Honeybee Venom Research Articles

Recombinant Expression and Chemical Amidation of Isotopically Labeled Native Melittin.

Post-translational modifications are ubiquitous in the eukaryotic proteome. However, these modifications are rarely incorporated in NMR studies of eukaryotic proteins, which are typically produced through recombinant expression in E. coli. Melittin is the primary peptide in honey bee venom. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and is thus a prerequisite for studying its native structure and function. Here, we present a method for producing triply isotopically labeled (2H, 13C, and 15N) native melittin through recombinant expression followed by chemical amidation. We then show that structural models produced with AlphaFold-Multimer are in even better agreement with experimental residual dipolar couplings than the 2.0 Å resolution X-ray crystal structure for residues G3-K23.

Identification of waprin and its microbicidal activity: A novel protein component of honeybee (Apis mellifera) venom

Bee venom is a rich source of biologically and pharmacologically active proteins. Waprin is a protein component of venoms; however, waprin has yet to be identified in bee venom. Moreover, the biological functions of waprin in venoms remain poorly characterized. Thus, in this study, we have identified and characterized waprin: a novel protein component from the venom of honeybees (Apis mellifera). The waprin in A. mellifera venom (Amwaprin) was found to consist of an 80-amino acid mature peptide, in which the whey acidic protein domain contains four conserved disulfide bonds. We discovered the presence of the Amwaprin protein in secreted venom by using an antibody against recombinant Amwaprin produced in baculovirus-infected insect cells. Recombinant Amwaprin exhibited inhibitory activity against microbial serine proteases and elastases but not thrombin or plasmin. It recognized carbohydrates in the microbial cell wall molecules and bound to the live microbial surfaces. The binding action of Amwaprin produced its microbicidal activity by inducing structural damage to bacterial and fungal cell walls. In addition, recombinant Amwaprin is heat-stable and contains no hemolytic activity. These findings demonstrate that Amwaprin acts as a microbicidal and anti-elastolytic agent.

Varied mechanisms for the neutralization of biological toxins using natural products.

Natural proteinaceous toxins in the pore-forming class and misfolded protein oligomers can interact with and perturb cell membranes, culminating in the death of the cell. Finding antidotes for such biologically toxic species is important towards the development of medical countermeasures for potential biological threat agents and treatments for neurodegenerative diseases. Using small molecule natural products, we have identified diverse mechanisms that result in the neutralization of model pore-forming toxins, including melittin from honeybee venom and alpha-hemolysin from Staphylococcus aureus. Through a combination of biophysical studies to interrogate the physico-chemical properties of the toxins in the presence of natural products and tissue culture experiments, we show that specific aminosterols and tea catechins act as therapeutics for these toxins. In particular, aminosterols attenuated toxin activity by targeting the cell membrane rather than the toxin directly, while catechins modulated the size-hydrophobicity-toxicity relationship of the proteins. These findings draw parallels to the mechanisms by which certain small molecules, including aminosterols, can suppress the toxicity caused by endogenous aggregates of misfolded proteins in a range of neurodegenerative diseases, in particular Alzheimer's and Parkinson's diseases, and they highlight modes by which biological toxins can be neutralized by natural products.

Tuning the size and solvent-exposed hydrophobicity of a pore-forming peptide eliminates its cytotoxicity using a natural product.

Pore-forming toxins (PFTs) are a diverse group of peptides and proteins that interact with the cell membrane to form transmembrane pores which lead to to ion dyshomeostasis and cell death. Our previous work showed that claramine, an aminosterol derivative of cholesterol, protects brain and kidney cells from prototypical PFTs. This effect was not induced by a change in protein structure, but instead by incorporating into and changing the physico-chemical properties of the plasma membrane to prevent the binding of these toxins. In the search for compounds that can target the structures of PFTs directly to eliminate their toxicity, we found that the polyphenol epigallocatechin gallate (EGCG) directly interacts with the monomeric PFT melittin, the active component of honeybee venom, resulting in the elimination of its binding to the cell membrane and neutralization of its cytotoxicity. EGCG achieved this effect by lowering the solvent-exposed hydrophobicity of melittin and promoting its oligomerization into larger aggregates. These biophysical properties have also shown to play an important role in the membrane binding and associated cytotoxicity of misfolded protein oligomers implicit in neurodegenerative diseases. Our results show that this relationship translates to the attenuation of melittin cytotoxicity by EGCG. These findings emphasize the importance of the physico-chemical properties of PFTs in controlling their binding to cell membranes and suggest a potential mechanism for therapeutic development against PFTs.

Polymer-Antimicrobial Peptide Constructs with Tailored Drug-Release Behavior

Microbial resistance is one of the main problems of modern medicine. Recently, antimicrobial peptides have been recognized as a novel approach to overcome the microbial resistance issue, nevertheless, their low stability, toxicity, and potential immunogenic response in biological systems have limited their clinical application. Herein, we present the design, synthesis, and preliminary biological evaluation of polymer-antibacterial peptide constructs. The antimicrobial GKWMKLLKKILK-NH2 oligopeptide (PEP) derived from halictine, honey bee venom, was bound to a polymer carrier via various biodegradable spacers employing the pH-sensitive or enzymatically-driven release and reactivation of the PEP's antimicrobial activity. The antibacterial properties of the polymer-PEP constructs were assessed by a determination of the minimum inhibitory concentrations, followed by fluorescence and transmission electron microscopy. The PEP exerted antibacterial activity against both, gram-positive and negative bacteria, via disruption of the bacterial cell wall mechanism. Importantly, PEP partly retained its antibacterial efficacy against Staphylococcus epidermidis, Escherichia coli, and Acinetobacter baumanii even though it was bound to the polymer carrier. Indeed, to observe antibacterial activity similar to the free PEP, the peptide has to be released from the polymer carrier in response to a pH decrease. Enzymatically-driven release and reactivation of the PEP antimicrobial activity were recognized as less effective when compared to the pH-sensitive release of PEP.

Increased bee venom production in Apis mellifera workers on the provision of probiotics and organic acids

Bee venom of honeybees has great importance in Apitherapy. It cures amyotrophic lateral sclerosis, diseases related to central nervous system (CNS), rheumatoid arthritis and inflammations. The potential of honeybee's can be exploited by improving their diets. Recently, the use of synbiotics (simultaneous use of probiotics and prebiotics) has been increased as apipromoters. The current research study was conducted to evaluate the potential of probiotics (Lactobacillus rhamnosus, Lactobacillus brevis and Bacillus clausii) and acidifying agents (Lactic acid and acetic acid) on development of bee sting and its associated gland. Changes in the morphology of this gland affect the amount of venom produced. Significant increases in stings lengths were observed when honeybees were fed with (Pollen + L. rhamnosus in 50% (w/v) sucrose in distilled water) and (Pollen + L. rhamnosus in 50% (w/v) sucrose in 2.91% acetic acid) with mean values of 2.36 ± 0.04 and 2.38 ± 0.05 respectively compared with control's 2.35 ± 0.06 were observed. Significant increase in sting width was observed when bees were fed with (Pollen + B. clausii in 50% w/v sucrose in 2.99% lactic acid), (Pollen + B. clausii in 50% w/v sucrose in1.96% acetic acid), (Pollen + L. brevis in 50% w/v sucrose in distilled water) and (Pollen + L. brevis in 50% w/v sucrose in 2.99% lactic acid) with mean values of 1.84 ± 0.07, 1.79 ± 0.05, 1.87 ± 0.04, 1.78 ± 0.04 compared to control's 1.71 ± 0.03. In addition to this, width of stylet also increased significantly when bees were provided with (Pollen + B. clausii in 50% w/v sucrose in distilled water), (Pollen + B. clausii in 50% w/v sucrose in 2.99% lactic acid), (Pollen + B. clausii in 50% w/v sucrose in 2.91% acetic acid), (Pollen + B. clausii in 50% w/v sucrose in1.96% acetic acid), (Pollen + L. brevis in 50% w/v sucrose in distilled water), (Pollen + L. brevis in 50% w/v sucrose in 2.99% lactic acid), (Pollen + L. brevis in 50% w/v sucrose in 2.91% acetic acid), and (Pollen + L. brevis in 50% w/v sucrose in1.96% acetic acid) with mean values of 65.00 ± 1.58, 57.50 ± 3.23, 62.00 ± 2.00, 65.00 ± 0.00, 65.00 ± 1.58, 60.00 ± 1.58 compared to control's 55.00 ± 3.54. Rest of the observed parameters also depicted reasonable increases. Supplementation of honeybee's feed with organic acids and probiotics resulted in overall sting development.

A simple self-assembling system of melittin for hepatoma treatment

BackgroundHepatoma is a serious public health concern. New attempts are urgently needed to solve this problem. Melittin, a host defense peptide derived from the venom of honeybees, has noteworthy hemolysis and non-specific cytotoxicity in clinical applications. Here, the self-assembly of melittin and vitamin E-succinic acid-(glutamate)12 (VG) was fabricated via noncovalent π-stacking and hydrogen bonding interactions using an environment-friendly method without “toxic” solvents.ResultsAs expected, the designed self-assembly (denoted as M/VG nanoparticles) exhibits a uniform morphology with a particle size of approximately 60 nm and a zeta potential of approximately − 26.8 mV. Furthermore, added VG significantly decreased hemolytic activity, increased tumor-targeted effects, and accelerated apoptosis.ConclusionOur research provides a promising strategy for the development of natural self-assembled biological peptides for clinical application, particularly for transforming toxic peptides into safe therapeutic systems.Graphical

An Overview on the Effects of Propolis Administration in Different Branches of Livestock Production

Recently, the increase in the awareness of nutrition with natural products has gradually increased the popularity of honey bee products. Popularity of honey bee products like honey pollen, propolis, bee bread, royal jelly, bee venom and apilarnil are beekeeping products. Propolis is one of the most popular bee product and has anticancer, anti-inflammatory, antibiotic, antioxidative, antibacterial, antiviral, antifungal, anesthetic, immunostimulant and cytotoxic effects. Approximately 300 compounds have been identified in the content of propolis, including polyphenols, phenolic aldehydes, sequiterpene quinines, coumarins, amino acids, steroids, and inorganic compounds. These properties have been used in folk medicine for centuries. The use of propolis in the livestock sector has become popular with the restriction on the use of antibiotics and synthetic drugs in this sector. Various studies were carried out to determine the effetcs of propolis administration in different branches of livestock sector. This study aimed to reveal the effects of propolis administration in animal husbandry by analyzing the current studies and provide brief information on the subject.

Characterization of honey bee venom from Spilje, Debar region, N. Macedonia

Characterization of honey bee venom from Spilje, Debar region, N. Macedonia

Isolation and Antibacterial Activity of Honey Bee Venom Bioactive from Apis cerana

Honey bee venom from Apis species has garnered considerable attention in medicine due to its bioactive, which have antimicrobial activity. This research aims to isolate bioactive melittin and phospholipase A2 (PLA2) of honey bee venom from Indonesian Apis cerana and examine the antibacterial activity of these bioactive. Honey bee venoms were isolated from four locations using an electric shock method and purified using Fast Protein Liquid Chromatography. Purified protein was analyzed by SDS PAGE and measured by the Lowry method. Further, the antibacterial activity of melittin and PLA2 against Gram-positive (Staphylococcus aureus and Salmonella typhi) and Gram-negative (Escherichia coli) bacteria was defined by inhibition zone. Our results show that the average of all bee venoms obtained from four locations with eight sampling times was 17.1 mg proteins. Melittin was purified using gradient steps with 55%, 90%, and 100% elution buffer, while PLA2 was purified using 55% elution buffer. Melittin and PLA2 showed molecular weights of less than 7.5 kDa and 19 kDa, respectively. Protein concentrations in melittin and PLA2 were 95

EGCG inactivates a pore-forming toxin by promoting its oligomerization and decreasing its solvent-exposed hydrophobicity

Natural proteinaceous pore-forming agents can bind and permeabilize cell membranes, leading to ion dyshomeostasis and cell death. In the search for antidotes that can protect cells from peptide toxins, we discovered that the polyphenol epigallocatechin gallate (EGCG) interacts directly with melittin from honeybee venom, resulting in the elimination of its binding to the cell membrane and toxicity by markedly lowering the extent of its solvent-exposed hydrophobicity and promoting its oligomerization into larger species. These physicochemical parameters have also been shown to play a key role in the binding to cells of misfolded protein oligomers in a host of neurodegenerative diseases, where oligomer-membrane binding and associated toxicity have been shown to correlate negatively with oligomer size and positively with solvent-exposed hydrophobicity. For melittin, which is not an amyloid-forming protein and has a very distinct mechanism of toxicity compared to misfolded oligomers, we find that the size-hydrophobicity-toxicity relationship also rationalizes the pharmacological attenuation of melittin toxicity by EGCG. These results highlight the importance of the physicochemical properties of pore forming agents in mediating their interactions with cell membranes and suggest a possible therapeutic approach based on compounds with a similar mechanism of action as EGCG.

Honey bee venom melittin increases the oxidant activity of cisplatin and kills human glioblastoma cells by stimulating the TRPM2 channel

Melittin (MLT) treatment is believed to enhance tumor cell death, apoptotic, and oxidative cytotoxic effects of cisplatin (CSP) via the modulation of Ca2+ channels in several cancer lines. The activation of TRPM2 mediated anticancer and CSP resistance actions via mitochondrial Ca2+ and Zn2+ accumulation-induced mitochondrial reactive free oxygen species (MitSOX) in the glioblastoma cells. The aim was to elucidate the effects of CSP and MLT combination via the TRPM2 stimulation on the tumor cell viability, cell number, cell death (propidium iodide/Hoechst rate), apoptosis, and MitSOX levels in the DBTRG-05MG cells.In the DBTRG-05MG cells, we induced four groups as control, MLT (2.5 μg/ml for 24 h), CSP (25 μM for 24 h), and CSP + MLT.The CSP-induced intracellular Ca2+ influxes to the TRPM2 activation were increased in the cells from coming H2O2 and ADP-Ribose. The influxes were decreased in the cells by the incubations of TRPM2 antagonists (ACA and carvacrol). The incubation of CSP increased the parameters of intracellular Ca2+ responses, mitochondria function, cytosolic free Zn2+ accumulation, apoptosis (caspase −3, −8, and −9), and MitSOX generation in the tumor cells. After the treatment of MLT with/without CSP, the parameters were further increased in the cells.In conclusion, the treatment of MLT increased the anticancer, tumor cell death, apoptotic, and oxidant effects of CSP in the glioblastoma tumor cells via activating the TRPM2. As a result, TRPM2 stimulation by MLT may be utilized as a successful agent in the CSP treatment of glioblastoma tumors.

Bioengineered peptibodies as blockers of ion channels

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

The Pharmacological Potential of Novel Melittin Variants from the Honeybee and Solitary Bees against Inflammation and Cancer.

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.

Antifungal and antibiofilm activities of bee venom loaded on chitosan nanoparticles: a novel approach for combating fungal human pathogens

The prevalence of opportunistic human fungal pathogens is increasing worldwide, and antimicrobial resistance is one of the greatest medical challenges the world faces. Therefore, this study aimed to develop a novel agent to control fungal pathogens. The honeybee products (honey, royal jelly, propolis, bee bread, and bee venom) were screened against unicellular fungal (UCF) pathogens (Cryptococcus neoformans, Kodamaea ohmeri, and Candida albicans) and the bee venom was only exhibited an inhibitory effect against them. The protein contents of crude bee venom were separated using the gel filtration technique into eight fractions which were visualized on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to confirm the presence of five bands with molecular weights of 65, 43, 21, 15, and 3 KDa. Bee venom (BV) of Apis mellifera loaded chitosan nanoparticles were prepared by the ionotropic gelation method. The encapsulation efficiency%, average size, zeta potentials, and surface appearance by Transmission electron microscope (TEM) were evaluated for the prepared nanoparticles. The minimum inhibitory concentration (MIC) of crude BV and BV loaded chitosan nanoparticles (BV-CNPs) was evaluated against the offer mentioned UCF where the MIC values of crude BV were 6.25, 3.12 & 6.25 while MIC values in the case of BV-CNPs were decreased to 3.12, 3.12 & 1.56 mg/ml against C. neoformans, K. ohmeri and C. albicans, respectively. Also, the results showed that BV-CNPs suppressed the biofilm formation as well as yeast to hyphal transition formed by the examined UCF. These results revealed that BV-CNPs are a promising natural compound for fungal pathogens treatment.Graphical abstract

Apis mellifera Venom Inhibits Bacterial and Fungal Pathogens in vitro.

<b>Background and Objective:</b> Bacterial and fungal infections are major public health problems. Emerging of drug-resistant microbial strains urges the need for the development of alternative untraditional antimicrobial agents. Bee venom is a rich source of secondary metabolites and antimicrobial agents. In this study, the antimicrobial and antifungal potential of <i>Apis mellifera</i> BV (<i>Am</i>BV) against some medically important bacterial and fungal pathogens was investigated. <b>Materials and Methods:</b> Broth microdilution method and Colony Forming Unit (CFU) assay were used to screen the antibacterial potential of <i>Am</i>BV. Similarly, the antifungal activity of <i>Am</i>BV was evaluated using the agar-well diffusion assay. Moreover, the minimum inhibitory concentration (MIC) values of <i>Am</i>BV against tested microorganisms were determined. <b>Results:</b> <i>Am</i>BV significantly inhibited bacterial and fungal growth. The MIC values of <i>Am</i>BV were 15.625, 31.25, 7.8, 7.8 μg mL<sup></sup><sup>1</sup> against <i>Escherichia coli</i> ATCC 8739, <i>Staphylococcus aureus</i> ATCC 6538P, <i>Serratia marcescens</i> AUH 98 and <i>Streptococcus mutans</i> ATCC 25175, respectively. Similarly, <i>Am</i>BV at concentrations of 300 and 600 μg mL<sup></sup><sup>1</sup> significantly inhibited the growth of <i>Aspergillus niger</i> ATCC 16404, <i>Alternaria alternata</i> MLBM09, <i>Fusarium oxysporum </i>MLBM212 and <i>Aspergillus flavus. </i><b>Conclusion:</b> These results indicated that<i> Am</i>BV could be used in future preclinical and clinical studies to develop cost-effective and efficient antibacterial and antifungal agents. Moreover, this study presents <i>Am</i>BV as an efficient alternative antimicrobial agent against medically important pathogens.

Effect of honey bee venom on the histological changes of testes and hormonal disturbance in diabetic mice.

Hyperglycemia associated with hyper- or hypo-insulinemia is a hallmark of type 2 diabetes mellitus, which is firmly linked to decreased male infertility. Recently, bee venom (BV) has shown potential health prosperities, including antidiabetic; however, no study focuses on the effect of BV on male fertility in diabetic conditions. This study aimed to detect the effect of BV on histological and hormonal alteration of the testis in diabetic mice. Twenty adult male mice were selected and assigned to four groups: Control, diabetic (150 mg/kg alloxan), BV1 (diabetic + 0.5 mg/kg BV), and BV2 (diabetic + 1 mg/kg BV). After 35 days, the serum levels of glucose, insulin, testosterone, follicular-stimulating hormone, luteinizing hormone, and prolactin were estimated. The histological structure of the testes was also evaluated. Alloxan-induced hyperglycemia and decreased insulin concentrations were reversed significantly by BV. Furthermore, diabetic mice exhibited various alterations in fertility hormones, while these disturbances were improved considerably to normal concentrations by BV. Similarly, alloxan-induced changes in sperm and testis histological parameters such as motility, viability, abnormality, sperm count, the number and diameter of seminiferous tubules, and the number of Leydig and Sertoli cells were significantly ameliorated to the normal condition by BV. Changes in the number, size, and shape of seminiferous tubules, the number of Leydig and Sertoli cells, and initial degeneration and vacuolization in interstitial cells and spermatogonia and spermatocyte were seen in diabetic mice. All these changes were shifted almost to normal structure by BV. The BV could be used as an alternative therapeutic agent that manages the markers related to diabetic conditions concomitant with the improved histological structure of the testes and hormone production to accelerate male fertility.

Adverse reactions in venom immunotherapy protocols: conventional versus ultra-rush

Background Venom immunotherapy (VIT) is an effective treatment in the patients at high risk of anaphylaxis or life-threatening systemic reactions due to Hymenoptera venom allergy. But, systemic and large local reactions can be observed, especially during the build-up phase of VIT. We evaluated the safety of conventional and ultra-rush build-up protocols. Materials and methods Two protocols in 71 patients (39 conventional and 32 ultra-rush protocols) with honeybee and wasp venom allergy were evaluated retrospectively. Patients were diagnosed and selected for VIT according to the criteria established by the European Academy of Allergy and Clinical Immunology. The severity of systemic reactions was evaluated according to the criteria of Mueller. Results Build-up phases were tolerated in 66.2% (n = 47) without any reaction. Allergic adverse reactions were observed in 33.8% (n = 24): large local reactions 22.5% (n = 16) and systemic reactions 11.3% (n = 8). There was no significant difference in the number of adverse reactions comparing patients receiving conventional and ultra-rush protocol. In addition, no association was found between allergic adverse reactions and the following factors: sex, previous systemic sting reactions, honeybee and wasp venom extract. Conclusion We found that both protocols were tolerated in patients with honeybee and wasp venom allergy. Ultra-rush protocol will be preferred for patients and clinicians because of its advantages in terms of time and costs. KEY MESSAGES VIT is the only curative treatment method that reduces the risk of severe reactions after a bee sting and improves the quality of life in patients with Hymenoptera venom allergy. Ultra-rush VIT protocol has advantages such as few injection and time savings. Both ultra-rush and conventional VIT are safe treatments to prevent potentially life-threatening reactions in patients with honeybee and wasp venom allergy.

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.

Antimicrobial activity of major royal jelly protein 8 and 9 of honeybee (Apis mellifera) venom

• Apis mellifera has major royal jelly protein 8 and 9 (AmMRJP 8 and 9) in its venom. • AmMRJP 8 and 9 inhibit microbial serine proteases. • AmMRJP 8 and 9 exhibit antibacterial and antifungal activities. • AmMRJP 8 and 9 act as antimicrobial agents in honeybee venom. Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee ( Apis mellifera ) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom.