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Michael D Wiese - One of the best experts on this subject based on the ideXlab platform.
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global view on Ant Venom allergy from allergenic components to clinical management
Clinical Reviews in Allergy & Immunology, 2021Co-Authors: Simon G A Brown, Troy Wanandy, Emily M Mulcahy, Wun Yee Lau, Michael D WieseAbstract:Hymenoptera Venom allergy is characterised by systemic anaphylactic reactions that occur in response to stings from members of the Hymenoptera order. Stinging by social Hymenoptera such as Ants, honeybees, and vespids is one of the 3 major causes of anaphylaxis; along with food and drug exposure, it accounts for up to 43% of anaphylaxis cases and 20% of anaphylaxis-related fatalities. Despite their recognition as being of considerable public health significance, stinging Ant Venoms are relatively unexplored in comparison to other animal Venoms and may be overlooked as a cause of Venom allergy. Indeed, the Venoms of stinging Ants may be the most common cause of anaphylaxis in Ant endemic areas. A better understanding of the natural history of Venom allergy caused by stinging Ants, their Venom components, and the management of Ant Venom allergy is therefore required. This article provides a global view on allergic reactions to the Venoms of stinging Ants and the contemporary approach to diagnose and manage Ant Venom allergy.
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factors influencing the quality of myrmecia pilosula jack jumper Ant Venom for use in in vitro and in vivo diagnosis of allergen sensitization and in allergen immunotherapy
Clinical & Experimental Allergy, 2017Co-Authors: Troy Wanandy, Simon G A Brown, Michael D Wiese, Noel W Davies, He Dwyer, Loyola Mclean, David Nichols, Nuri GuevenAbstract:SummaryBackground Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnosis in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. Objectives We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) Ant Venom (JJAV) in the form of Active Pharmaceutical Ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. Methods The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quAntification, HPLC-UV for JJAV allergen peptides quAntification, ELISA Inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and Immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quAntification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. Results Pharmaceutical grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure does not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. Conclusions and Clinical relevance We have identified factors which can influence the quality and consistency of JJAV AIs and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT. This article is protected by copyright. All rights reserved.
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causes of Ant sting anaphylaxis in australia the australian Ant Venom allergy study
The Medical Journal of Australia, 2011Co-Authors: Simon G A Brown, Pauline Van Eeden, Michael D Wiese, Raymond J Mullins, Graham O Solley, Robert Puy, Robert W TaylorAbstract:Objective: To determine the Australian native Ant species associated with Ant sting anaphylaxis, geographical distribution of allergic reactions, and feasibility of diagnostic Venom-specific IgE (sIgE) testing. Design, setting and participAnts: Descriptive clinical, entomological and immunological study of Australians with a history of Ant sting anaphylaxis, recruited in 2006–2007 through media exposure and referrals from allergy practices and emergency physicians nationwide. We interviewed participAnts, collected entomological specimens, prepared reference Venom extracts, and conducted serum sIgE testing against Ant Venom panels relevAnt to the species found in each geographical region. Main outcome measures: Reaction causation attributed using a combination of Ant identification and sIgE testing. Results: 376 participAnts reported 735 systemic reactions. Of 299 participAnts for whom a cause was determined, 265 (89%; 95% CI, 84%–92%) had reacted clinically to Myrmecia species and 34 (11%; 95% CI, 8%–16%) to green-head Ant (Rhytidoponera metallica). Of those with reactions to Myrmecia species, 176 reacted to jack jumper Ant (Myrmecia pilosula species complex), 18 to other jumper Ants (15 to Myrmecia nigrocincta, three to Myrmecia ludlowi) and 56 to a variety of bulldog Ants, with some participAnts reacting to more than one type of bulldog Ant. Variable serological cross-reactivity between bulldog Ant species was observed, and sera from patients with bulldog Ant allergy were all positive to one or more Venoms extracted from Myrmecia forficata, Myrmecia pyriformis and Myrmecia nigriceps. Conclusion: Four main groups of Australian Ants cause anaphylaxis. Serum sIgE testing enhances the accuracy of diagnosis and is a prerequisite for administering species
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stability of myrmecia pilosula jack jumper Ant Venom for use in immunotherapy
Journal of Pharmaceutical and Biomedical Analysis, 2011Co-Authors: Simon G A Brown, Michael D Wiese, Robert J Heddle, Noel W Davies, Tim Chataway, Robert W MilneAbstract:Allergy to Myrmecia pilosula (Jack Jumper Ant) Venom is common in Australia, affecting ∼2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the Venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant Venom must be demonstrated. HPLC–UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess Venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pH of 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100ug/mL Venom, but improved stability at concentrations of 1ug/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilising. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant Venom was stable at −18 C and 4 C for 12 months; following dilution to 100ug/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), Venom was stable for 7 days when stored at 4 ◦C. Concentrated Jack Jumper Ant Venom can be stored in 22% sucrose for 12 months, and after dilution to 100ug/mL for clinical use, it should be discarded after 7 days.
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myrmecia pilosula jack jumper Ant Venom validation of a procedure to standardise an allergy vaccine
Journal of Pharmaceutical and Biomedical Analysis, 2008Co-Authors: Simon G A Brown, Michael D Wiese, Robert J Heddle, Noel W Davies, Tim Chataway, Robert W MilneAbstract:Ant sting allergy is relatively common within south-eastern Australia and is predominAntly due to Myrmecia pilosula (Jack Jumper Ant, JJA). Venom immunotherapy has been shown to be effective in preventing anaphylaxis to the sting of the JJA, but analytical techniques to standardise the Venom have not been validated. The purpose of this study was to develop assays to analyse JJA Venom and apply these to the standardisation of Venom prior to new batches being used for the diagnosis and treatment of JJA sting allergy. Venom was analysed by protein content, HPLC-UV, enzyme-linked immunosorbent assay (ELISA) inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE immunoblot. The protein content in JJA Venom was adjusted so that all batches were equivalent. A HPLC-UV assay was used to quAntify the relative amount of the major allergen Myr p 2 and two minor allergens Myr p 1 and Myr p 3 and allergenic potency was determined by ELISA inhibition. SDS-PAGE and SDS-PAGE immunoblot were used as qualitative tools to determine the protein profile and presence or absence of additional high molecular weight allergens not quAntifiable by HPLC-UV. A standardisation procedure has been developed that complies with the requirements described in the European Pharmacopoeia. Techniques used to determine the content of some of the other minor allergens could be developed, which would further improve the standardisation methodology.
Simon G A Brown - One of the best experts on this subject based on the ideXlab platform.
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global view on Ant Venom allergy from allergenic components to clinical management
Clinical Reviews in Allergy & Immunology, 2021Co-Authors: Simon G A Brown, Troy Wanandy, Emily M Mulcahy, Wun Yee Lau, Michael D WieseAbstract:Hymenoptera Venom allergy is characterised by systemic anaphylactic reactions that occur in response to stings from members of the Hymenoptera order. Stinging by social Hymenoptera such as Ants, honeybees, and vespids is one of the 3 major causes of anaphylaxis; along with food and drug exposure, it accounts for up to 43% of anaphylaxis cases and 20% of anaphylaxis-related fatalities. Despite their recognition as being of considerable public health significance, stinging Ant Venoms are relatively unexplored in comparison to other animal Venoms and may be overlooked as a cause of Venom allergy. Indeed, the Venoms of stinging Ants may be the most common cause of anaphylaxis in Ant endemic areas. A better understanding of the natural history of Venom allergy caused by stinging Ants, their Venom components, and the management of Ant Venom allergy is therefore required. This article provides a global view on allergic reactions to the Venoms of stinging Ants and the contemporary approach to diagnose and manage Ant Venom allergy.
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factors influencing the quality of myrmecia pilosula jack jumper Ant Venom for use in in vitro and in vivo diagnosis of allergen sensitization and in allergen immunotherapy
Clinical & Experimental Allergy, 2017Co-Authors: Troy Wanandy, Simon G A Brown, Michael D Wiese, Noel W Davies, He Dwyer, Loyola Mclean, David Nichols, Nuri GuevenAbstract:SummaryBackground Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnosis in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. Objectives We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) Ant Venom (JJAV) in the form of Active Pharmaceutical Ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. Methods The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quAntification, HPLC-UV for JJAV allergen peptides quAntification, ELISA Inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and Immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quAntification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. Results Pharmaceutical grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure does not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. Conclusions and Clinical relevance We have identified factors which can influence the quality and consistency of JJAV AIs and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT. This article is protected by copyright. All rights reserved.
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Causes of Ant sting anaphylaxis in Australia: the Australian Ant Venom Allergy Study
Australasian Medical Association, 2016Co-Authors: Simon G A Brown, Van Eeden Pauline, Wiese, Michael D, Mullins Raymond, Solley Graham, Puy Robert, Taylor Robert, Heddle, Robert JAbstract:Objective: To determine the Australian native Ant species associated with Ant sting anaphylaxis, geographical distribution of allergic reactions, and feasibility of diagnostic Venom-specific IgE (sIgE) testing. Design, setting and participAnts: Descriptive clinical, entomological and immunological study of Australians with a history of Ant sting anaphylaxis, recruited in 2006-2007 through media exposure and referrals from allergy practices and emergency physicians nationwide. We interviewed participAnts, collected entomological specimens, prepared reference Venom extracts, and conducted serum sIgE testing against Ant Venom panels relevAnt to the species found in each geographical region. Main outcome measures: Reaction causation attributed using a combination of Ant identification and sIgE testing. Results: 376 participAnts reported 735 systemic reactions. Of 299 participAnts for whom a cause was determined, 265 (89%; 95% CI, 84%-92%) had reacted clinically to Myrmecia species and 34 (11%; 95% CI, 8%-16%) to green-head Ant (Rhytidoponera metallica). Of those with reactions to Myrmecia species, 176 reacted to jack jumper Ant (Myrmecia pilosula species complex), 18 to other jumper Ants (15 to Myrmecia nigrocincta, three to Myrmecia ludlowi) and 56 to a variety of bulldog Ants, with some participAnts reacting to more than one type of bulldog Ant. Variable serological cross-reactivity between bulldog Ant species was observed, and sera from patients with bulldog Ant allergy were all positive to one or more Venoms extracted from Myrmecia forficata, Myrmecia pyriformis and Myrmecia nigriceps. Conclusion: Four main groups of Australian Ants cause anaphylaxis. Serum sIgE testing enhances the accuracy of diagnosis and is a prerequisite for administering species- specific Venom immunotherapy
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Ant Venom immunotherapy in australia the unmet need
The Medical Journal of Australia, 2014Co-Authors: Raymond J Mullins, Simon G A BrownAbstract:Jack jumper Ant (JJA) Venom allergy is an importAnt cause of anaphylaxis in south-eastern Australia. The efficacy and real-world effectiveness of JJA Venom immunotherapy (VIT) to prevent anaphylaxis in allergic patients are now well established, with an evidence base that is at least equivalent to that supporting VIT for allergy to other insect species. The tolerability and safety of JJA VIT are comparable with those of honeybee VIT.
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causes of Ant sting anaphylaxis in australia the australian Ant Venom allergy study
The Medical Journal of Australia, 2011Co-Authors: Simon G A Brown, Pauline Van Eeden, Michael D Wiese, Raymond J Mullins, Graham O Solley, Robert Puy, Robert W TaylorAbstract:Objective: To determine the Australian native Ant species associated with Ant sting anaphylaxis, geographical distribution of allergic reactions, and feasibility of diagnostic Venom-specific IgE (sIgE) testing. Design, setting and participAnts: Descriptive clinical, entomological and immunological study of Australians with a history of Ant sting anaphylaxis, recruited in 2006–2007 through media exposure and referrals from allergy practices and emergency physicians nationwide. We interviewed participAnts, collected entomological specimens, prepared reference Venom extracts, and conducted serum sIgE testing against Ant Venom panels relevAnt to the species found in each geographical region. Main outcome measures: Reaction causation attributed using a combination of Ant identification and sIgE testing. Results: 376 participAnts reported 735 systemic reactions. Of 299 participAnts for whom a cause was determined, 265 (89%; 95% CI, 84%–92%) had reacted clinically to Myrmecia species and 34 (11%; 95% CI, 8%–16%) to green-head Ant (Rhytidoponera metallica). Of those with reactions to Myrmecia species, 176 reacted to jack jumper Ant (Myrmecia pilosula species complex), 18 to other jumper Ants (15 to Myrmecia nigrocincta, three to Myrmecia ludlowi) and 56 to a variety of bulldog Ants, with some participAnts reacting to more than one type of bulldog Ant. Variable serological cross-reactivity between bulldog Ant species was observed, and sera from patients with bulldog Ant allergy were all positive to one or more Venoms extracted from Myrmecia forficata, Myrmecia pyriformis and Myrmecia nigriceps. Conclusion: Four main groups of Australian Ants cause anaphylaxis. Serum sIgE testing enhances the accuracy of diagnosis and is a prerequisite for administering species
Donald R. Hoffman - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of sol i 2 a major allergen from fire Ant Venom
Journal of Molecular Biology, 2012Co-Authors: Aline S Borer, Margit Schmidt, Donald R. Hoffman, Paul Wassmann, Jingjiang Zhou, Christine S Wright, Tilman Schirmer, Zora MarkovichousleyAbstract:Sol i 2 is a potent allergen from the Venom of red imported fire Ant, which contains allergens Sol i 1, Sol i 2, Sol i 3, and Sol i 4 that are known to be powerful triggers of anaphylaxis. Sol i 2 causes IgE Antibody production in about one-third of individuals stung by fire Ants. Baculovirus recombinAnt dimeric Sol i 2 was crystallized as a native and selenomethionyl-derivatized protein, and its structure has been determined by single-wavelength anomalous dispersion at 2.6 A resolution. The overall fold of each subunit consists of five helices that enclose a central hydrophobic cavity. The structure is stabilized by three intramolecular disulfide bridges and one intermolecular disulfide bridge. The nearest structural homologue is the sequence-unrelated odorAnt binding protein and pheromone binding protein LUSH of the fruit fly Drosophila, which may suggest a similar biological function. To test this hypothesis, we measured the reversible binding of various pheromones, plAnt odorAnts, and other ligands to Sol i 2 by the changes in N-phenyl-1-naphthylamine fluorescence emission upon binding of ligands that compete with N-phenyl-1-naphthylamine. The highest binding affinity was observed for hydrophobic ligands such as aphid alarm pheromone (E)-β-farnesene, analogs of Ant alarm pheromones, and plAnt volatiles decane, undecane, and β-caryophyllene. Conceivably, Sol i 2 may play a role in capturing and/or transporting small hydrophobic ligands such as pheromones, odors, fatty acids, or short-living hydrophobic primers. Molecular surface analysis, in combination with sequence alignment, can explain the serological cross-reactivity observed between some Ant species.
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crystal structure of the major allergen from fire Ant Venom sol i 3
Journal of Molecular Biology, 2008Co-Authors: Sivaraman Padavattan, Margit Schmidt, Donald R. Hoffman, Zora MarkovichousleyAbstract:Fire Ant Venom is an extremely potent allergy-inducing agent containing four major allergens, Sol i 1 to Sol i 4, which are the most frequent cause of hypersensitivity reactions to hymenoptera in the southern USA. The crystal structure of recombinAnt (Baculovirus) major fire Ant allergen Sol i 3 has been determined to a resolution of 3.1 A by the method of molecular replacement. The secondary-structure elements of Sol i 3 are arranged in an alpha-beta-alpha sandwich fold consisting of a central Antiparallel beta-sheet surrounded on both sides by alpha helices. The overall structure is very similar to that of the homologous wasp Venom allergen Ves v 5 with major differences occurring in the solvent-exposed loop regions that contain amino acid insertions. Consequently, the limited conservation of surface chemical properties and topology between Sol i 3 and Ves v 5 may explain the observed lack of relevAnt cross-reactivity. It is concluded that Sol i 3 recognizes immunoglobulin E Antibodies with a distinct set of its own epitopes, which are different from those of Ves v 5. Indeed, the molecular area in Sol i 3 covered by non-conserved residues is large enough to accommodate four unique Sol i 3 epitopes.
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sol i 1 the phospholipase allergen of imported fire Ant Venom
The Journal of Allergy and Clinical Immunology, 2005Co-Authors: Donald R. Hoffman, Rhonda H Sakell, Margit SchmidtAbstract:Background Sol i 1, the Venom phospholipase of imported fire Ant Venom is an importAnt allergen and exhibits some cross-reactivity with IgE Antibodies from patients sensitized to other Hymenoptera Venoms. Objective To determine the primary structure of Sol i 1 and evaluate the roles of protein and carbohydrate epitopes in its cross-reactivity. Methods Sol i 1 was purified from Venom, proteolytic peptides prepared and amino acid sequences obtained. The cDNA for Sol i 1 was cloned, sequenced, and compared with sequences of other wasp Venom phospholipases. The role of carbohydrate epitopes in the cross-reactivity with other Hymenoptera Venoms was studied by RAST inhibition. Results The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid Venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. Mannosylated N-glycans could completely inhibit binding of IgE from honeybee Venom sensitized patients to Sol i 1. Inhibition by glycan of IgE binding from yellow jacket Venom sensitized patients was low or absent for three of eight sera and substAntial, but not complete for five sera. Conclusions Sol i 1 is related to wasp Venom phospholipases. Cross-reactivity with honeybee Venom is caused by carbohydrate, whereas cross-reactivity with yellow jacket Venom involves reactivity with both carbohydrate determinAnts of hyaluronidase and high molecular weight proteins and phospholipase protein determinAnts.
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immunologic characterization of the recombinAnt fire Ant Venom allergen sol i 3
Allergy, 2003Co-Authors: Margit Schmidt, Thomas J. Mcconnell, Donald R. HoffmanAbstract:Individuals sensitized to fire Ant stings show immunoglobulin (Ig)E Antibodies against the Venom protein Sol i 3. We determined the full-length complementary DNA (cDNA) sequence of this protein and expressed recombinAnt Sol i 3 in immunogenic form. The complete cDNA of Sol i 3 was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR + 1 reactions using gene-specific oligonucleotides, and oligonucleotides designed from the amino acid sequence of this protein. The encoding cDNA is 705 bp in length corresponding to 235 amino acids. The first 22 amino acids are a leader sequence. The protein with an added C-terminal hexahistidine tag was expressed in insect cells using a baculovirus system. The recombinAnt protein was secreted into the supernatAnt and affinity purified with a cobalt chelating resin. The recombinAnt fire Ant Venom allergen Sol i 3 showed similar IgE binding activity to the native protein in radioallergosorbent test (RAST) and RAST inhibition assays. It was produced in both a glycosylated and an unglycosylated form. A three-dimensional reconstruction of Sol i 3 was compared with the experimentally determined structure of the related allergen Ves v 5. This model is supported by results of circular dichroism spectroscopy.
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Production of a recombinAnt imported fire Ant Venom allergen, Sol i 2, in native and immunoreactive form
The Journal of allergy and clinical immunology, 1996Co-Authors: Margit Schmidt, Thomas J. Mcconnell, Donald R. HoffmanAbstract:Abstract Background: The complementary DNA encoding for the importAnt imported fire Ant Venom allergen, Sol i 2, has previously been cloned. The binding of human IgE Antibodies to Sol i 2 has been demonstrated to be conformation-dependent. Methods: A complete cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinAnt baculovirus was isolated by plaque purification. The recombinAnt baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinAnt Sol i 2 was isolated and characterized. Results: RecombinAnt (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 μg/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was identical to that of the natural molecule. No significAnt increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal Antibody was lost by heating in sodium dodecylsulfate and reduction. Conclusions: A recombinAnt Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinAnt protein is produced with the native conformation. (J ALLERGY CLIN IMMUNOL 1996;98:82-8.)
Robert J Heddle - One of the best experts on this subject based on the ideXlab platform.
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stability of myrmecia pilosula jack jumper Ant Venom for use in immunotherapy
Journal of Pharmaceutical and Biomedical Analysis, 2011Co-Authors: Simon G A Brown, Michael D Wiese, Robert J Heddle, Noel W Davies, Tim Chataway, Robert W MilneAbstract:Allergy to Myrmecia pilosula (Jack Jumper Ant) Venom is common in Australia, affecting ∼2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the Venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant Venom must be demonstrated. HPLC–UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess Venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pH of 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100ug/mL Venom, but improved stability at concentrations of 1ug/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilising. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant Venom was stable at −18 C and 4 C for 12 months; following dilution to 100ug/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), Venom was stable for 7 days when stored at 4 ◦C. Concentrated Jack Jumper Ant Venom can be stored in 22% sucrose for 12 months, and after dilution to 100ug/mL for clinical use, it should be discarded after 7 days.
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myrmecia pilosula jack jumper Ant Venom validation of a procedure to standardise an allergy vaccine
Journal of Pharmaceutical and Biomedical Analysis, 2008Co-Authors: Simon G A Brown, Michael D Wiese, Robert J Heddle, Noel W Davies, Tim Chataway, Robert W MilneAbstract:Ant sting allergy is relatively common within south-eastern Australia and is predominAntly due to Myrmecia pilosula (Jack Jumper Ant, JJA). Venom immunotherapy has been shown to be effective in preventing anaphylaxis to the sting of the JJA, but analytical techniques to standardise the Venom have not been validated. The purpose of this study was to develop assays to analyse JJA Venom and apply these to the standardisation of Venom prior to new batches being used for the diagnosis and treatment of JJA sting allergy. Venom was analysed by protein content, HPLC-UV, enzyme-linked immunosorbent assay (ELISA) inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE immunoblot. The protein content in JJA Venom was adjusted so that all batches were equivalent. A HPLC-UV assay was used to quAntify the relative amount of the major allergen Myr p 2 and two minor allergens Myr p 1 and Myr p 3 and allergenic potency was determined by ELISA inhibition. SDS-PAGE and SDS-PAGE immunoblot were used as qualitative tools to determine the protein profile and presence or absence of additional high molecular weight allergens not quAntifiable by HPLC-UV. A standardisation procedure has been developed that complies with the requirements described in the European Pharmacopoeia. Techniques used to determine the content of some of the other minor allergens could be developed, which would further improve the standardisation methodology.
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proteomic analysis of myrmecia pilosula jack jumper Ant Venom
Toxicon, 2006Co-Authors: Simon G A Brown, Michael D Wiese, Noel W Davies, Tim Chataway, Robert W Milne, Wei Ping Gai, Robert J HeddleAbstract:Ant sting allergy in Australia is predominAntly due to the Myrmecia pilosula species complex. Gel separation of M. pilosula Venom is necessary so that the allergenic importance of each component can be defined by western blotting. However, previous PAGE methods produced suboptimal resolution and the components of each band were not precisely defined. Venom was resolved in both non-reduced and reduced form by one-dimensional acid urea PAGE, SDS-PAGE and two-dimensional acid urea-SDS PAGE. Resolved peptides were extracted and analysed by HPLC–MS. Acid urea PAGE and acid urea-SDS PAGE proved more effective than SDS-PAGE for resolution of peptides smaller than 10 kDa. All of the major peptides previously observed in M. pilosula Venom were observed in gel resolved Venom. Venom was found to primarily consist of peptides with molecular weight <10 kDa, most of which contain disulfide bridges. SDS-PAGE of non-reduced Venom clearly defined six higher molecular weight proteins between 26 and 90 kDa. An 8546 Da dimer named pilosulin 5 was observed, but pilosulin 4, a peptide recently proposed to be present in Venom was not. A variAnt of pilosulin 4 here named pilosulin 4.1a, existing as an 8198 Da dimer, was observed and has been characterised.
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prevention of anaphylaxis with Ant Venom immunotherapy
Current Opinion in Allergy and Clinical Immunology, 2003Co-Authors: Simon G A Brown, Robert J HeddleAbstract:Purpose of reviewWorldwide, eight genera of Ants have been associated with sting allergy. Until recently only whole Ant body extracts have been used for immunotherapy. The purpose of this review is to examine recent advances in the understanding of Ant Venom allergy and treatment using Venom immunot
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Ant Venom immunotherapy a double blind placebo controlled crossover trial
The Lancet, 2003Co-Authors: Simon G A Brown, Michael D Wiese, Konrad E Blackman, Robert J HeddleAbstract:Summary Background The jack jumper Ant Myrmecia pilosula is responsible for about 90% of Ant Venom anaphylaxis in southeastern Australia. We aimed to establish whether M pilosula Venom immunotherapy (VIT) prevents lifethreatening sting anaphylaxis in otherwise healthy adults. Methods We did a double-blind, placebo-controlled crossover trial of M pilosula VIT. ParticipAnts were randomly allocated either immunotherapy, in accordance with the semirush hyposensitisation regimen, or placebo. The primary endpoint was systemic reaction after a deliberate sting challenge. Analysis was per protocol. Findings We randomly allocated 68 healthy volunteers (aged 20–63 years) who were allergic to M pilosula Venom to placebo (33) and VIT (35). Four on placebo were stopped early and 12 on VIT had their treatment allocations revealed before the sting challenge, thus 29 on placebo and 23 on VIT were included in the primary analysis. Objectively defined systemic reactions to sting challenges arose in 21 of 29 participAnts (72%) on placebo (8 reactions were associated with hypotension) and none of 23 on VIT (p Interpretation In well motivated, highly allergic, but otherwise healthy adults, VIT is highly effective in prevention of M pilosula sting anaphylaxis. The risk of systemic reactions during VIT means that treatment should be given where there is immediate access to resuscitation facilities.
Axel Touchard - One of the best experts on this subject based on the ideXlab platform.
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heterodimeric insecticidal peptide provides new insights into the molecular and functional diversity of Ant Venoms
bioRxiv, 2020Co-Authors: Axel Touchard, Helen C Mendel, Isabelle Boulogne, Volker Herzig, Braga N Emidio, Glenn F King, Mathilde Triquigneaux, Lucie Jaquillard, Remy BeroudAbstract:Ants use Venom for predation, defence and communication, however, the molecular diversity, function and potential applications of Ant Venom remains understudied compared to other Venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modelling, stability studies, and a series of in vitro and in vivo bioassays to investigate the molecular diversity of the Venom of the Amazonian Pseudomyrmex penetrator Ants. We isolated a potent insecticidal heterodimeric peptide {Delta}-pseudomyrmecitoxin-Pp1a ({Delta}-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain crosslinked by two disulfide bonds in an Antiparallel orientation. We chemically synthesised {Delta}-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that {Delta}-PSDTX-Pp1a had the most potent insecticidal effects in blow fly assays (LD50 = 3 nM). Molecular modelling and circular dichroism studies revealed strong alpha-helical features, indicating its cytotoxic effects could derive from membrane disruption, which was further supported by insect cell calcium assays. The native heterodimer was also substAntially more stable against proteolytic degradation (t1/2 =13 h) than its homodimers or monomers (t1/2 <20 min), indicating an evolutionary advAntage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator Venom and in-depth characterisation of {Delta}-PSDTX-Pp1a provide novel insights in the structural complexity of Ant Venom, and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advAntage via improved stability; a concept that is also highly relevAnt for the design and development of peptide therapeutics, molecular probes and bioinsecticides.
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An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.
Toxins, 2020Co-Authors: Samira R. Aili, Axel Touchard, Regan J. Hayward, Samuel D. Robinson, Sandy S. Pineda, Hadrien Lalagüe, Mrinalini, Irina Vetter, Eivind A. B. Undheim, R. Manjunatha KiniAbstract:A critical hurdle in Ant Venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of Venom proteins and peptides. To resolve this, we used Venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of Venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the Venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
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an integrated proteomic and transcriptomic analysis reveals the Venom complexity of the bullet Ant paraponera clavata
Toxins, 2020Co-Authors: Samira R. Aili, Axel Touchard, Regan J. Hayward, Samuel D. Robinson, Sandy S. Pineda, Hadrien Lalagüe, Irina Vetter, Eivind A. B. UndheimAbstract:A critical hurdle in Ant Venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of Venom proteins and peptides. To resolve this, we used Venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of Venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the Venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata Venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
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Venom peptide repertoire of the european myrmicine Ant manica rubida: identification of insecticidal toxins
Journal of Proteome Research, 2020Co-Authors: Axel Touchard, Samira R. Aili, Mrinalini, R. Manjunatha Kini, Alain Dejean, Nathan Téné, Valentine Barasse, Christophe Klopp, Laurent Coquet, Thierry JouenneAbstract:Using an integrated transcriptomic and proteomic approach, we characterized the Venom peptidome of the European red Ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified Ant Venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC Venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic Venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.
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the peptide Venom composition of the fierce stinging Ant tetraponera aethiops formicidae pseudomyrmecinae
Toxins, 2019Co-Authors: Valentine Barasse, Axel Touchard, Alain Dejean, Nathan Téné, Elsa Bonnafé, Maurice Tindo, Martin Kenne, Christophe Klopp, Michel TreilhouAbstract:In the mutualisms involving certain pseudomyrmicine Ants and different myrmecophytes (i.e., plAnts sheltering colonies of specialized “plAnt-Ant” species in hollow structures), the Ant Venom contributes to the host plAnt biotic defenses by inducing the rapid paralysis of defoliating insects and causing intense pain to browsing mammals. Using integrated transcriptomic and proteomic approaches, we identified the Venom peptidome of the plAnt-Ant Tetraponera aethiops (Pseudomyrmecinae). The transcriptomic analysis of its Venom glands revealed that 40% of the expressed contigs encoded only seven peptide precursors related to the Ant Venom peptides from the A-superfamily. Among the 12 peptide masses detected by liquid chromatography-mass spectrometry (LC–MS), nine mature peptide sequences were characterized and confirmed through proteomic analysis. These Venom peptides, called pseudomyrmecitoxins (PSDTX), share amino acid sequence identities with myrmeciitoxins known for their dual offensive and defensive functions on both insects and mammals. Furthermore, we demonstrated through reduction/alkylation of the crude Venom that four PSDTXs were homo- and heterodimeric. Thus, we provide the first insights into the defensive Venom composition of the Ant genus Tetraponera indicative of a streamlined peptidome.