Pure Venom Research Articles

Diagnosis of Apis dorsata venom allergy: use of recombinant allergens of Apis mellifera and a passive basophil activation test

BackgroundAllergy to Apis dorsata (Giant Asian Honeybee) venom is the commonest insect allergy in Sri Lanka and South East Asia. However, laboratory diagnosis is difficult as the pure venom and diagnostic reagents are not commercially available.ObjectiveThis study assessed the use of four recombinant allergens of A. mellifera venom and the passive basophil activation test in the diagnosis of A. dorsata venom anaphylaxis.MethodsSerum IgE levels to four recombinant allergens of A. mellifera, rApi m 1, 2, 5 and 10 were assessed and compared with serum IgE to the crude venom of A. mellifera or V. vulgaris by Phadia ImmunoCAP, in patients who developed anaphylaxis to A. dorsata stings. Basophil activation in response to venom of A. dorsata or V. affinis was assessed using a passive basophil activation test. Association of the severity of the reaction with basophil activation was compared.ResultsrApi m 1 and 10 combinedly had significant correlation (r = 0.722; p < 0.001) with the crude venom of A. mellifera (Western honeybee) and a higher positivity rate of 90% (27/30). Whereas, IgE reactivity to rApi m 2 or 5 had significant correlation (p = 0.02 and p = 0.005 respectively) with V. vulgaris crude venom. All 30 (100%) were positive to A. dorsata venom in passive BAT; 70% (21/30) had over 80% activation, 96.7% (29/30) had over 60% activation and 100% had over 50% activation. Percentage activation of basophils in patients who had mild or moderate reactions (n = 20) was significantly low (p = 0.02) from that of patients who had severe reactions (n = 10).ConclusionsrApi m 1 and 10 when combined was sensitive for the diagnosis of A. dorsata allergy. This combination had the lowest cross-reactivity rate with Vespula vulgaris. The passive BAT is highly sensitive in A. dorsata allergy. The basophil reactivity was significantly higher in severe anaphylaxis compared to mild/moderate anaphylaxis. This finding should be further explored in further studies.

Venom collection and analysis in the pseudoscorpion Chelifer cancroides (Pseudoscorpiones: Cheliferidae)

Pseudoscorpions are very small arthropods with almost worldwide distribution. They possess a unique venom delivery system in the chelal hands of their pedipalps that has evolved independently from that of scorpions and spiders. Studies on the venom composition of pseudoscorpions are very rare. Recently, the potential venom composition of the pseudoscorpion Synsphyronus apimelus Harvey, 1987 (Pseudoscorpiones: Garypidae) has been studied by transcriptome analysis. However, a proteome analysis of venom to identify the genuine venom compounds of pseudoscorpions has not yet been performed. In our study, we have developed a non-invasive approach for extracting minute amounts of venom, which for the first time allowed collecting pure venom samples of pseudoscorpions with minimal contaminations and high reproducibility. These experiments first required a morphological investigation of the venom delivery system with a focus on the role of the lamina defensor in the release of venom. Likely, the venom delivery system of pseudoscorpions has a mechanism that prevents the release of venom if the prey is not successfully penetrated by a venom tooth. Electrical stimulation of a gland-containing chelal hand in combination with a mechanical stimulation of the lamina defensor at the base of the venom tooth resulted in an average of 5 nl of collected venom. The utility of the method was then validated by repeated venom extractions and subsequent analysis of the venom composition using MALDI-TOF mass fingerprinting. Subsequent proteomics analysis in combination with transcriptome analyses of chelal hand tissue has identified the first genuine venom compounds of pseudoscorpions with putative antimicrobial peptides. For our experiments, we used the house pseudoscorpion Chelifer cancroides (Linnaeus, 1758) (Pseudoscorpiones: Cheliferidae).

Safety and Efficacy of a Progressively Prolonged Maintenance Interval of Venom Immunotherapy

Background: The long-term protection provided by venom immunotherapy (VIT) is related to the dose administered and to its long duration; the latter, however, becomes inconvenient for patients in countries like Greece, with many islanders or inhabitants of distant mountainous areas. Maintenance interval prolongation reduces the number of office visits – saving time and money – and as a consequence contributes to the patients’ compliance. The aim of this prospective study was to evaluate the safety and efficacy of VIT on a progressively prolonged maintenance interval (PPMI). Method: 450 venom-allergic patients were reviewed for participation in our study; all of them were initially treated with a modified rush or an ultrarush protocol using freshly reconstituted, pure venoms. Upon reaching the maintenance dose, the VIT interval was scheduled to be gradually prolonged – by 1 week each time – aiming at a maximal interval of 26 weeks. Results: 267/450 patients consented to participate in our VIT PPMI protocol: 98 were treated with vespid(s) venom, 142 with honeybee venom, and 27 with both. The mean duration of patient follow-up was 9.1 ± 4.2 years. The majority of systemic reactions due to VIT injections occurred up to the 8-weeks PPMI; few additional reactions were documented in a small fraction (2.9%) of our patient population beyond 9 weeks and up to 16 weeks; all were caused by honeybee VIT. No reactions were observed during VIT administration at the 26-week interval. Ninety-six patients reported 204 field sting occurrences by the culprit insect. Ten systemic reactions (8 mild and 2 moderate in severity) were registered between the 9- and 18-week PPMI; the honeybee was the culprit insect in all cases. 108 field stings by the offending insect were sustained beyond the 20- and up to the 26-week PPMI; there were no reactions at all. Conclusions: Progressively prolonging the VIT maintenance interval up to 26 weeks appears to be safe and efficacious.

Component-resolved evaluation of the content of major allergens in therapeutic extracts for specific immunotherapy of honeybee venom allergy

ABSTRACT Allergen-specific immunotherapy is the only curative treatment of honeybee venom (HBV) allergy, which is able to protect against further anaphylactic sting reactions. Recent analyses on a molecular level have demonstrated that HBV represents a complex allergen source that contains more relevant major allergens than formerly anticipated. Moreover, allergic patients show very diverse sensitization profiles with the different allergens. HBV-specific immunotherapy is conducted with HBV extracts which are derived from pure venom. The allergen content of these therapeutic extracts might differ due to natural variations of the source material or different down-stream processing strategies of the manufacturers. Since variations of the allergen content of therapeutic HBV extracts might be associated with therapeutic failure, we adressed the component-resolved allergen composition of different therapeutic grade HBV extracts which are approved for immunotherapy in numerous countries. The extracts were analyzed for their content of the major allergens Api m 1, Api m 2, Api m 3, Api m 5 and Api m 10. Using allergen-specific antibodies we were able to demonstrate the underrepresentation of relevant major allergens such as Api m 3, Api m 5 and Api m 10 in particular therapeutic extracts. Taken together, standardization of therapeutic extracts by determination of the total allergenic potency might imply the intrinsic pitfall of losing information about particular major allergens. Moreover, the variable allergen composition of different therapeutic HBV extracts might have an impact on therapy outcome and the clinical management of HBV-allergic patients with specific IgE to particular allergens.

A simple non-invasive technique for venom milking from a solitary wasp Delta conoideum Gmelin (Hymenoptera: Vespidae)

Prospecting wasp, ant and bee venom for active bio-molecules has gained considerable interest among researchers in recent years. Collecting sufficient quantity of venom from solitary wasps without sacrificing them is often difficult. Here we describe a non-invasive technique for collecting venom from a solitary wasp Delta conoideum Gmelin (Red-backed potter wasp). Venom was milked by presenting an agar block to a single female wasp for stinging. The venom was extracted from the agar block using ACN: water solvent system. The total protein in venom was estimated quantitatively and the presence of peptides in the venom was confirmed by MALDI-TOF analysis. The proposed technique is non-invasive and pure venom can be repeatedly ‘milked’ using this method from other wasps and also bees without the need for sacrificing a large number of individuals.

Fairy Bad Day (review)

Reviewed by: Fairy Bad Day Claire Gross Ashby, Amanda . Fairy Bad Day. Speak/Penguin, 2011. [352p]. Paper ed. ISBN 978-0-14-241259-6 $7.99 Reviewed from galleys R Gr. 7-10. Emma, a student at a school for those who can see (and thus slay) elementals (think goblins, demons, harpies, etc.), is certain that she's destined to be a dragon slayer, [End Page 455] just like her deceased mother. The indignity of her assignment as a fairy slayer—they're tiny! they're harmless! they keep mooning her!—is only compounded by that fact that she's paired with newly minted dragon slayer Curtis for a team learning assignment. That proves more interesting than she expected, though: Curtis has his own secrets, and while regular fairies pose little threat, a heretofore-unheard-of giant fairy called a darkhel ("seven feet of pure venom . . . sinister red eyes . . . razor-sharp teeth squished into a wide, misshapen mouth") proves a worthy foe and may even shed some new light on Emma's mother's death. Curtis is a low-key, good-natured man of mystery, and while his romance with Emma is nothing if not expected, it—along with the deadpan incorporation of generic high-school concerns—is a welcome balance to the seemingly unstoppable demonic peril. The littler fairies have a whiff of the Wee Free Men about them—that is, if Pratchett's bantering tricksters were mall rats instead of drinkers. There's a slight emotional tug in Emma's relationship with her father and revelations about her mother, but the demon-fighting high-school hijinks are where this book is really at. Hand it to fans of Hawkins' Hex Hall (BCCB 5/10) or Clement-Moore's Prom Dates from Hell (BCCB 7/07). Copyright © 2011 The Board of Trustees of the University of Illinois

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

BackgroundParasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.ResultsAbout 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein.An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins.ConclusionsThe use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

Secretion Profiles of Venom Alkaloids inSolenopsis geminata(Hymenoptera: Formicidae) in Taiwan

Solenopsis geminata (F.) was introduced into southern Taiwan decades ago and has continued to threaten the residents. Although the venom compositions of various fire ant species have been studied, the effects of environmental cues on the secretion pattern have received relatively little attention in an area with subtropical climate and high humidity, such as Taiwan. This study characterizes the effects of temperature and season on the venom compositions of S. geminata in Taiwan. Pure venom was sampled by using a microcapillary pipette and immersing the whole ant in hexane and subjected to gas chromatography-mass spectrometry analysis. The results showed that the ratio of cis C(11) to trans C(11) alkaloids in major workers was significantly higher than that in minor workers. No significant differences could be found in either the relative alkaloids content or the ratio of cis C(11) to trans C(11) alkaloids in venom of minor workers while rearing at four temperature conditions. Nevertheless, the ratio of cis C(11) to trans C(11) alkaloids in the venom of minor workers was the highest in spring and the lowest in winter. The results also showed that the body length, abdomen length, head length, head width, and venom volume differed significantly between major workers and minor workers of S. geminata. The venom volumes of these two castes were positively correlated with their body sizes.

An easy method for handling the genus Phoneutria (Araneae, Ctenidae) for venom extraction

Abstract This paper describes an easy, cheap, and safe method of capturing and handling the medically important spider Phoneutria for venom extraction. The method does not injure or kill the spider and allows the extraction of pure venom.

Sublingual Desensitization in Patients with Wasp Venom Allergy: Preliminary Results

The aim of this paper is to assess in an open prospective pilot case-control study the tolerability, safety and efficacy of an ultra-rush sublingual immunotherapy (SLIT) protocol with Vespula venom in wasp allergic patients compared to subcutaneous immunotherapy (SCIT). Forty-one wasp allergic patients were treated with sublingual (SLIT group) or subcutaneous (SCIT group) ultrarush immunotherapy with Vespula venom extract. All patients underwent skin tests and serum specific IgE and IgG4 detection before enrollment and after 6, 12 and 24 months of immunotherapy. The SLIT group consisted of 21 (6 females and 15 males) patients who received increasing doses of Vespula venom (Aquagen, ALK-Abellò) until the final dose of 30 drops of extract in 3 hours, containing 100,000 SQ-U/ml. The maintenance dose was of 10 drops of pure venom extract 3 times a week, for a total dose of 100,000 SQ-U weekly (corresponding to 100 microgram of venom extract). The SCIT group consisted of 20 patients (16 males and 4 females) who were treated with subcutaneous ultrarush immunotherapy with Vespula venom extract (Pharmalgen, Alk-Abellò). Patients received 101.1 microgram of Vespula venom in 3 hours and were treated with 100 microgram of wasp venom monthly. During the ultrarush sublingual treatment 2 patients (9.5%) experienced mild side-effects. Specific IgE and specific IgG to wasp venom did not show any significant modification. Four patients were field-stung by a wasp during the treatment (for a total of 6 stings). Two patients (3 stings), with a previous clinical history of a grade III and IV reaction, did not experience any reaction. One patient, with a previous grade II reaction, showed a large local reaction. The fourth patient, with a previous grade III reaction, was re-stung twice (after 12 and 24 months) with two systemic reactions (SR) (mild throat constriction). During the ultrarush SCIT phase, 3 (15%) patients experienced side-effects: 2 of them showed a large local reaction and 1 had headache and stomach ache. Specific IgE showed a significant (P = 0.001) increase after 6 months of treatment and then returned to baseline levels while specific IgG showed a significant (P = 0.001) increase after 6, 12 and 24 months in comparison with baseline. Nine patients were field-stung during the treatment: 8 of them experienced large local reactions; one patient (11%) experienced an SR (dizziness). Our results, even if in a small number of patients, suggest that in patients with Hymenoptera sting allergy SLIT could be efficacious with a good tolerability profile when compared to SCIT. Larger studies are needed to assess efficacy, safety and tolerability profile of wasp venom SLIT.

Insektengiftallergie bei Kindern

The prevalence of insect venom allergy in the European population is about 5%. In Germany, 20 - 40 deaths are reported every year. Clinical manifestations of the IgE-mediated disease range from local to systemic reactions to anaphylactic shock. Diagnosis is established based on anamnesis, skin testing and specific IgE antibodies in serum. Systemic reactions require preventive and therapeutic measures, especially an emergency set. Repetitive education is indispensable. Immunotherapy treatment of patients with insect venom allergy is one of the most advantageous uses of VIT (venom immunotherapy) with a reported effectiveness of 80% to nearly 100%. Since the first immunotherapy with pure venom was carried out in 1974, protocols of various duration have been devised in an effort to maximize protection and minimize side-effects. For children, a modified ultrarush immunotherapy protocol is appropriate. In contrast to conventional dose increase, schedules lasting a minimum of 5 days, shorter protocols reduce the patients stay in the hospital and provide an earlier protection towards stings. The risks of insect venom allergy are still underestimated not only in the general population, but also by many physicians.

Pharmacologically distinct cardiovascular effects of box jellyfish ( Chironexfleckeri) venom and a tentacle-only extract in rats

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the in vivo cardiovascular effects of Chironex fleckeri venom and tentacle extract (devoid of nematocysts). In anaesthetised rats, venom (10 μg/kg, i.v.) produced a transient pressor response (23 ± 4 mmHg) followed, in two of five animals, by cardiovascular collapse. Tentacle extract (100 μg/kg, i.v.) produced a more prolonged hypertensive effect (31 ± 3 mmHg) without cardiovascular collapse. Prazosin (50 μg/kg, i.v.) did not have any significant effect on the cardiovascular effects produced by venom. However, prazosin significantly attenuated the pressor response produced by tentacle extract. Ketanserin (1 mg/kg, i.v.) did not have any significant effect on the cardiovascular response of the anaesthetised rat to venom (10 μg/kg, i.v.; 25 ± 1 mmHg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to compare the two jellyfish samples used in the present study. In addition to ensuring reproducibility of future studies and allow comparison with previous research. We show, for the first time, that a pure venom sample extracted from C. fleckeri nematocysts and a tentacle extract have cardiovascular effects in the anaesthetised rat which are different and pharmacologically distinct.

Identification of allergens in the venom of the common striped scorpion

The common striped scorpion, Centruroides vittatus, is endemic to the southwestern United States and causes thousands of human stings annually. Immediate hypersensitivity reactions to C. vittatus venom have been reported. To identify specific IgE in 11 patients with immediate hypersensitivity to C. vittatus and to characterize the allergens present in the venom. Skin testing to dialyzed, filtered venom was performed in 5 patients. Immunoglobulin E immunoblots to whole milked venom was accomplished with serum samples from 8 patients. Enzymatic properties of whole venom were also determined. C. vittatus venom was found to contain 150 microg/microL of protein. Four of 5 patients tested had positive skin test reactions to the purified venom extract, with no late reactions. In all 8 patients, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated multiple proteins, 9 of which were identified as allergens on IgE immunoblots, ranging in size from 30 to 170 kD. Enzymatic activity was found to include phospholipase A, alkaline phosphatase, esterase, esterase lipase, and acid phosphatase. C. vittatus envenomation may result in immediate hypersensitivity reactions in susceptible individuals. Venom specific IgE can be identified by using skin tests and IgE immunoblots. The allergens identified in these patients had molecular weights distinct from those of known scorpion neurotoxins. A safe and effective skin testing extract can be prepared from dialyzed pure venom and may lead to the widespread ability to diagnose C. vittatus venom allergy.

A simple technique for milking the venom of a small parasitic wasp, Melittobia digitata (Hymenoptera: Eulophidae)

We describe a simple method for obtaining good a quantity of pure venom from a small parasitoid wasp, Melittobia digitata. Crushing the insect's head causes venom to be extruded from the ovipositor that dries rapidly as it is collected onto an insect pin. This technique may be applicable to other parasitic Hymenoptera.

The in vitro effects of two chirodropid ( Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 μg/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1 U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5 U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5 U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 μg/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 μg/ml). Antivenom (5 U/ml) added 1 h after the addition of C. fleckeri venom (50 μg/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 μg/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin. Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically.

Allergy to bumblebee venom.

Allergy to bumblebee venom is a rare form of Hymenoptera venom allergy. Because bumblebees are increasingly used for the pollination of greenhouse plants, the prevalence of this Hymenoptera allergy has increased during the past decade. The clinical presentation, diagnosis and therapy of bumblebee venom allergy are similar to other Hymenoptera venom allergies. There is a significant immunological cross-reactivity between bumblebee and honeybee venom. It has been claimed that immunotherapy with honeybee venom can protect patients with bumblebee venom allergy. This concept, however, has been called into question after the finding of bumblebee venom-specific IgE lacking cross-reactivity to honeybee venom, and three cases of bumblebee venom-allergic patients in whom immunotherapy with honeybee venom was unsuccessful. Immunotherapy with pure bumblebee venom has been shown to be effective and safe, and is currently the treatment of choice in individuals who cannot avoid contact with bumblebees. Immunotherapy with honeybee venom, however, should be considered in patients with severe reactions to bumblebee stings and concurrent sensitization to honeybee venom.

A simple method to obtain pure venom from small spiders

A simple method to obtain pure venom from small spiders

Characterization of peptides in sea anemone venom collected by a novel procedure

Peptide neurotoxins were isolated from the venom obtained by electrical stimulation of the sea anemone Bunodosoma caissarum. This technique allows almost pure venom to be collected, and the animals to survive. Three neurotoxins (assayed on crustacean nerves) were isolated by gel filtration and reversed-phase high performance liquid chromatography. Hemolysins were also detected in the venom. The amino acid sequence of a major neurotoxin BcIII was determined. BcIII has 48 amino acid residues with six half-cystine residues. This sequence has homology with the type 1 long sea anemone neurotoxins. Two minor toxins (BcI and II) have similar amino acid composition and amino-terminal sequences to BcIII.

Analysis of human sperm antigens obtained by affinity chromatography with polyclonal antisperm antibodies

Pure venoms from yellow jackets and bees were used to evaluate denaturing and nondenaturing immunoblot techniques and to compare results IgE antibody-stained immunoblots to RAST with highly purified allergens. Significant differences in IgE antibody binding were observed among all three techniques, suggesting that tertiary conformation is a major factor in the allergenic determinants of venom allergens. IgG antibodies from hyperimmunized animals and from specimens obtained from allergic patients before and after venom immunotherapy appeared to exhibit less conformational dependence for binding. The conformational dependence of IgE antibody binding to bee and yellow jacket phospholipases was confirmed by direct and inhibition RAST studies. The allergens studied were all single polypeptide chains.

Inhibition of human platelet aggregation and secretion by ant venom and a compound isolated from venom

Venom from the tropical ant, Pseudomyrmex triplarinus, has activity against rheumatoid arthritis. Since platelets are involved in inflammatory responses, they were employed to study the effects of venom on prostaglandin-dependent human platelet aggregation and secretion. The assay is very sensitive and uses microliter volumes, which makes it useful as a screen during isolation and characterization of venom components. Whole venom inhibited arachidonic acid- and U46619-induced platelet aggregation with IC50s of 45 and 39 micrograms/ml, respectively. This suggested that venom prevented the action of prostaglandins. Pure venom was fractionated by gel filtration and at least three materials with antiplatelet activity were detected. The smallest component (factor F) was most active and was purified by additional molecular filtration and characterized by UV absorbance, thin-layer chromatography, nuclear magnetic resonance spectra, and activity to platelets. Factor F was identified as adenosine, which is known to stimulate platelet adenylate cyclase and has not been previously reported to be a component of insect venom.