Melittin-Carrying Nanoparticle Suppress T Cell-Driven Immunity in a Murine Allergic Dermatitis Model.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are the most common human skin disorders. Although corticosteroids have been widely used to treat ACD and AD, the side effects of corticosteroids encourage researchers to explore new immunoregulatory treatments. Here, an immunomodulatory approach based on lipid nanoparticles carrying α-helical configurational melittin (α-melittin-NP) is developed to overcome T cell-mediated inflammatory reactions in an oxazolone (OXA)-induced contact hypersensitivity mouse model and OXA-induced AD-like mouse model. Intradermal injection of low-dose α-melittin-NPs prevents the skin damage caused by melittin administration alone and efficiently targeted lymph nodes. Importantly, melittin and α-melittin-NPs restrain RelB activity in dendritic cells (DCs) and further suppresses dendritic cell activation and maturation in lymph nodes. Furthermore, low-dose α-melittin-NPs leads to relief of antigen recognition-induced effector T cell arrest in the dermis and inhibited allergen-specific T cell proliferation and activation. Significantly, this approach successfully controls Th1-type cytokine release in the ACD model and restricts Th2-type cytokine and IgE release in the AD-like model. Overall, intradermal delivery of low-dose α-melittin-NPs efficiently elicits immunosuppression against T cell-mediated immune reactions, providing a promising therapeutic strategy for treating skin disorders not restricted to the lesion region.

Bio-nano scale modifications of melittin for improving therapeutic efficacy.

INTRODUCTION: Melittin (MLT), a natural membrane-active component, is the most prominent cytolytic peptide from bee venom. Remarkable biological properties of MLT, including anti-inflammatory, antimicrobial, anticancer, anti-protozoan, and antiarthritic activities, make it an up-and-coming therapeutic candidate for a wide variety of human diseases. Therapeutic applications of MLT may be hindered due to low stability, high toxicity, and weak tissue penetration. Different bio-nano scale modifications hold promise for improving its functionality and therapeutic efficacy. AREAS COVERED: In the current review, we aimed to provide a comprehensive insight into strategies used for MLT conjugations and modifications, cellular delivery of modified forms, and their clinical perspectives by reviewing the published literature on PubMed, Scopus, and Google Scholar databases. We also emphasized the MLT structure modifications, mechanism of action, and cellular toxicity. EXPERT OPINION: Developing new analogs and conjugates of MLT as a natural drug with improved functions and fewer side effects is crucial for the clinical translation of this approach worldwide, especially where the chemicals and synthetic drugs are more expensive or unavailable in the healthcare system. MLT-nanoconjugation may be one of the best-optimized strategies for improving peptide delivery, increasing its therapeutic efficacy, and providing minimal nonspecific cellular lytic activity. [Figure: see text].

Anaphylactic shock with pulmonary eosinophilic infiltration due to honeybee attack in a donkey: case report / [Choque anafilático com infiltração eosinófila pulmonar devido a ataque de abelhas em um burro: relato de caso]

A case of a donkey attacked by Africanized honeybee is reported here with clinical signs of agitation, dehydration, congestion of the ocular mucous membranes, tongue edema, tachycardia and inspiratory dyspnea, and progression to death. At necropsy, diffuse, severe subcutaneous edema at face and cervical regions and severe diffuse pulmonary hyperemia with abundant edema without parenchymal collapse were observed. Microscopically, marked, diffuse deep dermis and panniculus carnosus edema and marked diffuse alveolar edema, with moderate population of eosinophils predominantly around larger caliber vessels were noted. The final diagnosis of anaphylactic shock was supported by history, clinical signs, and anatomic pathology findings. This is the first report of a honeybee attack with pulmonary eosinophilic infiltration in a mammal.(AU)

Descreve-se um caso de ataque de abelha africanizada em um burro, com sinais clínicos de agitação, desidratação, mucosas oculares congestas, edema de língua, taquicardia e dispneia inspiratória, com progressão e morte. Na necropsia, foram verificados edema subcutâneo difuso grave nas regiões de face e cervical, hiperemia pulmonar difusa grave com edema abundante e sem colapso do parênquima. Microscopicamente, foram observados edema marcado difuso na derme profunda e panículo carnoso e edema alveolar difuso acentuado, com população moderada de eosinófilos predominantemente em torno de vasos de maior calibre. O diagnóstico de choque anafilático foi baseado no histórico, em sinais clínicos e em achados anatomopatológicos. Este é o primeiro relato de ataque de abelhas com infiltração eosinofílica pulmonar em um mamífero.(AU)

Single dose eradication of extensively drug resistant Acinetobacter spp. In a mouse model of burn infection by melittin antimicrobial peptide.

Bacterial infections caused by antibiotic resistant bacteria are the leading cause of morbidity and mortality after burn injuries. This issue has driven the need for promising antimicrobial drugs to eradication of bacterial pathogens. Accordingly, we aimed to determine the therapeutic value of melittin, as a natural Antimicrobial peptide (AMP), in eradication of extensively drug-resistant (XDR) Acinetobacter spp. on a mouse model of third degree burn infection. Melittin killed all examined XDR isolates at 4 µg/mL up to 3 h. Melittin caused significant fluorescence release from XDR isolates at the minimum dose of 0.062 µg/mL. Vesicle formation on the membrane and squeezing of bacteria followed by cell lysis indicated the membranolytic effect of melittin. Melittin at 32 µg/mL completely eradicated the colonized XDR bacteria on infected burn mice during 2 h. No toxicity was observed on injured or healthy derma, as well as circulating Red Blood Cells (RBCs) in the examined mice. Potent promising antibacterial activity of melittin and the lack of toxicity at the therapeutic dose can clarify that melittin can be implemented as a topical drug lead in a preclinical trial of third degree burn infections.

Melittin, a major peptide component of bee venom, and its conjugates in cancer therapy.

Melittin (MEL), a major peptide component of bee venom, is an attractive candidate for cancer therapy. This agent has shown a variety of anti-cancer effects in preclinical cell culture and animal model systems. Despite a convincing efficacy data against variety of cancers, its applicability to humans has met with challenges due to several issues including its non-specific cytotoxicity, degradation and hemolytic activity. Several optimization approaches including utilization of nanoparticle based delivery of MEL have been utilized to circumvent the issues. Here, we summarize the current understanding of the anticancer effects of bee venom and MEL on different kinds of cancers. Further, we also present the available information for the possible mechanism of action of bee venom and/or MEL.

A novel melittin nano-liposome exerted excellent anti-hepatocellular carcinoma efficacy with better biological safety.

Melittin is the main effective component of bee venom and has extensive biological functions; however, serious side effects have restricted its clinical application. Preclinical and clinical studies showed that the main adverse events were allergic reaction and pain at the administration site. To decrease the toxicity, we prepared melittin nano-liposomes by encapsulating melittin with poloxamer 188 and explored the inhibitory activities on liver cancer together with biological safety. Here, we showed that melittin nano-liposomes significantly inhibited the survival of hepatocellular carcinoma (HCC) cells in vitro and prominently suppressed the growth of subcutaneous and orthotopic HCC transplantation tumors in vivo. It was important that it induced less inflammation and allergy in mice compared with melittin. Overall, melittin nano-liposomes would have a better application in HCC therapy due to its significant anti-tumor activity and better biological safety.

Anti-Inflammatory Applications of Melittin, a Major Component of Bee Venom: Detailed Mechanism of Action and Adverse Effects.

Inflammation is a pervasive phenomenon triggered by the innate and adaptive immune systems to maintain homeostasis. The phenomenon normally leads to recovery from infection and healing, but when not properly phased, inflammation may cause immune disorders. Bee venom is a toxin that bees use for their protection from enemies. However, for centuries it has been used in the Orient as an anti-inflammatory medicine for the treatment of chronic inflammatory diseases. Bee venom and its major component, melittin, are potential means of reducing excessive immune responses and provide new alternatives for the control of inflammatory diseases. Recent experimental studies show that the biological functions of melittin could be applied for therapeutic use in vitro and in vivo. Reports verifying the therapeutic effects of melittin are accumulating in the literature, but the cellular mechanism(s) of the anti-inflammatory effects of melittin are not fully elucidated. In the present study, we review the current knowledge on the therapeutic effects of melittin and its detailed mechanisms of action against several inflammatory diseases including skin inflammation, neuroinflammation, atherosclerosis, arthritis and liver inflammation, its adverse effects as well as future prospects regarding the use of melittin.

[Mellitin induced acute kidney injury and continuous renal replacement therapy in goat model].

OBJECTIVE: To establish a goat model of melittin induced acute kidney injury (AKI), and to evaluate the therapeutic effect of continuous veno-venus heamofiltration (CVVH) in melittin- induced AKI. METHODS: Twelve male goats were randomized into three groups: control group, melittin induced AKI group (melittin group), and CVVH intervention group (CVVH group). The AKI goat model was established by the injection of melittin via the auricular vein for four times in 48 h to reach a total dose of 0.5 mg/kg, then serum creatinine (Cr) and creatine kinase (CK) were tested every 6 h and urine output was record each hour. AKI was diagnosed when Cr level increased to the double value of control group, or the urine output decreased to less than 0. 5 mL/(kg x h) in 6 h. After the diagnosis of AKI, the animals in CVVH group received CVVH treatment for 12 h. At the end, the goats in all groups were sacrificed by anesthesia and kidney tissue samples were collected. Light microscopy and telectron microscopy observation were performed. Apoptosis was detected by immunohistochemistry and TUNEL technique. RESULTS: AKI was successfully induced by melittin in the goats. The Cr level in control group was (43.95 +/- 1.59) micromol/L, while (100.75 +/- 7.87) micromol/L in AKI group and (102.10 +/- 5.06) micromol/L in CVVH group. Cr level was lowered significantly after CVVH treatment [(45.02 +/- 2.41) micromol/L in control group vs. (108.60 +/- 9.40) micromol/L in AKI group vs. (64.13 +/- 5.82) micromol/L in CVVH group, P < 0.001]. Swelling and reduction of mitochondrial crests in AKI group were more obvious than those in CVVH group. Expression of caspase-3 and apoptosis cells percentage of renal tubules in AKI group were significantly higher than those in CVVH group. CONCLUSION: Melittin induced AKI model could be established in goats. CVVH could alleviate melittin induced AKI, probably in the mechanism to reduce the apoptosis of renal tubular cells.

Cytolytic nanoparticles attenuate HIV-1 infectivity.

BACKGROUND: We investigated whether cytolytic melittin peptides could inhibit HIV-1 infectivity when carried in a nanoparticle construct that might be used as a topical vaginal virucide. Free melittin and melittin-loaded nanoparticles were prepared and compared for cytotoxicity and their ability to inhibit infectivity by CXCR4 and CCR5 tropic HIV-1 strains. METHODS: TZM-bl reporter cells expressing luciferase under the control of the HIV-1 promoter were incubated with HIV-1 NLHX (CXCR4) or HIV-1 NLYU2 (CCR5) viral strains and different doses of soluble CD4 (positive control) or free melittin to determine infectivity and viability. Melittin-loaded nanoparticles were formulated and different doses tested against VK2 vaginal epithelial cells to determine cell viability. Based on VK2 viability, melittin nanoparticles were tested for prevention of CXCR4 and CCR5 tropic HIV-1 infectivity and viability of TZM-bl reporter cells. Low-speed centrifugation was used to compare the ability of blank non-melittin nanoparticles and melittin nanoparticles to capture CCR5 tropic HIV-1. RESULTS: As expected, the soluble CD4 positive control inhibited CXCR4 (50% inhibitory concentration [IC50] 3.7 µg/ml) and CCR5 (IC50 0.03 µg/ml) tropic HIV-1 infectivity. Free melittin doses <2 µM were not cytotoxic and were highly effective in reducing HIV-1 infectivity for both CXCR4 and CCR5 strains in TZM-bl reporter cells, while VK2 vaginal cell viability was adversely affected at all free melittin doses tested. However, VK2 cell viability was not affected at any dose of melittin-loaded nanoparticles. Melittin nanoparticles safely and significantly decreased CXCR4 (IC50 2.4 µM and IC90 6.9 µM) and CCR5 (IC50 3.6 µM and IC90 11.4 µM) strain infectivity of TZM-bl reporter cells. Furthermore, melittin nanoparticles captured more HIV-1 than blank nanoparticles. CONCLUSIONS: These data illustrate the first proof-of-concept for therapeutic and safe nanoparticle-mediated inhibition of HIV-1 infectivity. Future investigations appear warranted to explore the antiviral prophylactic potential of melittin nanoparticles to capture, disrupt and prevent initial infection with HIV-1 or potentially other enveloped viruses.

Episodic hemorrhage during honeybee venom anaphylaxis: potential mechanisms.

Episodic hemorrhage is not a typical symptom of anaphylactic reaction to insect stings. Cases of reactions to honeybee (HB) sting or venom immunotherapy in which the uterus is the main target organ are very rare. Hemorrhage can be induced by HB venom components, especially melittin, which interfere with complement cleavage and bradykinin release. Both mechanisms are directly or indirectly associated with coagulation, thrombolysis, hemolysis, and smooth muscle tone. Induction of episodic hemorrhage through pathway destabilization in a defective bradykinin system or vulnerable organ may not be compensated by appropriate regulatory mechanisms. The pathological role of effectors is generally offset by the interaction of various regulatory systems, and the probability of hemorrhage is minimized thanks to this compensatory capability. In endometrial bleeding, the uterus becomes more vulnerable as a result of postmenstrual vascular fragility and additional induction of anaphylaxis-related uterine contractions. Episodic hemorrhage, especially metrorrhagia, as a consequence of HB venom activity may be suspected by an allergologist, but not by a physician. Melittin-free or recombinant allergens of HB venom, as well as modulators of the biochemical systems involved, could help to reduce the likelihood of hemorrhage. However, further investigation is required before these strategies can be introduced in clinical practice.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis.

We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.

Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes.

Melittin-polyethylenimine (PEI) conjugates have been shown to enhance gene transfer efficiency of polyplexes due to their membrane-destabilizing properties. Inherent lytic activity at neutral pH however also provokes high cytotoxicity due to plasma membrane damage. In order to shift the lytic activity towards the endosomal membrane, several melittin analogs were designed. Acidic modification of melittin by replacing neutral glutamines (Gln-25 and Gln-26) with glutamic acid residues greatly improved the lytic activity of C-terminally linked PEI conjugates at the endosomal pH of 5. This activity correlated well with the gene transfer efficiency of polyplexes in four different cell lines. Melittin-PEI conjugates with high lytic activities at endosomal pH were then incorporated into EGF receptor-targeted and polyethylene glycol-shielded polyplexes. The resulting particles had virus-like dimension (150 nm) with a neutral surface charge and were subsequently purified by size exclusion chromatography to remove unbound toxic PEI conjugate. These purified polyplexes mediated EGF-receptor-specific gene transfer with up to 70-fold higher activity compared to the corresponding PEI polyplexes without melittin.

Secondary heat hyperalgesia induced by melittin in humans.

Melittin, which is a principal protein of honeybee venom, can induce mechanical hyperalgesia in humans. The characteristics of the melittin induced mechanical hyperalgesia are quantitatively and qualitatively different from those evoked by capsaicin. The aim of the present study was to investigate in detail secondary heat hyperalgesia induced by melittin in humans. In six healthy volunteers, 10 microg of melittin was injected intradermally on the volar forearm, and VAS score to radiant heat stimuli (focused light from a xenon lamp) was assessed around the injection site 5, 30, and 60 min after injection. For normalization purposes, a pain rating index was calculated as the individual heat evoked VAS scores obtained after melittin divided by the individual baseline VAS scores. A two-way ANOVA revealed a significant increase of the pain rating index over time (F=3.6; P=0.03). The pain rating index at 60 min was significantly larger than at 5 min (P=0.04) and at 30 min (P=0.03). These results demonstrated slowly developing secondary heat hyperalgesia after injection of melittin. A possible contribution of peripheral inflammatory responses to the manifestation of secondary heat hyperalgesia is suggested, which in reality render the distinction between the primary and secondary area of heat hyperalgesia unnecessary.

C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes.

BACKGROUND: One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency. METHODS: The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH. RESULTS: All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines. CONCLUSIONS: The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model.

A comparison of hyperalgesia and neurogenic inflammation induced by melittin and capsaicin in humans.

Melittin (a main compound of bee venom) and capsaicin were injected intradermally in healthy human volunteers: (1) to study secondary mechanical hyperalgesia (static hyperalgesia and dynamic hyperalgesia) around the injection site; and (2) to correlate the sensory changes to the neurogenic inflammation assessed by laser-doppler blood flowmetry. Melittin 50 microg and capsaicin 10 microg induced comparable spontaneous pain and increased blood flow (neurogenic inflammation). Intradermal injection of melittin induced regions of secondary mechanical hyperalgesia around the injection site, however, they were not as large as the hyperalgesia induced by capsaicin. This is the first report studying mechanical hyperalgesia induced by melittin in humans, and the results were in agreement with the previous observations in rats. Melittin seems to be a valuable model to study a possible contribution of neurogenic inflammation to hyperalgesia in humans.