves for evaluation of smooth muscle function in the obstructed guinea pig jejunum. Partial and sham obstruction of the jejunum in guinea pigs was created surgically, with guinea pigs not being operated on served as normal controls. The animals survived 2, 4, 7, and 14 days, respectively. The jejunal segment was distended to 10 cm H2O. The pressure and outer diameter changes were recorded. Passive conditions were obtained by using papaverine. Total phasic, tonic, and passive circumferential stress and strain were computed from the diameter and pressure data with reference to the zero-stress-state geometry. The active phasic and tonic stresses were defined as the total phasic and tonic stress minus the passive stress. The thickness of intestinal muscle layers increased in a time-dependent manner after obstruction. The amplitude of passive, total phasic, total tonic, active phasic, and active tonic circumferential stresses increased as function of strain 7 days after obstruction. However, when normalized to muscle layer thickness, the amplitude of active stresses did not differ among the groups. In conclusion, the long-term-obstructed intestine exhibits increased total smooth muscle contraction force. However, the contraction force per smooth muscle unit did not increase.

scholarly journals Citrullinated vimentin mediates development and progression of lung fibrosis

2021 ◽  
Vol 13 (585) ◽  
pp. eaba2927
Author(s):  
Fu Jun Li ◽  
Ranu Surolia ◽  
Huashi Li ◽  
Zheng Wang ◽  
Gang Liu ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Lung Fibrosis ◽  
Ex Vivo ◽  
Lavage Fluid ◽  
Surface Expression ◽  
Lung Fibroblasts ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Molecular Pattern ◽  
Citrullinated Vimentin

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)–dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2−/− and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

scholarly journals A Novel Ex Vivo Model of Aortic Valve Calcification. A Preliminary Report

2020 ◽  
Vol 11 ◽  
Author(s):  
Arsenii Zabirnyk ◽  
Maria del Mar Perez ◽  
Marc Blasco ◽  
Kåre-Olav Stensløkken ◽  
Miguel D. Ferrer ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Growth Medium ◽  
Ex Vivo ◽  
Growth Media ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Aortic Valves ◽  
Valve Calcification ◽  
Calcium Accumulation ◽  
Aortic Valve Leaflets

Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs.Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at −80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal–Wallis test with Dunn’s post-test was applied.Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM.Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

scholarly journals The Novel TRPA1 Antagonist BI01305834 Inhibits Ovalbumin-Induced Bronchoconstriction in Guinea Pigs

2020 ◽  
Author(s):  
Mariska van den Berg ◽  
Susan Nijboer - Brinksma ◽  
Sophie Bos ◽  
Maarten van den Berge ◽  
David Lamb ◽  
...  
Keyword(s):  
Pilot Study ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Optimal Dose ◽  
Pig Model ◽  
The Novel ◽  
Airway Narrowing ◽  
Guinea Pig Model

Abstract Background: We hypothesized that TRPA1 channels contribute to airway hyperresponsiveness (AHR) and inflammation in asthma. We evaluated the efficacy of the novel TRPA1 antagonist BI01305834 in a guinea pig model of asthma. Methods: First a pilot study was performed in a guinea pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), magnitude of EAR and LAR and airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett’s post-hoc test where appropriate.Results: A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea pig trachea strips.Conclusions: TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction, independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

scholarly journals Three Protein Cocktails Mediate Delayed-Type Hypersensitivity Responses Indistinguishable from That Elicited by Purified Protein Derivative in the Guinea Pig Model ofMycobacterium tuberculosisInfection

2010 ◽  
Vol 79 (2) ◽  
pp. 716-723 ◽  
Author(s):  
Hongliang Yang ◽  
JoLynn Troudt ◽  
Ajay Grover ◽  
Kimberly Arnett ◽  
Megan Lucas ◽  
...  
Keyword(s):  
T Cells ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Interleukin 10 ◽  
Pig Model ◽  
Delayed Type Hypersensitivity ◽  
Necrosis Factor Alpha ◽  
Purified Protein Derivative ◽  
Guinea Pig Model

ABSTRACTPurified protein derivative (PPD) is a widely used reagent for the diagnosis ofMycobacterium tuberculosisinfection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4+/CD8+T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4+and CD8+T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis ofM. tuberculosisinfection.

Effects of interleukin 5-induced pulmonary eosinophilia on airway reactivity in the guinea pig

1996 ◽  
Vol 270 (3) ◽  
pp. L368-L375 ◽  
Author(s):  
C. M. Lilly ◽  
R. W. Chapman ◽  
S. J. Sehring ◽  
P. J. Mauser ◽  
R. W. Egan ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Airway Hyperresponsiveness ◽  
Histological Grade ◽  
Platelet Activating Factor ◽  
Inflammatory Cells ◽  
Dependent Manner ◽  
Interleukin 5 ◽  
Dose Dependent

Administration of interleukin 5 (IL-5) to guinea pigs by tracheal injection was associated with increased recovery of eosinophils and neutrophils from bronchoalveolar lavage (BAL) fluid. The number of eosinophils recovered from BAL fluid increased in a dose-dependent manner from 9 +/- 2 X 10(3)/ml to a plateau of 143 +/- 29 X 10(3)/ml after the administration of recombinant human IL-5 (rhIL-5). Tracheal administration of recombinant guinea pig IL-5 (gpIL-5) also increased eosinophil recovery but was less potent than rhIL-5. Histological analysis confirmed the presence of inflammatory cells in the lung; there were higher grades of inflammation in airway than in parenchymal tissue after gpIL-5 administration. In addition, the histological grade of airway inflammation was greater 24 and 72 h after gpIL-5 administration than it was 6 days after administration. Airway hyperresponsiveness is reported to occur in guinea pigs exposed to rhIL-5 by intraperitoneal cellular production. It is surprising that airway infiltration with eosinophils induced by the topical application of IL-5 was not associated with hyperresponsiveness to substance P, histamine, or platelet-activating factor in intact animals or to methacholine in tracheally perfused lungs. Furthermore, the microvascular leakage induced by substance P was not altered by rhIL-5 administration. These findings indicate that the presence of eosinophils alone is not sufficient for the expression of airway hyperresponsiveness. Our ability to separate eosinophil recruitment and retention in the tissues from airway hyperresponsiveness indicates that these two processes are distinct and that the presence of eosinophils in lung tissue, by itself, is not sufficient to alter airway contractile responses.

scholarly journals Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James M. McCarthy ◽  
Jasmeet Virdee ◽  
Jessica Brown ◽  
Daniel Ursu ◽  
Zeshan Ahmed ◽  
...  
Keyword(s):  
Hippocampal Slice ◽  
Ex Vivo ◽  
Direct Access ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Concentration Dependent Manner ◽  
Hippocampal Slice Cultures ◽  
Organotypic Hippocampal Slice Cultures

AbstractIntracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

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