Allergenic extracts to diagnose and treat sensitivity to insect venoms and inhaled allergens.

OBJECTIVE: To review allergenic extracts used to diagnose or treat insect allergies, including how the extracts are manufactured and their measurements of potency or concentration. DATA SOURCES: Peer-reviewed articles derived from searching PubMed (National Center for Biotechnology Information) about insect allergies and extract preparation. Encyclopedia of Life (http://www.eol.org/) and http://allergome.org/ were also referenced for background information on insects and associated allergens. STUDY SELECTIONS: Search terms used for the PubMed searches included insect allergens and allergies, Apidae, Vespidae, fire ants, cockroach allergies, insect allergen extract preparation, and standardization. RESULTS: Humans may be sensitized to insect allergens by inhalation or through stings. Cockroaches and moths are predominantly responsible for inhalation insect allergy and are a major indoor allergen in urban settings. Bees, fire ants, and wasps are responsible for sting allergy. In the United States, there are multiple insect allergen products commercially available that are regulated by the US Food and Drug Administration. Of those extracts, honeybee venom and insect venom proteins are standardized with measurements of potency. The remaining insect allergen extracts are nonstandardized products that do not have potency measurements. CONCLUSION: Sensitization to inhalational and stinging insect allergens is reported worldwide. Crude insect allergen extracts are used for diagnosis and specific immunotherapy. A variety of source materials are used by different manufacturers to prepare these extracts, which may result in qualitative differences that are not reflected in measurements of potency or protein concentration.

Isolation and characterization of an anti-insect ß-toxin from the venom of the scorpion Isometrus maculatus.

Im-3 was isolated from the venom of the scorpion Isometrus maculatus through several steps of HPLC fractionation based on the insect paralytic activity. Injecting Im-3 into crickets induced paralysis, but no toxicity was apparent in mice after an intracerebroventricular injection. Im-3 shares sequence similarity to scorpion ß-toxins that specifically affect insect sodium channels.

A family of orthologous proteins from centipede venoms inhibit the hKir6.2 channel.

Inhibitors targeting ion channels are useful tools for studying their functions. Given the selectivity of any inhibitor for a channel is relative, more than one inhibitor of different affinities may be used to help identify the channel in a biological preparation. Here, we describe a family of small proteins in centipede venoms that inhibit the pore (hKir6.2) of a human ATP-sensitive K+ channel (hKATP). While the traditional peptide-sequencing service gradually vanishes from academic institutions, we tried to identify the sequences of inhibitory proteins purified from venoms by searching the sequences of the corresponding transcriptomes, a search guided by the key features of a known hKir6.2 inhibitor (SpTx1). The candidate sequences were cross-checked against the masses of purified proteins, and validated by testing the activity of recombinant proteins against hKir6.2. The four identified proteins (SsdTx1-3 and SsTx) inhibit hKATP channels with a Kd of <300 nM, compared to 15 nM for SpTx1. SsTx has previously been discovered to block human voltage-gated KCNQ K+ channels with a 2.5 µM Kd. Given that SsTx inhibits hKir6.2 with >10-fold lower Kd than it inhibits hKCNQ, SsTx may not be suitable for probing KCNQ channels in a biological preparation that also contains more-SsTx-sensitive KATP channels.

Understanding toxicological implications of accidents with caterpillars Megalopyge lanata and Podalia orsilochus (Lepidoptera: Megalopygidae).

Megalopygids Megalopyge lanata and Podalia orsilochus are common causative agents of accidents in agricultural workers. These accidents are provoked by dermal contact at their larval stage and are characterized by cutaneous reactions, such as burning pain, edema and erythema, typically mild and self-limited. There is very little information about their venoms and their toxicological implications on human health. Thus, we employed proteomic techniques and biological assays to characterize venoms (bristle extracts) from caterpillars of both species collected from Misiones, Argentina. The electrophoretic profiles of both venoms were substantially different, and they presented proteins related to toxicity, such as serinepeptidases, serpins and lectins. P. orsilochus venom exhibited higher caseinolytic activity than M. lanata venom, agreeing with the fact that only P. orsilochus venom hydrolyzed human fibrin(ogen). In addition, the latter shortened the clotting time triggered by calcium. While the venom of M. lanata induced a mild inflammatory lesion in mouse skin, P. orsilochus venom caused prominent necrosis, inflammatory infiltration and hemorrhage at the site of venom injection. On the other hand, P. orsilochus venom was better recognized by Lonomia obliqua antivenom, although many of its proteins could not be cross-reacted, what may explain the difference in the clinical manifestations between accidents by Podalia and those by Lonomia. Altogether, this study provides relevant information about the pathophysiological mechanisms whereby both caterpillars can induce toxicity on human beings, and paves the way for novel discovery of naturally occurring bioactive compounds.

Speedy milking of fresh venom from aculeate hymenopterans.

A straightforward method for extracting aculeate arthropod venoms by centrifugation is described, based on adapting a glass insert containing a piece of metal mesh or glass wool into a centrifuge tube. Venom apparatuses are centrifuged for 30 s intervals at ≈2000-6000 g, with samples being dislodged between cycles. Venom from fire ants, honeybees, and a social wasp were extracted within minutes. The method is suited for small-scale bioassays and allows for faithful descriptions of unmodified toxin cocktails.

Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

Low cost venom extractor based on Arduino(®) board for electrical venom extraction from arthropods and other small animals.

Extracting venom from small species is usually challenging. We describe here an affordable and versatile electrical venom extractor based on the Arduino(®) Mega 2560 Board, which is designed to extract venom from arthropods and other small animals. The device includes fine tuning of stimulation time and voltage. It was used to collect venom without apparent deleterious effects, and characterized for the first time the venom of Zoropsis spinimana, a common spider in French Mediterranean regions.

Isolation and characterisation of glutamate receptor antagonists from venoms of orb-web spiders.

Two distinct families of low-molecular-weight toxins (argiotoxins) have been isolated from the venom of the orb-web spider. Argiope trifasciata. The toxins have been purified to homogeneity and characterised by spectroscopic, mass spectrometric and microchemical analysis. The major biologically active member of the first family of toxins is 2,4-dihydroxyphenylacetyl-asparagine linked through a C11-tetra-amine to N-terminal arginine; other members of this family are methylene homologues. The second family of toxins possesses an indolic group in place of the 2,4-dihydroxyphenyl chromophore. The toxins act as non-competitive inhibitors at quisqualate-type glutamatergic receptors on a metathoracic retractor unguis nerve-muscle preparation of Schistocerca gregaria. The loss of the N-terminal arginine reduces biological activity of the first family of toxins, but not of the second. The nature of the polyamine appears to be less important, perhaps acting as a spacer between the cationic arginine and the more hydrophobic aromatic tail of the toxins.

Effect on platelet FXIII and partial characterization of Lonomin V, a proteolytic enzyme from Lonomia achelous caterpillars.

Contact with Lonomia achelous caterpillars venom induces a severe bleeding syndrome in humans. A constant finding in all reported cases is a marked decrease of blood coagulation factor XIII (FXIII), which has been attributed to the presence of a proteolytic enzyme, isolated and named Lonomin V, in the hemolymph and hair secretion. In this study, the effect of Lonomin V on transglutaminase activity from human plasma, rabbit plasma, and platelet FXIII was analyzed. The decrease of activity was more pronounced in platelet (A2) when compared with rabbit plasma (AB) and human plasma FXIII (A2B2). This finding might be explained by the differences in FXIII molecular structure. In addition, platelet FXIII molecule was degraded by Lonomin to several fragments of low molecular mass. Lonomin V was stable over a wide range of pH (6-8.5) and temperatures of -70 degrees C, -20 degrees C and between 4 to 24 degrees C, with a progressive decrease at 37 degrees C and total inactivation at 60 degrees C after 2 hours incubation. Diisopropyl fluoro-phosphate, phenylmethylsulfonyl fluoride, tosyl-l-lysine chloromethyl ketone, and iodoacetamide abolished the effect of Lonomin V on FXIII; in contrast dithiothreitol and EDTA-Na enhance the activity. We concluded that Lonomin V is a serine proteinase with a free Cys essential for the enzymatic activity. Due to its proteolytic activity on FXIII, with concomitant impairment of fibrin cross-linking, Lonomin V might be useful in association with thrombolytic drugs for preventing rethrombosis.

A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap) biochemical characterization.

Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to a Factor X and to a prothrombin activating activity. Here, we describe a 69 kDa prothrombin activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C(4) column). The purified protein was able to activate prothrombin in a dose-dependent manner, and calcium ions increased this activity. The prothrombin-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtained for this substrate were Kmapp of 4.5 microM, kcat of 5.32 s(-1), and a kcat/Kmapp of 1.2 x 10(6) M(-1) s(-1). The prothrombin fragments generated by the purified enzyme corresponded to the molecular masses of prethrombin 2, fragment 1, fragment 2, and thrombin as seen in SDS-PAGE. The thrombin generated was able to clot purified fibrinogen. The partial amino acid sequence of the purified protein, named Lopap (L. obliqua prothrombin activator protease), showed no similarity to any known prothrombin activator.

In vivo characterization of Lopap, a prothrombin activator serine protease from the Lonomia obliqua caterpillar venom.

Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.

A comparative study of the spicule venom of Euproctis caterpillars.

Caterpillar spicule venoms were extracted and studied for the following activities: arginine ester (BAEE) hydrolase, tyrosine ester (ATEE) hydrolase, protease (casein digestion) and phospholipase A (indirect hemolytic activity). Crude spicule venom of E. chrysorrhoea preferably hydrolyzed BAEE in contrast to E. subflava venom, which hydrolyzed ATEE in preference to BAEE. This difference was confirmed by Sephadex G-100 elution profiles. The esterase activity in E. chrysorrhoea venom was separated into two peaks with average mol. wts. of 96,000 and 44,000. The first peak demonstrated optimal BAEE hydrolysis at pH 8.6 and 37 degrees C, whereas the second peak optimally hydrolyzed both BAEE and ATEE at pH 8.45 and pH 8.6 at 45 degrees C respectively. The esterase activity in E. subflava venom was separated into two peaks with average mol. wts of 63,000 and 32,000 showing optimal hydrolysis of BAEE at pH 8.9 and 37 degrees C, and of ATEE at pH 7.75 and pH 8.5 at 40 degrees C. The column fractions showed comparable proteolytic activity, irrespective of differences between their esterase activities. The presence of phospholipase A (PLA) enzyme in crude spicule venom of both species was evident from their indirect hemolytic activities. The PLA activity eluted with the void volume and seems to be associated with some high molecular weight protein. Under the assay conditions used, E. subflava venom contained 50-100 times less PLA activity than E. chrysorrhoea venom.

Purification and characterization of two forms of antigen 5 from polybia scutellaris venom.

Two polypeptides from the venom of Polybia scutellaris were purified to homogeneity by RP-HPLC. They differ very slightly in mol. wt (both are about 23,000) and hydrophobicity, and have isoelectric points greater than 9. Amino acid analyzes show close similarity between them and with antigen 5 of vespids from different species. The two polypeptides have an identical N-terminal sequence (18 amino acids) which shows a high degree of homology with those of other vespids. Owing to the fact that the venom of this species is non-allergenic, the data for the mol. wt, isoelectric point, amino acid composition and N-terminal sequence allow us to identify the isolated polypeptides as two forms of antigen 5. Amino acids at positions 5 and 11 in P. scutellaris antigen 5 differ from those of the previously known sequences for antigen 5, suggesting that one or other might be responsible for the lack of allergenicity of the P. scutellaris venom.

Isolation and structure analysis of components from venom of the spider Argiope lobata.

Homologous low molecular weight compounds, blocking postsynaptic glutamate receptors, were isolated from the venom of the spider Argiope lobata by ion exchange chromatography and reverse phase HPLC. Structures of nine blocking agents were determined by 1H-NMR and mass spectroscopy. Two-dimensional COSY spectra were used to identify spin systems of the protons coupled via two or three chemical bonds. The spin systems were combined into the sequence by means of the nuclear Overhauser effect between spatially close protons. Ionogenic groups were identified from pH dependences of the proton chemical shifts. Molecular structures thus obtained were checked against amino acid analysis and mass spectrometry data. The antagonists can be divided into three groups: argiopin, argiopinins and pseudoargiopinins depending on features of the chromophoric moiety (2,4-dihydroxyphenylacetic acid, 4-hydroxy-indole-3-acetic acid or indole-3-acetic acid).

Toxins produced by arthropod parasites: salivary gland proteins of human body lice and venom proteins of chelonine wasps.

A review is presented of our ongoing research projects on the protein components of the saliva of human body lice and of the non-paralyzing venom of wasps in the subfamily Cheloninae. Sodium dodecyl sulfate-polyacryamide gel electrophoretic analysis of lice salivary gland proteins showed a predominance of high and intermediate mol. wt proteins. Immunoblotting with a low titer polyclonal antiserum to lice salivary proteins indicated that some, but not all, of the predominant high mol. wt salivary gland proteins are injected into the host during feeding. The venom of a Chelonus sp. wasp contains a chitinase, and a 33,000 mol. wt protein with a primary structure composed mostly of a series of 12 tandem repeats of a 14-residue sequence. The N-terminus of this protein and its homologs in a related species of Ascogaster share a conserved adjacent pair of acidic residues. Epitope mapping/immunoprecipitation experiments now in progress will provide information on which linear motifs are on the surface of the protein, and will thereby provide information on the tertiary structure of the protein.

A microinjection technique using Drosophila melanogaster for bioassay-guided isolation of neurotoxins in arthropod venoms.

Modern analytical techniques permit isolation and structural determination of neurotoxins at the picomole level. However, bioassay-guided fractionation of the sample often relies on simple injection assays using insects, vertebrates or crustaceans of a fairly large size, thus consuming quite a large amount of the samples being investigated. In order to investigate samples of very small size, we have devised an insect microinjection method using glass micropipettes and Drosophila melanogaster adults as test insects. The validity of the method was tested with a series of six buthoid scorpion venoms (Androctonus australis, Buthotus judaicus, Buthus tamulus, Centruroides sculpturatus, Leiurus quinquestriatus hebraeus, Tityus serrulatus) and one chactoid scorpion (Scorpio maurus palmatus) as standards. The LD50S of the venoms were determined using both the microinjection method and a classical injection assay with crickets (Gryllus bimaculatus) as test insects. Results demonstrated that the new method can successfully be applied to the study of insect neurotoxic activity in arthropod venoms. The Gryllus:Drosophila ratio in amount of sample utilized is 100. However, for all Buthoid venoms tested, except L. quinquestriatus, Drosophila showed less sensitivity, thus reducing the gain by a factor of 2-10. Drosophila were several times more sensitive to the only chactoid venom tested. These results clearly demonstrate the advantage of using this microtechnique, when limited amounts of material are available for both chemical and biological work.

The Bushman arrow toxin, Diamphidia toxin: isolation from pupae of Diamphidia nigro-ornata.

The Bushmen of the Kalahari Desert in Botswana use the pupae of the beetle Diamphidia nigro-ornata Ståhl to poison their arrows. Sequential aqueous extraction, ammonium sulfate precipitation, ultrafiltration and chromatofocusing have given an apparently homogeneous active protein from these pupae with an approximate mol. wt of 54,000, an isoelectric point of about 8.0 pH and a lethal potency (minimum lethal dose, MLD) between 5 and 20 micrograms/kg (i.p. mouse). Preliminary pharmacological studies on less purified material show that, after a delay, this Diamphidia toxin causes sustained contraction of isolated intestinal smooth muscle. This contraction is not blocked by atropine or mepyramine and, therefore, is not due to release of acetylcholine or histamine. Results on the phrenic nerve - hemidiaphragm preparation demonstrate that in the presence of the toxin, contraction in response to indirect stimulation gradually fails and is accompanied by contracture. Since direct stimulation of the muscle still elicits a contraction, the toxin apparently does not affect the contractile mechanism itself. We conclude that Diamphidia pupae contain a protein toxin that is responsible for its lethality. Although this toxin appears to differ in some properties from the toxins reported by Mebs et al., de la Harpe et al. and Kündig, these protein preparations undoubtedly correspond to each other. We did not find any evidence of the low molecular weight toxic component reported by Mebs et al.

[Isolation of alpha-latrotoxin from karakurt spider venom using monoclonal antibodies]. / Vydelenie alpha-latrotoksina iz iada pauka karakurta s pomoshchiu monoklonalnykh antitel.

A method of alpha-latrotoxin (LT) isolation from the venom of Latrodectus mactans tredecimguttatus by means of immunoaffinity chromatography on sepharose conjugated with monoclonal antibodies against LT has been developed. This one-step, high-yield, relatively simple and rapid procedure yields active LT for structural and functional studies of its receptor.

[Hemolysis of Scolopendra toxins].

The hemolysis of toxins from alive Scolopendra subspinipes mutilans, medicinal material of Scolopendra subspimipes mutilans and S. multidens have been compared. The result shows that all the toxins have hemolytic activity. The hemolytic activity of the toxin from the medicinal materials of S. subspinipes mutilans is obviously lower than that from alive ones, and that from fresh medicinal materials are twice as high that from old ones, and that from S. multidens is higher than that from S. subspinipes multilans.