Enhancement of target toxin neutralization effect in vivo by PEGylation of multifunctionalized lipid nanoparticles.

Protein-protein (e.g., antibody-antigen) interactions comprise multiple weak interactions. We have previously reported that lipid nanoparticles (LNPs) bind to and neutralize target toxic peptides after multifunctionalization of the LNP surface (MF-LNPs) with amino acid derivatives that induce weak interactions; however, the MF-LNPs aggregated after target capture and showed short blood circulation times. Here we optimized polyethylene glycol (PEG)-modified MF-LNPs (PEG-MF-LNPs) to inhibit the aggregation and increase the blood circulation time. Melittin was used as a target toxin, and MF-LNPs were prepared with negatively charged, hydrophobic, and neutral amino-acid-derivative-conjugated functional lipids. In this study, MF-LNPs modified with only PEG5k (PEG5k-MF-LNPs) and with both PEG5k and PEG2k (PEGmix-MF-LNPs) were prepared, where PEG5k and PEG2k represent PEG with a molecular weight of 5000 and 2000, respectively. PEGylation of the MF-LNPs did not decrease the melittin neutralization ability of nonPEGylated MF-LNPs, as tested by hemolysis assay. The PEGmix-MF-LNPs showed better blood circulation characteristics than the PEG5k-MF-LNPs. Although the nonPEGylated MF-LNPs immediately aggregated when mixed with melittin, the PEGmix-MF-LNPs did not aggregate. The PEGmix-MF-LNPs dramatically increased the survival rate of melittin-treated mice, whereas the nonPEGylated MF-LNPs increased slightly. These results provide a fundamental strategy to improve the in vivo toxin neutralization ability of MF-LNPs.

Melittin ameliorates inflammation in mouse acute liver failure via inhibition of PKM2-mediated Warburg effect.

Acute liver failure (ALF) is a fatal clinical syndrome with no special drug. Recent evidence shows that modulation of macrophage to inhibit inflammation may be a promising strategy for ALF treatment. In this study we investigated the potential therapeutic effects of melittin, a major peptide component of bee venom both in mice model of ALF and in LPS-stimulated macrophages in vitro, and elucidated the underlying mechanisms. ALF was induced in mice by intraperitoneal injection of D-galactosamine/LPS. Then the mice were treated with melittin (2, 4, and 8 mg/kg, ip). We showed that melittin treatment markedly improved mortality, attenuated severe symptoms and signs, and alleviated hepatic inflammation in D-galactosamine/LPS-induced ALF mice with the optimal dose being 4 mg/kg. In addition, melittin within the effective doses did not cause significant in vivo toxicity. In LPS-stimulated RAW264.7 macrophages, melittin (0.7 µM) exerted anti-oxidation and anti-inflammation effects. We showed that LPS stimulation promoted aerobic glycolysis of macrophages through increasing glycolytic rate, upregulated the levels of Warburg effect-related enzymes and metabolites including lactate, LDHA, LDH, and GLUT-1, and activated Akt/mTOR/PKM2/HIF-1α signaling. Melittin treatment suppressed M2 isoform of pyruvate kinase (PKM2), thus disrupted the Warburg effect to alleviate inflammation. Molecular docking analysis confirmed that melittin targeted PKM2. In LPS-stimulated RAW264.7 macrophages, knockdown of PKM2 caused similar anti-inflammation effects as melittin did. In D-galactosamine/LPS-induced ALF mice, melittin treatment markedly decreased the expression levels of PKM2 and HIF-1α in liver. This work demonstrates that melittin inhibits macrophage activation-mediated inflammation via inhibition of aerobic glycolysis by targeting PKM2, which highlights a novel strategy of using melittin for ALF treatment.

A Biomimetic Nanoparticle to "Lure and Kill" Phospholipase†A2.

Inhibition of phospholipase A2 (PLA2) has long been considered for treating various diseases associated with an elevated PLA2 activity. However, safe and effective PLA2 inhibitors remain unavailable. Herein, we report a biomimetic nanoparticle design that enables a "lure and kill" mechanism designed for PLA2 inhibition (denoted "L&K-NP"). The L&K-NPs are made of polymeric cores wrapped with modified red blood cell membrane with two inserted key components: melittin and oleyloxyethyl phosphorylcholine (OOPC). Melittin acts as a PLA2 attractant that works together with the membrane lipids to "lure" in-coming PLA2 for attack. Meanwhile, OOPC acts as inhibitor that "kills" PLA2 upon enzymatic attack. Both compounds are integrated into the L&K-NP structure, which voids toxicity associated with free molecules. In the study, L&K-NPs effectively inhibit PLA2-induced hemolysis. In mice administered with a lethal dose of venomous PLA2, L&K-NPs also inhibit hemolysis and confer a significant survival benefit. Furthermore, L&K-NPs show no obvious toxicity in mice. and the design provides a platform technology for a safe and effective anti-PLA2 approach.

Role of the inflammasome in defense against venoms.

Venoms consist of a complex mixture of toxic components that are used by a variety of animal species for defense and predation. Envenomation of mammalian species leads to an acute inflammatory response and can lead to the development of IgE-dependent venom allergy. However, the mechanisms by which the innate immune system detects envenomation and initiates inflammatory and allergic responses to venoms remain largely unknown. Here we show that bee venom is detected by the NOD-like receptor family, pyrin domain-containing 3 inflammasome and can trigger activation of caspase-1 and the subsequent processing and unconventional secretion of the leaderless proinflammatory cytokine IL-1ß in macrophages. Whereas activation of the inflammasome by bee venom induces a caspase-1-dependent inflammatory response, characterized by recruitment of neutrophils to the site or envenomation, the inflammasome is dispensable for the allergic response to bee venom. Finally, we find that caspase-1-deficient mice are more susceptible to the noxious effects of bee and snake venoms, suggesting that a caspase-1-dependent immune response can protect against the damaging effects of envenomation.

IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

Melittin-The principal toxin of honeybee venom-Is also produced in the honeybee fat body.

Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.

Experimental envenomation with honeybee venom melittin and phospholipase A2 induced multiple ultrastructural changes in adrenocortical mitochondria.

Bee stings represent a public health subject, but the mechanisms involved in bee venom toxicity are not yet fully understood. To evaluate the reactions of adrenocortical cells, through which organisms respond to stress, two honeybee venom components: melittin (Mlt) and phospholipase A2 (PLA2) were tested as potential chemical stressors. Modifications were investigated with transmission electron microscopy and microanalysis. A single dose of Mlt (31 mg/kg) or PLA2 (9.3 mg/kg) was injected in rats of groups ML and PL; daily doses of Mlt (350 µg/kg) or PLA2 (105 µg/kg) were injected 30 days in rats of groups M30 and P30. Adrenocortical cells in ML group showed ultrastructural degenerative alterations of nuclei, endoplasmic reticulum, and mitochondria that exhibited lipid inclusions and mitochondrial cristae (MC) re-organized into mono- or multimembrane large vesicles, and whorls of membranes. Many MC were degenerated. In the M30 group, similar ultrastructural changes, but of lower amplitude were noted; lipid cytosolic droplets were heterogenous. MC diameters in Mlt groups (melittin treated groups) were significantly higher than in control (C) group. In PL group, mitochondria contained large lipid inclusions, vesicular MC of different sizes and multiple membranes, and debris, or whorl structures. In P30 group MC were tubular with increased diameters. In both PLA2 groups (PLA2 treated groups) MC were significantly larger than in C group. We concluded that Mlt and PLA2 were powerful stressors, toxic at the tested doses, cellular reactions concerning in all groups mainly mitochondria, but also other cellular compartments. Apart from degenerative regression of MC, the rearrangement of tubular MC occurred into one or multiple large multimembrane vesicular MC. Reactions to the high doses were more pronounced, with the highest amplitude in ML group, and the lowest in P30 group.

Involvement of peripheral NMDA receptor in melittin-induced thermographic flare.

Intradermal injection of an active compound of European honeybee toxin, melittin, into the forearm in humans produces temporary pain and evokes sustained increase of local skin temperature. This increase of skin temperature is suppressed by the pretreatment of a voltage gated sodium channel blocker, lidocaine, suggesting that neurogenic inflammation is involved in the skin temperature increase after the melittin treatment. In this study, we tested a hypothesis that the melittin-induced skin temperature increase is augmented by an N-methyl-D-aspartate (NMDA) glutamate receptor that is present on the peripheral terminals of cutaneous primary afferents. Skin temperature was examined after the local application of incremental doses of melittin by a computer-assisted-thermography in pentobarbital-anesthetized rats. Local subcutaneous glutamate was collected through a microdialysis probe and glutamate levels were measured by a high pressure liquid chromatography with electrochemical detection method. Intraplantar injection of melittin resulted in the increase of subcutaneous glutamate levels and the increase of local skin temperature, which was partially attenuated by co-injection of an NMDA receptor antagonist, MK-801. In addition, intraplantar injection of NMDA itself increased the local skin temperature. Our data suggest that melittin-induced increase of skin temperature is enhanced through the activation of peripheral NMDA receptors by locally released glutamate. We suggest that topical administration of NMDA receptor antagonists could be an effective treatment of neuro-inflammatory pain.

Membrane Cholesterol Content and Lipid Organization Influence Melittin and Pneumolysin Pore-Forming Activity.

Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than interacting with a specific receptor, melittin interacts with the lipid components, disrupting the lipid bilayer and inducing ion leakage and osmotic shock. This mechanism of action is shared with pneumolysin and other members of the cholesterol-dependent cytolysin family. In this manuscript, we investigated the inverse correlation for cholesterol dependency of these two toxins. While pneumolysin-induced damage is reduced by pretreatment with the cholesterol-depleting agent methyl-ß-cyclodextrin, the toxicity of melittin, after cholesterol depletion, increased. A similar response was also observed after a short incubation with lipophilic simvastatin, which alters membrane lipid organization and structure, clustering lipid rafts. Therefore, changes in toxin sensitivity can be achieved in cells by depleting cholesterol or changing the lipid bilayer organization.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of ß-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Bottlebrush Polymer-Conjugated Melittin Exhibits Enhanced Antitumor Activity and Better Safety Profile.

Despite potency against a variety of cancers in preclinical systems, melittin (MEL), a major peptide in bee venom, exhibits non-specific toxicity, severe hemolytic activity, and poor pharmacological properties. Therefore, its advancement in the clinical translation system has been limited to early-stage trials. Herein, we report a biohybrid involving a bottlebrush-architectured poly(ethylene glycol) (PEG) and MEL. Termed pacMEL, the conjugate consists of a high-density PEG arrangement, which provides MEL with steric inhibition against protein access, while the high molecular weight of pacMEL substantially enhances plasma pharmacokinetics with a ∼10-fold increase in the area under the curve (AUC∞) compared to free MEL. pacMEL also significantly reduces hepatic damage and unwanted innate immune response and all but eliminated hemolytic activities of MEL. Importantly, pacMEL passively accumulates at subcutaneously inoculated tumor sites and exhibits stronger tumor-suppressive activity than molecular MEL. Collectively, pacMEL makes MEL a safer and more appealing drug candidate.

In vitro and in vivo toxicity and antibacterial efficacy of melittin against clinical extensively drug-resistant bacteria.

BACKGROUND: Melittin is one of the most studied antimicrobial peptides, and several in vitro experiments have demonstrated its antibacterial efficacy. However, there is evidence showing melittin has non-promising effects such as cytotoxicity and hemolysis. Therefore, concerns about unwanted collateral toxicity of melittin lie ahead in the path toward its clinical development. With these considerations, the present study aimed to fill the gap between in vitro and in vivo studies. METHODS: In the first step, in vitro toxicity profile of melittin was assessed using cytotoxicity and hemolysis tests. Next, a maximum intraperitoneal (i.p.) sub-lethal dose was determined using BALB/c mice. Besides toxicity, antimicrobial efficacy of melittin against extensively drug-resistant (XDR) Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and KPC-producing Klebsiella pneumonia (KPC-KP) pathogens were tested using both in vitro and in vivo methods. RESULTS: Melittin showed extensive hemolysis (HD50 = 0.44 µg/mL), and cytotoxicity (IC50 = 6.45 µg/mL) activities with i.p. LD50 value of 4.98 mg/kg in BALB/c mice. In vitro antimicrobial evaluation showed melittin MIC range from 8 to 32 µg/mL for the studied pathogens. Treatment of infected mice with repeated sub-lethal doses of melittin (2.4 mg/kg) displayed no beneficial effect on their survival and peritoneal bacterial loads. CONCLUSIONS: These results indicate that melittin at its safe dose could not exhibit antimicrobial activity, which hinders its application in clinical practice.

Molecular characterization of the anticancer properties associated with bee venom and its components in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a frequent form of malignant glioma. Strategic therapeutic approaches to treat this type of brain tumor currently involves a combination of surgery, radiotherapy and chemotherapy. Nevertheless, survival of GBM patients remains in the 12-15 months range following diagnosis. Development of novel therapeutic approaches for this malignancy is therefore of utmost importance. Interestingly, bee venom and its components have shown promising anti-cancer activities in various types of cancer even though information pertaining to GBMs have been limited. The current work was thus undertaken to better characterize the anti-cancer properties of bee venom and its components in Hs683, T98G and U373 human glioma cells. MTT-based cell viability assays revealed IC50 values of 7.12, 15.35 and 7.60 µg/mL for cell lines Hs683, T98G and U373 treated with bee venom, respectively. Furthermore, melittin treatment of these cell lines resulted in IC50 values of 7.77, 31.53 and 12.34 µg/mL, respectively. Cell viability assessment by flow cytometry analysis confirmed signs of late apoptosis and necrosis after only 1 h of treatment with either bee venom or melittin in all three cell lines. Immunoblotting-based quantification of apoptotic markers demonstrated increased expression of Bak and Bax, while Caspsase-3 levels were significantly lower when compared to control cells. Quantification by qRT-PCR showed increased expression levels of long non-coding RNAs RP11-838N2.4 and XIST in glioma cells treated with either bee venom or melittin. Overall, this study provides preliminary insight on molecular mechanisms via which bee venom and its main components can impact viability of glioma cells and warrants further investigation of its anticancer potential in gliomas.

Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides.

In order to evaluate their antibacterial activities and toxicities, the cecropins-melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp's 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.

Prolonged melittin release from polyelectrolyte-based nanocomplexes decreases acute toxicity and improves blood glycemic control in a mouse model of type II diabetes.

Gating modifier toxins (GMTs) from animal venom have shown great potential in controlling blood glucose levels in type II diabetes (T2D), but their high acute toxicity and quick clearance in the body hamper their potential therapeutic use. Inspired by their highly positive charge, we have developed a nanocomplex system based on polyelectrolytes, in which strong interactions form between positively charged GMTs and negatively charged dextran sulfate (DS). Using melittin as a model GMT and adapting flash nanocomplexation (FNC) technology for complex preparation, uniform nanocomplexes (polydispersity index: ~0.1) with high melittin encapsulation efficiency (~100%), high payload capacity (~30%), and tunable release profiles were formulated. In contrast to the high acute liver toxicity and low survival rate (60% after 8 days) observed after a single intraperitoneal (i.p.) injection of 3 mg/kg free melittin, melittin-loaded nanocomplexes displayed improved safety (100% survival after 8 days) due to prolonged melittin release. In a mouse model of T2D, a single i.p. injection of nanocomplexes decreased the blood glucose level to 12 mmol/L within 12 h and maintained it within the therapeutic range (<15 mmol/L) for 48 h. In addition, body weight decreased following treatment. This GMT/DS binary system shows great promise due to its simple components, facile preparation method, and enhanced potential druggability, including a decreased dosing frequency, decreased acute toxicity, and improved pathological indicators.

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action.

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells.

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

Ultrastructural variability of mitochondrial cristae induced in vitro by bee (Apis mellifera) venom and its derivatives, melittin and phospholipase A2, in isolated rat adrenocortical mitochondria.

We tested the ability of bee venom (BV), melittin (Mlt), and phospholipase A2 (PLA) - used in 5 concentrations each (5, 10, 15, 20 and 40 µg/100 µl) - to promote ultrastructural changes and reorganization of cristae in vitro in mitochondria isolated from rat adrenal cortex after a protocol optimized by us. Thus, apart from two control grups (CI and CS), in which the mitochondria were suspended into saline buffer and isolation medium respectively, 15 more groups of mitochondria were constituted, corresponding to the five different doses of the three substance tested (BV5 to M40; M5 to M40 and P5 to P40). The ultrastructural effects were quantified on transmission electron micrographs using a morphometry software. Values of 84.49 nm and 95.45 nm were calculated for median diameters of mitochondrial cristae in two control groups. Large and very large vesicular cristae, many with 2 or 3 membranes, were generated depending on dose among normal cristae in all treated groups. In the BV and Mlt treated groups, after an initial increase (up to 127.27 nm in V15 group and 151.2 nm in M10 group) due to stimulation of cristae fusion, the cristae diameter diminished as the doses increased, mainly by the collapse of the cristae. In the PLA treated groups, the cristae diameter increased continuously from 83.84 nm to 136.01 nm, by stimulated fusion of cristae, only the two largest doses promoting the collapse of cristae in some mitochondria. The highest percentage of abnormal cristae was found in the Mlt treated groups and next in BV treated groups. All substances tested produced pronounced ultrastructural variability of mitochondrial cristae in vitro: they also changed (depending on dose) mitochondrial shapes, generated matrix debris and the highest concentrations of BV and Mlt were responsible for mitochondrial breakdown. These ultrastructural alterations of mitochondrial criste in the presence of the BV molecules suggest a reduced capacity of adrenocortical mitochondria to synthetize steroid hormones consequently to BV envenomations and partially explain the toxic effects of the BV.

A biomimetic nanoparticle-enabled toxoid vaccine against melittin.

BACKGROUND: Melittin, the main active peptide ingredient of bee venom, can cause severe cell membrane lysis due to its robust interaction with negatively charged phospholipids. So far, no effective anti-melittin vaccine has been developed to protect people from undesired melittin intoxication. METHODS: Herein, we prepared a polydiacetylene (PDA) nanoparticle with cell membrane-mimic surface to complex melittin, forming an anti-melittin vaccine (PDA-melittin). RESULTS: PDA nanoparticles could effectively combine with melittin and neutralize its toxicity. PDA-melittin nanocomplex is demonstrated to enhance melittin uptake by DCs and stimulate strong melittin-specific immunity. Mice immunized with PDA-melittin nanocomplex showed higher survival rate after exposion to melittin than untreated mice. CONCLUSION: The PDA-melittin nanocomplex can efficiently and safely generate a specific immunity against melittin to protect body from melittin intoxication, providing a new method with potential clinical application for the treatment of melittin intoxication.

Ultrastructural analysis of early toxic effects produced by bee venom phospholipase A2 and melittin in Sertoli cells in rats.

In this study, we aimed to investigate the testicular toxicity of two molecules derived from bee venom (BV): phospholipase A2 (PlA2) and melittin (Mlt). Ultrastructural effects of purified BV PlA2 and Mlt were assessed consecutive to repeated dose (30 days) and acute toxicity studies. For the subchronic treatment, PlA2 and Mlt were injected in daily doses equivalent to those released by a bee sting (105 µg PlA2/kg/day and 350 µg Mlt/kg/day), while in the acute treatment their doses corresponded to those released by 100 bee stings (9.3 mg PlA2/kg and 31 mg Mlt/kg). Both PlA2 and Mlt affected the Leydig cells and the cells in seminiferous tubules, the Sertoli cells first of all. PlA2 injection resulted in detachment of the Sertoli cells from the surrounding cells, and extracellular vacuolations, cytoplasmic vacuolations in their basal region and in branches as well, detachment of spermatids, residual bodies and sometimes even spermatocytes into the lumen, changes that had a higher magnitude after the acute treatment. Mlt injection induced similar ultrastructural alterations, but more severe, including degeneration of cellular organelles and cellular necrosis, resulting into rarefaction of the seminiferous epithelium; the ultrastructural changes had a higher magnitude after the 30 repeated dose treatment. We concluded that either of the two molecules tested here, PlA2 and Mlt, were Sertoli cells toxicants at the used doses, and they participated both in the BV testicular toxicity. We consider the observed changes as part of a preceding mechanism of the more severe alterations produced by the BV. It also remains possible that these early unspecific changes reported here could represent the response of the SCs not only to the components of bee venom, but to molecules of other venoms as well. The Sertoli cells were the primary target of PlA2 and Mlt in the spermatogenic epithelium, and their alteration led to further degenerative changes of the germ cells. Since the exposure to PlA2 and Mlt caused severe alteration, including cell death and detachment of immature germ cells into the lumen, we may also conclude that the bee venom molecules had a potential to interfere with normal progression of spermatogenesis. All the degenerative changes observed in the Sertoli cells were accompanied with changes of the Leydig cells.