Honeybee Venom Research Articles

Honeybee venom: influence of collection on quality and cytotoxicity

ABSTRACT: Apitoxin is the venom produced by bees. It is a complex chemical compound, rich in protein substances and with pharmacological effects. This study was carried out with the objective of comparing the quality of apitoxin extracted in an apiary in different parts of the collector in relation to moisture content, protein analysis and cytotoxicity assay with Artemia salina L. Type 1 apitoxin was collected from glass slabs at the entrance to the hive, while type 2 apitoxin was collected from the waste accumulated in the collection rods and treated by rinsing in distilled water. Both apitoxins presented significant differences (P>0.05) in relation to protein profile, with type 1 showing a higher content (77.8%) than type 2 (51.9%), and presented polypeptide bands with more than 50% of their nitrogenous components having molecular weight below 10KDa. Regarding cytotoxicity assays, type 1 apitoxin had LD50 of 71.5μg mL-1, while type 2 had LD50 of 191.6μg mL-1. Thus, the region where apitoxin accumulates in the collector does influence the product quality if moisture and protein contents are in accordance with the standards recommended in specific legislation, and so it can be commercialized by the beekeeper.

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

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

Wound healing: time to look for intelligent, \u2018natural\u2019 immunological approaches?

There is now good evidence that cytokines and growth factors are key factors in tissue repair and often exert anti-infective activities. However, engineering such factors for global use, even in the most remote places, is not realistic. Instead, we propose to examine how such factors work and to evaluate the reparative tools generously provided by ‘nature.’ We used two approaches to address these objectives. The first approach was to reappraise the internal capacity of the factors contributing the most to healing in the body, i.e., blood platelets. The second was to revisit natural agents such as whey proteins, (honey) bee venom and propolis. The platelet approach elucidates the inflammation spectrum from physiology to pathology, whereas milk and honey derivatives accelerate diabetic wound healing. Thus, this review aims at offering a fresh view of how wound healing can be addressed by natural means.

Evaluation of the skin phototoxicity and photosensitivity of honeybee venom.

Bee (Apis mellifera L.) venom (BV) has been used as a cosmetic ingredient owing to its anti-aging, anti-inflammatory, and antibacterial effects. The aim of this study was to assess the skin safety of BV. For this purpose, skin phototoxicity and sensitization tests were conducted in healthy male Hartley guinea pigs. The animals were divided into three groups (n=5) for the phototoxicity test: G1 (negative control), G2 (BV gel treatment), and G3 (positive control). After specified treatments, the animals were irradiated with ultraviolet A (15 J/cm2 ). The photosensitivity test was also performed in three groups: G4 (negative control, n=5), G5 (BV gel treatment, n=10), and G6 (positive control, n=5). Erythema and edema were observed after 24, 48, and 72 hours in the positive control group, but not in the negative control and BV gel groups. Application of BV to the guinea pig skin had no toxic effects on any clinical signs, body weight, or mortality. In addition, it did not evoke a skin reaction in both either the skin phototoxicity and skin photosensitization tests. Therefore, it can be concluded that BV has the potential to be developed as a drug ingredient for topical uses.

Component Resolved Diagnosis in Hymenoptera Anaphylaxis

Hymenoptera anaphylaxis is one of the leading causes of severe allergic reactions and can be fatal. Venom-specific immunotherapy (VIT) can prevent a life-threatening reaction; however, confirmation of an allergy to a Hymenoptera venom is a prerequisite before starting such a treatment. Component resolved diagnostics (CRD) have helped to better identify the responsible allergen. Many new insect venom allergens have been identified within the last few years. Commercially available recombinant allergens offer new diagnostic tools for detecting sensitivity to insect venoms. Additional added sensitivity to nearly 95% was introduced by spiking yellow jacket venom (YJV) extract with Ves v 5. The further value of CRD for sensitivity in YJV and honey bee venom (HBV) allergy is more controversially discussed. Recombinant allergens devoid of cross-reactive carbohydrate determinants often help to identify the culprit venom in patients with double sensitivity to YJV and HBV. CRD identified a group of patients with predominant Api m 10 sensitization, which may be less well protected by VIT, as some treatment extracts are lacking this allergen. The diagnostic gap of previously undetected Hymenoptera allergy has been decreased via production of recombinant allergens. Knowledge of analogies in interspecies proteins and cross-reactive carbohydrate determinants is necessary to distinguish relevant from irrelevant sensitizations.

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

ObjectiveTo review allergenic extracts used to diagnose or treat insect allergies, including how the extracts are manufactured and their measurements of potency or concentration. Data SourcesPeer-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 SelectionsSearch terms used for the PubMed searches included insect allergens and allergies, Apidae, Vespidae, fire ants, cockroach allergies, insect allergen extract preparation, and standardization. ResultsHumans 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. ConclusionSensitization 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.

Cellular toxicity and biological activities of honey bee (Apis mellifera L.) venom

Ari zehri kanser dahil cesitli hastaliklar icin bir apiterapi araci olarak onerilmektedir. Ancak, ari zehrinin etki mekanizmasi ve tumorojenik ve tumorojenik olmayan hucreler uzerindeki toksisitesi cok fazla anlasilamamistir. Biz burada bal arisi (Apis mellifera L.) zehrinin tumorojenik olmayan, tumorojenik olan kanser hucreleri ve Genislemis Spektrumlu Beta Laktamaz ureten Escherichia coli and Vankomisin-direncli Enterococcus faecium gibi coklu direncli patojenler (MDRP) uzerinde antiproliferatif, sitotoksik ve antibakteriyel aktivitesini arastirdik. Ari zehri uygulamasi onemli antiproliferatif, sitotoksik ve antibakteriyel aktivite gostermistir. Bizim sonuclarimiz ari zehrinin sadece kanser hucrelerine degil nontumorojenik hucre hattina da toksik oldugunu gostermektedir. Biz ayrica etki mekanizmasini arastirdigimiz ari zehrinin apoptozisi isaret eden genomik DNA kirilmasina ve hucre gocu inhibisyonuna neden oldugunu gorduk. Immunokimyasal calismalar ari zehrinin Bcl-2 ve P16 ifadelerinde azalmaya neden oldugu gostermistir. Ari zehri test edilen bazi coklu direncli bakterilere karsi antimikrobiyal aktivite gostermistir. Bizim sonuclarimiz malignensilerin tedavisi icin ari zehrinin klinik degerlendirilmesinin normal hucrelere karsi toksik etkisine bagli olarak yeniden degerlendirilmesine ihtiyac oldugunu gostermektedir.

EFFECT OF SOME FACTORS ON HONEY BEE VENOM PRODUCTION FROM DIFFERENT STRAINS

These experiments were carried out at the apiary of honeybee research center of Faculty of Environmental Agricultural Sciences, Arish University, North Sinai, Egypt. To study some factors that affecting on the honey bee venom collection (bee race, season of the year and supplemental protein nutrition) on collected venom quantity .The results showed that the Italian hybrid gave the largest quantity of venom. Spring and summer are the best seasons for collecting the largest quantity of the bee venom. The pollen grain and dried Moringa leaves diets treatments gave the best results for bee venom production comparing with the other treatments (mixture of soybean flour + pollen grains and sugar solution).

Simultaneous Determination of Absolute Configuration and Quantity of Lipopeptides Using Chiral Liquid Chromatography/Mass Spectrometry and Diastereomeric Internal Standards.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

Honey bee venom combined with 1,25-dihydroxyvitamin D3as a highly efficient inducer of differentiation in human acute myeloid leukemia cells.

Most cancer cells exhibit a defect in their capacity to mature into nonreplicating adult cells and existing in a highly proliferating state. Differentiation therapy by agents such as 1,25-dihydroxyvitamin D3(1,25-(OH)2 VD3) represents a useful approach for the treatment of cancer including acute myeloid leukemia. Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-(OH)2 VD3. However, usage of these findings in the clinical trials is limited by calcemic effects of 1,25-(OH)2 VD3. Attempts to overcome this problem have focused on a combination of low concentrations 1,25-(OH)2 VD3 with other compounds to induce differentiation of HL-60 cells. In this study, the effect of honey bee venom (BV) and 1,25-(OH)2 VD3, individually and in combination, on proliferation and differentiation of human myeloid leukemia HL-60 cells were assayed. In this in vitro study, toxic and nontoxic concentrations of BV and 1,25-(OH)2 VD3 were tested using Trypan blue stained cell counting and (3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In addition, differentiation of cells was assayed using a Wright-Giemsa staining and nitroblue tetrazolium reduction test. Data were analyzed by a one-way analysis of the variance test using SPSS software. Our findings showed that both the BV and 1,25-(OH)2 VD3, in a dose and time-dependent manner, caused cell death at high concentrations and inhibited cell proliferation at lower concentrations. About 5 nM of 1,25-(OH)2 VD3 induced differentiation of HL-60 cells to monocytes after 72 h. 2.5 μg/ml of BV suppressed proliferation of HL-60 cells but had not any effects on their differentiation, whereas in combination with 5 nM of 1,25-(OH)2 VD3, it enhanced antiproliferative and differentiation potency of 1,25-(OH)2 VD3. These results indicate that BV potentiates the 1,25-(OH)2 VD3-induced HL-60 cell differentiation into monocytes.

One-Step Purification of Melittin Derived from Apis mellifera Bee Venom.

The concern over the use of melittin in honey bee venom due to its adverse reaction caused by allergens such as phospholipase A2 (PLA2) and hyaluronidase (HYA) has been an obstacle towards its usage. We developed a novel single-step method for melittin purification and the removal of PLA2 and HYA. This study explores the influence of pH, buffer compositions, salt concentration, and types of cation-exchange chromatography resins on the recovery of melittin and the removal of both HYA and PLA2. Melittin was readily purified with a strong cation-exchange resin at pH 6.0 with sodium phosphate buffer. It resulted in a recovery yield of melittin up to 93% (5.87 mg from a total of 6.32 mg of initial melittin in crude bee venom), which is higher than any previously reported studies on melittin purification. PLA2 (99%) and HYA (96%) were also successfully removed. Our study generates a single-step purification method for melittin with a high removal rate of PLA2 and HYA, enabling melittin to be fully utilized for its therapeutic purposes.

Risk factors in Hymenoptera venom allergy.

. Risk factors should be part of the decision, of which patient should be offered venom immunotherapy (VIT) and how VIT should be performed. Risk factors for a severe systemic anaphylactic reaction (SAR) after a Hymenoptera field sting include a preceding less severe sting reaction, a wasp sting, an increased baseline serum tryptase concentration (BSTC), mastocytosis, older age, ACE inhibitor medication, and male gender. During VIT, treatment with honey bee venom is the most important risk factor for a SAR. Further risk factors include a high BSTC (for vespid VIT only), presence of venom specific IgE in serum, any antihypertensive medication during therapy, and an ultra-rush protocol for build-up. Treatment failure is more common in patients suffering from honey bee venom allergy, high BSTC (for vespid VIT only) or mastocytosis, and in those who had experienced side effects during VIT. Besides discontinuing antihypertensive medication or switching to a moderate type of dose increase during build-up, little can be done to minimize the risks associated with VIT. Increasing the maintenance dose may improve the efficacy of VIT. In patients with a particularly high risk for treatment failure, or in case of treatment failure, VIT should include an increased maintenance dose right from the beginning. Usually, 200 µg will be sufficient.

Synthetic secapin bee venom peptide exerts an anti-microbial effect but not a cytotoxic or inflammatory response

Our previous study demonstrated that the secapin peptide from the venom of the Asiatic honeybee (Apis cerana, AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. In the present study, we investigated the anti-microbial activity and cytotoxicity of a synthetic AcSecapin-1 peptide. Seven synthetic AcSecapin-1 peptides (AcSecapin-S1 to AcSecapin-S7) were synthesized based on the peptide sequence of AcSecapin-1. AcSecapin-S1, which consists of the 25-amino acid sequence identical to that of the mature AcSecapin-1 peptide, exhibited the highest anti-microbial activity against bacteria and fungi. This was followed by AcSecapin-S6, which was missing 10 N-terminal amino acids and 6 C-terminal amino acids from AcSecapin-S1. Furthermore, AcSecapin-S1 was not cytotoxic and did not activate macrophages. Taken together, our data demonstrated that a synthetic AcSecapin-1 peptide could exhibit anti-microbial activity without producing a cytotoxic or inflammatory response, suggesting that this synthetic AcSecapin-1 peptide can be used as an anti-microbial agent for biomedical applications.

EFFECT OF SOME TYPES OF PROTEIN NUTRITION ON THE PRODUCTIVITY OF HONEY BEE VENOM

Experiments were conducted in the apiary of Honey Bee Research Center. A field experiment was carried out during 2014 and 2015 seasons, at the Experimental Research Station of the Agriculture Research Center, El-Arish, North Sinai, to study the impact of different sources of nutritive protein on honey bee worker venom gland secretion. The aim of this study is to determine the effects of some factors that could increase the productivity of honeybee venom. This investigation discusses improving the production of the venom from honeybee's colonies by using the electrical impulses technique. This study includes several essential factors, such as using different types of nutrition proteins 1:1 W: W of honey bee to beer yeast or soya beans or natural pollen grain, respectively. Results indicated that the natural pollen grain is the best because it is mainly provide structural elements of muscles, glands and other tissues.

Bee Venom (Apis Mellifera) an Effective Potential Alternative to Gentamicin for Specific Bacteria Strains: Bee Venom an Effective Potential for Bacteria.

Objectives:Mellitine, a major component of bee venom (BV, Apis mellifera), is more active against gram positive than gram negative bacteria. Moreover, BV has been reported to have multiple effects, including antibacterial, antivirus, and anti-inflammation effects, in various types of cells. In addition, wasp venom has been reported to have antibacterial properties. The aim of this study was to evaluate the antibacterial activity of BV against selected gram positive and gram negative bacterial strains of medical importance.Methods:This investigation was set up to evaluate the antibacterial activity of BV against six grams positive and gram negative bacteria, including Staphylococcus aureus (S. aureus), Salmonella typhimurium, Escherichia coli (E. coli) O157:H7, Pseudomonas aeruginosa, Burkholderia mallei and Burkholderia pseudomallei. Three concentrations of crude BV and standard antibiotic (gentamicin) disks as positive controls were tested by using the disc diffusion method.Results:BV was found to have a significant antibacterial effect against E. coli, S. aureus, and Salmonella typhyimurium in all three concentrations tested. However, BV had no noticeable effect on other tested bacteria for any of the three doses tested.Conclusion:The results of the current study indicate that BV inhibits the growth and survival of bacterial strains and that BV can be used as a complementary antimicrobial agent against pathogenic bacteria. BV lacked the effective proteins necessary for it to exhibit antibacterial activity for some specific strains while being very effective against other specific strains. Thus, one may conclude, that Apis mellifera venom may have a specific mechanism that allows it to have an antibacterial effect on certain susceptible bacteria, but that mechanism is not well understood.

Melittin ameliorates CVB3-induced myocarditis via activation of the HDAC2-mediated GSK-3β/Nrf2/ARE signaling pathway

Viral myocarditis (VMC) is characterized as an inflammatory process of the myocardium and can be fatal in infants and children. Melittin is a major polypeptide in honey bee venom that has been traditionally used against inflammation. However, its effect on VMC and the underlying molecular mechanism has not been reported. In this study, BALB/c mice were intraperitoneally injected with CVB3 to build a VMC model and treated with melittin. The results showed that melittin increased the mice's body weight and inhibited CVB3 replication. HE staining also showed that melittin alleviated myocardial injury in the VMC model. Flow cytometry showed that melittin inhibited myocardial cell apoptosis; in addition, real-time PCR showed that melittin decreased the expression of bax and caspase-3, and increased the expression of bcl-2. The results of echocardiographic examination showed that melittin improved cardiac function. Moreover, melittin decreased the activity of AST, CK, HBDH and LDH, and decreased the production of IL-1β, IL-6, TNF-α and MCP-1 in CVB3-induced myocardial tissues. Finally, we also found that melittin increased the expression of HDAC2 and activated the GSK-3β/Nrf2/ARE signaling pathway, whereas these changes were reversed by inhibition of HDAC2 in VMC model mice. In conclusion, our results suggested that melittin ameliorates CVB3-induced myocarditis via activation of the HDAC2-mediated GSK-3β/Nrf2/ARE signaling pathway.

Use of sIgE/T-IgE in Predicting Systemic Reactions: Retrospective Analysis of 54 Honeybee Venom Allergy Cases in North China.

Background:Venom allergy is significantly underestimated in China. Venom-specific IgE may not provide accurate clinical reactions. Our conducted retrospective analysis observes alternative diagnostic considerations in assessing confirmation and severity of honeybee venom allergy.Methods:Retrospective review of honeybee venom allergy versus nonallergy patients presented with positive honeybee venom (i1) sIgE results. According to clinically observed reactions caused by a honeybee sting, patients were divided into three groups. Patient residence and exposure types were analyzed. The sIgE/T-IgE among allergy and control groups was compared.Results:Gender ratio male:female was 32:22; median age was 39 years (31, 50). 48% (26/54) of patients live in urban areas, 52% (28/54) in rural areas. Based on bee sting reactions, patients were divided into common localized reactions (32/54), large localized reactions (7/54), and systemic reactions (15/54). In the systemic reaction group, patients presented as Type II (6/15), Type III (6/15). There is significant difference (P < 0.001) between the three groups in regards to exposure types. In the systemic reaction group, 8.7% (13/15) of patients are beekeepers. A significant difference (P < 0.001) was observed between allergic and control groups based on sIgE/T-IgE results. As well as significant difference observed between the systemic reaction group to the other two reaction groups in regards to sIgE/T-IgE results. Six systemic reaction patients presented with large localized reactions before onset of system symptoms 1 month to 1 year of being stung.Conclusions:Occupational exposure is the most common cause in honeybee venom allergy induced systemic reactions. The use of sIgE/T-IgE results is a useful diagnostic parameter in determining honeybee venom allergy.

Phospholipase A2 isolated from the venom of honey bees prevents viral attachment in mammalian cells

The belief that honey bee venom (BV) can be used to treat certain immune-related diseases, such as arthritis and rheumatic conditions, goes back to antiquity. A growing number of reports have demonstrated that BV contains at least 18 pharmacologically active components, including phospholipase A2 (PLA2). Recent research has shown that bee venom PLA2 (bvPLA2) induces protective immune responses against several diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. However, the antiviral properties of bvPLA2 have not been well investigated. Hence, we examined the potential inhibitory effects of bvPLA2 and its possible mechanism of action against a broad panel of pathogenic viruses in vitro. Pre-treatment with bvPLA2 significantly inhibited the replication of vesicular stomatitis virus (VSV), coxsackie virus (H3), enterovirus-71 (EV-71), herpes simplex virus (HSV) and Adenovirus (AdV) dramatically. However, bvPLA2 did not show antiviral activity against Influenza A virus (PR8) and Newcastle disease virus (NDV). Such inhibitory effects were explained by blocking of the attachment of the virus to cells upon bvPLA2 treatment. Additionally, we observed that Heparan sulfate (HS) has an inhibitory effect on the attachment of HSV to the cell surface dose dependently, which was inconsistent with bvPLA2 treatment. These findings suggest that bvPLA2 has an inhibitory effect on the replication of diverse viruses by blocking their attachment to the cell surface and could be a promising source of natural antiviral agents.

Activities of Venom Proteins and Peptides with Possible Therapeutic Applications from Bees and WASPS.

The variety of proteins and peptides isolated from honey bee venom and wasp venom includes melittin, adiapin, apamine, bradykinin, cardiopep, mast cell degranulating peptide, mastoparan, phospholipase A2 and secapin. Some of the activities they demonstrate may find therapeutic applications.

Molecular characterization of a venom acid phosphatase from the Asiatic honeybee Apis cerana

Bee venom contains a variety of toxic components, including enzymes, peptides, and biogenic amines. An acid phosphatase Acph-1-like protein has been identified from Asiatic honeybee (Apis cerana) venom. However, no molecular information is currently available for acid phosphatases from A. cerana venom. In this study, an A. cerana venom acid phosphatase (AcVAP) was identified. The amino acid sequence analysis of the predicted AcVAP protein revealed high identity with other bee venom acid phosphatases. An anti-AcVAP antibody was produced against a recombinant AcVAP (46-kDa) expressed in in baculovirus-infected insect cells. Northern and Western blot analyses showed that AcVAP was expressed in the venom gland and was present as a 46-kDa protein in the secreted bee venom. The enzymatic properties of recombinant AcVAP were determined using p-nitrophenyl phosphate (p-NPP) as a substrate and exhibited the highest activity at pH4.8 and 45°C. Taken together, our data demonstrated that AcVAP functions as a bee venom acid phosphatase.