Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom.

We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood-brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8-10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8-10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8-10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of P. nigriventer.

Biotechnological Trends in Spider and Scorpion Antivenom Development.

Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology.

Brown Recluse spider bite mediated hemolysis: clinical features, a possible role for complement inhibitor therapy, and reduced RBC surface glycophorin A as a potential biomarker of venom exposure.

BACKGROUND: The venom of Loxosceles reclusa (Brown Recluse spider) can cause a severe, life-threatening hemolysis in humans for which no therapy is currently available in the USA beyond supportive measures. Because this hemolysis is uncommon, relatively little is known about its clinical manifestation, diagnosis, or management. Here, we aimed to clarify the clinical details of envenomation, to determine the efficacy of the complement inhibitor eculizumab to prevent the hemolysis in vitro, and to investigate markers of exposure to Brown Recluse venom. STUDY DESIGN AND METHODS: We performed a 10-year chart review of cases of Brown Recluse spider bite-mediated hemolysis at our institution. We also designed an in vitro assay to test the efficacy of eculizumab to inhibit hemolysis of venom exposed red blood cells. Finally, we compared levels of CD55, CD59 and glycophorin A on venom exposed versus venom-naïve cells. RESULTS: Most victims of severe Brown Recluse spider mediated hemolysis at our institution are children and follow an unpredictable clinical course. Brown Recluse spider bite mediated hemolysis is reduced by 79.2% (SD=18.8%) by eculizumab in vitro. Erythrocyte glycophorin A, but not CD55 or CD59, is reduced after red blood cells are incubated with venom in vitro. CONCLUSION: Taken together, our laboratory data and clinical observations indicate that L. reclusa venom exposure results in non-specific antibody and complement fixation on red blood cells, resulting in complement mediated hemolysis that is curtailed by the complement inhibitor eculizumab in vitro. Glycophorin A measurement by flow cytometry may help to identify victims of L. reclusa envenomation.

Regional brain c-fos activation associated with penile erection and other symptoms induced by the spider toxin Tx2-6.

Brain areas expressing c-fos messenger RNA were mapped by quantitative in situ hybridization after 1-2 h of intoxication with 10 µg/kg Tx2-6, a toxin obtained from the venom of the spider Phoneutria nigriventer. Relative to saline-treated controls, brains from toxin-treated animals showed pronounced c-fos activation in many brain areas, including the supraoptic nucleus, the paraventricular nucleus of the hypothalamus, the motor nucleus of the vagus, area postrema, paraventricular and paratenial nuclei of the thalamus, locus coeruleus, central amydaloid nucleus and the bed nucleus of the stria terminalis. The paraventricular hypothalamus and the bed nucleus of the stria terminalis have been implicated in erectile function in other studies. A possible role for central NO is considered. Acute stress also activates many brain areas activated by Tx2-6 as well as with NOstimulated Fos transcription. Brain areas that appear to be selectively activated by Tx2-6, include the paratenial and paraventricular thalamic nuclei, the bed nucleus of the stria terminalis and the area postrema and the dorsal motor n. of vagus in the medulla. However, direct injections of different doses of the toxin into the paraventricular hypothalamic n. failed to induce penile erection, arguing against CNS involvement in this particular effect.

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom.

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24h after venom injection. In addition, higher levels of PGE(2) were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1 beta, TNF-alpha, TXB(2) and LTB(4). Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE(2)), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom.

Venomous spiders, snakes, and scorpions in the United States.

Venomous bites and stings are complex poisonings that have local and systemic effects. Mild envenomations can be treated with supportive care. Severe envenomations can be treated definitively with species-specific antivenom, although the use of these products has potential risk of immediate and a more delayed onset form of hypersensitivity reactions. Consultation with a toxicologist is recommended to help guide therapy. Field treatments such as tourniquets and incision likely cause more harm than benefit and should be avoided.

Diagnosis of loxoscelism in a child confirmed with an enzyme-linked immunosorbent assay and noninvasive tissue sampling.

BACKGROUND: Confirmation of mild bites caused by Loxosceles reclusa with swab testing has not been previously documented, to our knowledge. METHODS: We report a case using an enzyme-linked immunosorbent assay (ELISA) test. RESULTS: A lesion lacking necrosis or other specific signs of loxoscelism was confirmed by identification of the Loxosceles venom and further confirmed by identification of a spider found in the patient's bed. LIMITATIONS: This is a pilot single-case report for this enzyme-linked immunosorbent assay test. CONCLUSIONS: A sensitive and specific enzyme-linked immunosorbent assay designed to detect Loxosceles venom, using a specimen obtained by swabbing the lesion, can aid in diagnosis of loxoscelism.

Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

Variations in Loxosceles spider venom composition and toxicity contribute to the severity of envenomation.

Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.

Direct vs. mediated effects of scorpion venom: an experimental study of the effects of a second challenge with scorpion venom.

OBJECTIVE: To assess the respective roles of venom and of catecholamines following scorpion envenomation and to verify whether a second challenge with scorpion venom induces the same consequences than a first one. DESIGN AND SETTING: Controlled animal study in a university research laboratory. SUBJECTS: Anesthetized and ventilated dogs. INTERVENTIONS: Fifteen dogs received intravenously a sublethal dose of scorpion venom (0.05 mg/kg). In the reenvenomated group (n=5) a second venom challenge with one-half sublethal venom dose was performed 30 min after the first one. The control group (n=10) received saline. Five additional animals served as sham. MEASUREMENTS AND RESULTS: Plasma toxin and catecholamine levels and a set of usual hemodynamic measurements were repeatedly measured in the first hour following envenomation. In the reenvenomated group another set of measurements was performed 5 min after the second challenge. Changes in toxin, catecholamines, and the main hemodynamic parameters were compared between the study groups. Initial peak toxin levels were similar in the two groups. They induced a striking increase in circulating catecholamines, a fall in heart rate, and an increase in mean arterial and pulmonary artery occluded pressures and in systemic vascular resistance. In the reenvenomated group the second challenge with scorpion venom achieved a toxin blood level similar to the first peak. However, it was not associated with a significant effect either on catecholamines release or on hemodynamics. Subsequent trends in hemodynamic changes were similar to those observed in the control group. CONCLUSIONS: These data emphasize the limited role of direct effects of scorpion venom on the cardiovascular system and the key role of catecholamines.

Tarantulas: eight-legged pharmacists and combinatorial chemists.

Tarantula venoms represent a cornucopia of novel ligands for a variety of cell receptors and ion channels. The diversity of peptide toxin pharmacology has been barely explored as indicated by pharmacological, toxicological and mass spectrometry investigations on more than 55 tarantula venoms. MALDI-TOF MS analysis reveals that the pharmacological diversity is based on relatively small size peptides, which seem to fall into a limited number of structural patterns. Properties and biological activities of the 33 known peptide toxins from tarantula venoms are described. Most known toxins conform to the Inhibitory Cystine Knot (ICK) motif, with differences in the length of intercysteine loops. Recently described peptides show that tarantula toxins can fold according to an elaboration of the Disulfide-Directed beta-Hairpin (DDH) motif which is also the canonical motif for the ICK fold. The ICK fold itself offers many variations leading to differing toxin properties. Examination of pharmacological data gives insights on the possible conserved site of action of toxins acting on voltage-gated ion channels and other toxins acting by a pore-blocking mechanism. Structure-activity data shows the versatility of the toxin scaffolds and the importance of surface features in the selectivity and specificity of these toxins. Tarantulas appear to be a good model for the discovery of novel compounds with important therapeutic potential, and for the study of the molecular evolution of peptide toxins.