27 research outputs found
Effects of Tityus stigmurus (Thorell 1876) (Scorpiones: Buthidae) venom in isolated perfused rat kidneys
Study of Optimum Condition for Rapid Preparation of Thrombin using Russell’s Viper Venom Factor X Activator
Recommended from our members
Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
BackgroundBeta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein.Methodsβ-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration.ResultsPurified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis.ConclusionWe present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX
Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom
Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations
Immunity to LuloHya and Lundep, the salivary spreading factors from <i>Lutzomyia longipalpis</i>, protects against <i>Leishmania major</i> infection
<div><p>Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from <i>Lutzomyia longipalpis</i> have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from <i>Lu</i>. <i>longipalpis</i>. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized <i>in vitro</i>. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of <i>Leishmania</i> infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the <i>Lu</i>. <i>longipalpis—Leishmania major</i> combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity <i>in vitro</i>. Moreover, <i>in vivo</i> experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-<i>Leishmania</i> vaccine.</p></div
Validation of gene expression of cytokines and chemokines from HMVEC cells in the presence of LuloHya, Lundep or SGE.
<p>RT-PCR for validation of gene expression results obtained with the Human Cytokines & Chemokines RT<sup>2</sup> Profiler PCR Array PAHS-150ZD. Specific set of primers were used to amplify <b>(A)</b> CSF2; <b>(B)</b> CSF3; <b>(C)</b> LIF; <b>(D)</b> CXCL1; <b>(E)</b> CXCL2 and <b>(F)</b> CXCL8. Biological triplicates and technical duplicates were analyzed. Negative controls consisted of cDNA isolated from HMVEC cells incubated with incomplete medium. Results are expressed as the fold change of the gene expression normalized against the standard gene HPRT1 (NM_000194). Multiple comparisons were done by one-way ANOVA (****: p<0.0001; **: p<0.01; *: p<0.05). Bars indicate the SEM.</p
Vaccination studies with LuloHya and Lundep against <i>L</i>. <i>major</i> infection.
<p><b>(A)</b> Lesion size of C57BL/6 mice due to <i>L</i>. <i>major</i> infection steadily increased from the second week of the follow-up period until it stabilized or started to decrease after week 7 in all animals. For control animals (only immunized with Magic Mouse Adjuvant) lesion size was higher than vaccinated groups from week 4 onwards. The symbols represent the lesion size mean of 10 ears ± SEM analyzed by analysis of variance. <b>(B)</b> Lesion size values of C57BL/6 mice were converted to the area under the curve (AUC) showing that mice immunized against either LuloHya and Lundep presented significantly reduced lesions. <b>(C)</b> Parasite load of ears from C57BL/6 mice vaccinated with LuloHya or Lundep was significantly lower than control group (P<0.05). <b>(D)</b> <i>L</i>. <i>major</i> lesion size in B-cell-deficient B6.129S2-<i>Ighm</i><sup><i>tm1Cgn</i></sup>/J mice. <b>(E)</b> There are no statistical significant differences in lesion size of B6.129S2-<i>Ighm</i><sup><i>tm1Cgn</i></sup>/J mice immunized with either LuloHya, Lundep or adjuvant. <b>(F)</b> Parasite load of ears from B6.129S2-<i>Ighm</i><sup><i>tm1Cgn</i></sup>/J mice vaccinated with LuloHya or Lundep showed no differences with the control group. Multiple comparisons were done by one-way ANOVA (****: p<0.0001; ***: p<0.001; **: p<0.01; *: p<0.05; ns: non-significant). Bars indicate SEM.</p
Specificity of hyaluronidase activity of LuloHya.
<p><b>(A)</b> Five micrograms of high molecular weight HA (H), chondroitin sulfate B (CS), dextran sulfate (DS) and heparin (Hep) were fractionated on a 1.2% agarose gel alone or after incubation with LuloHya or <i>Lu</i>. <i>longipalpis</i> SGE. As molecular weight markers, Select-HA HiLadder, Select-HA 250k (Hyalose) and GeneRuler 1kb DNA ladder were used (Thermo Scientific). <b>(B)</b> Five micrograms of high, medium and low molecular weight HA (H, M and L, respectively) were separated on a 1.2% agarose gel alone of after incubation with LuloHya, bovine hyaluronidase (BovHya), <i>Streptomyces hyalurolyticus</i> hyaluronidase (BactHya) and <i>Lu</i>. <i>longipalpis</i> SGE.</p
<i>In vivo</i> recruitment of polymorphonuclear leukocytes by LuloHya, Lundep or SGE in mice.
<p>Recruitment of neutrophils to the inoculation site was determined by flow cytometry analysis. (<b>A</b>) Gating strategy analysis based on the CD11b expression to identify neutrophils (Ly6G<sup>+</sup>Ly6C<sup>int</sup>) and inflammatory monocytes (Ly6G<sup>-</sup>Ly6C<sup>hi</sup>). Mice (C57BL/6) were intradermally inoculated with LuloHya (10 μg and 1 μg), Lundep (10 μg and 1 μg), <i>Lu</i>. <i>longipalpis</i> SGE (equivalent to 2 SG pairs) and PBS alone as a negative control. Ears injected with 1 μg of non-related salivary protein from <i>Ae</i>. <i>aegypti</i> (ASP) were also included. After 2 h, the ears were collected and processed for flow cytometry analysis. <b>(B)</b> Total count of neutrophils per ear. (<b>C</b>) Frequency of neutrophils in mouse ears. Results (of at least 2 independent experiments) are shown as mean +/- SEM. Comparisons with PBS group were done by Mann-Whitney U test (**: p<0.01; *: p<0.05).</p
Cytokine and chemokine gene expression in the presence of salivary proteins LuloHya and Lundep.
<p>Cytokine and chemokine gene expression in the presence of salivary proteins LuloHya and Lundep.</p