melitten and Disease-Models--Animal

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases and a major contributor to dementia. Although the cause of this condition has been identified long ago as aberrant aggregations of amyloid and tau proteins, effective therapies for it remain elusive. The complexities of drug development for AD treatment are often compounded by the impermeable blood-brain barrier and low-yield brain delivery. In addition, the use of high drug concentrations to overcome this challenge may entail side effects. To address these challenges and enhance the precision of delivery into brain regions affected by amyloid aggregation, we proposed a transferrin-conjugated nanoparticle-based drug delivery system. The transferrin-conjugated melittin-loaded L-arginine-coated iron oxide nanoparticles (Tf-MeLioNs) developed in this study successfully mitigated melittin-induced cytotoxicity and hemolysis in the cell culture system. In the 5XFAD mouse brain, Tf-MeLioNs remarkably reduced amyloid plaque accumulation, particularly in the hippocampus. This study suggested Tf-LioNs as a potential drug delivery platform and Tf-MeLioNs as a candidate for therapeutic drug targeting of amyloid plaques in AD. These findings provide a foundation for further exploration and advancement in AD therapeutics.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Animals; Brain; Disease Models, Animal; Magnetic Iron Oxide Nanoparticles; Melitten; Mice; Mice, Transgenic; Plaque, Amyloid; Transferrin

Triple-negative breast cancer (TNBC) accounts for approximately 10-15% of all breast cancer cases and is characterized by high invasiveness, high metastatic potential, relapse proneness, and poor prognosis. M2-like tumor-associated macrophages (TAMs) contribute to tumorigenesis and are promising targets for inhibiting breast cancer metastasis. Therefore, we investigated whether melittin-conjugated pro-apoptotic peptide (TAMpepK) exerts therapeutic effects on breast cancer metastasis by targeting M2-like TAMs. TAMpepK is composed of M2-like TAM binding peptide (TAMpep) and pro-apoptotic peptide d(KLAKLAK)

Topics: Animals; Apoptosis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Female; Lymphatic Metastasis; Melitten; Mice; Mice, Inbred BALB C; Neoplasm Recurrence, Local; Peptides; Phagocytosis; Triple Negative Breast Neoplasms; Tumor Microenvironment; Tumor-Associated Macrophages

Ulcerative colitis (UC) is considered one of the most prevalent inflammatory bowel diseases (IBDs). However, due to the lack of satisfying efficacy of conventional therapies and their side effects, there is still a need for more efficient therapeutic agents. Melittin is a small peptide derived from bee venom, which shows potent anti-inflammatory activity. The present investigation aimed to assess the anti-inflammatory effect of melittin peptide alone and in co-therapy with sulfasalazine as a standard therapy on dextran sulfate sodium (DSS)-induced colitis models.. We used DSS to induce UC in C57BL/6 male mice. We investigated the effect of melittin peptide alone and in combination with sulfasalazine on improving the clinical symptoms among DSS-induced colitis models. Finally, we employed histological investigation to show the therapeutic effect of melittin on attenuating the pathological damage of colon tissue caused due to DSS-induced inflammation in colitis models.. Our findings demonstrated that melittin peptide alone and in combination with sulfasalazine dramatically cured the clinical UC. Moreover, we observed that this peptide almost eliminated the histological damage of colon tissue in colitis, while significantly reducing the inflammation in colon tissue. Meanwhile, our results demonstrated that this peptide had an antioxidant effect through the disruption of the oxidant/antioxidant balance.. All these findings suggest that melittin peptide has an anti-inflammatory effect and can probably be considered a novel therapeutic agent for UC. Furthermore, our results demonstrated that this peptide can enhance the therapeutic effects of conventional therapy while attenuating the adverse effects of conventional agents.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Inflammation; Male; Melitten; Mice; Mice, Inbred C57BL; Sulfasalazine

Much research has shown Bee venom to be an effective neuroprotective agent. However, the usual transdermal injection of bee venom poses many pharmacokinetic disadvantages. Here, we compared the administration of bee venom via subcutaneous injection (SC) and via Microneedle patch (MN). Both administrated routes produce significant recovery effects, however: the MN significantly prolongs the bio-significant-and-yet-lower concentration of bee venom in mice bodies. In contrast, SC could produce only a short period of much higher bee venom levels in the blood and brain. We also see that due to the concentration-response-curve of bee venom (represented by melittin): mice bodies do not require much higher bee venom concentration (seen in the SC group) to produce a much more significant neuroprotective effect (than seen in those treated with the MN method). Therefore, a MN could maintain bee venom levels in mice bodies at lower-yet-more-efficient concentrations. This is important, as bee venom can cause more adverse effects and pain sensations, at higher concentrations. For the first time, we confirmed that the pharmacokinetic advantages of MN delivered bee venom also guarantee a holistic neuroprotection effect (which was shown by SC delivered bee venom in previous research). This was proven via the results of the water maze experiments for long-term learning memory assessment and protein analysis of key neuronal regulatory proteins: BDNF, p-CREB, iNOS, and mArhR 1. In conclusion, for situations where we ought to administrate drugs at a more downward amount, such as bee venom, MN can keep the therapeutic concentrations at a lower, yet interestingly, more-efficient level.

Topics: Animals; Bee Venoms; Disease Models, Animal; Drug Delivery Systems; Melitten; Mice; Neuroprotective Agents; Scopolamine

High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1-7)-Melittin (CAMA) against three multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilization. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antimicrobial Peptides; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Infections; Larva; Melitten; Microbial Sensitivity Tests; Moths; Survival Rate

Acute pancreatitis is a disease associated with suffering and high lethality. Although the disease mechanism is unclear, phospholipase A2 (PLA2) produced by pancreatic acinar cells is a known pathogenic trigger. Here, we show macrophage membrane-coated nanoparticles with a built-in 'lure and kill' mechanism (denoted 'MΦ-NP(L&K)') for the treatment of acute pancreatitis. MΦ-NP(L&K) are made with polymeric cores wrapped with natural macrophage membrane doped with melittin and MJ-33. The membrane incorporated melittin and MJ-33 function as a PLA2 attractant and a PLA2 inhibitor, respectively. These molecules, together with membrane lipids, work synergistically to lure and kill PLA2 enzymes. These nanoparticles can neutralize PLA2 activity in the sera of mice and human patients with acute pancreatitis in a dose-dependent manner and suppress PLA2-induced inflammatory response accordingly. In mouse models of both mild and severe acute pancreatitis, MΦ-NP(L&K) confer effective protection against disease-associated inflammation, tissue damage and lethality. Overall, this biomimetic nanotherapeutic strategy offers an anti-PLA2 treatment option that might be applicable to a wide range of PLA2-mediated inflammatory disorders.

Topics: Acute Disease; Animals; Cytokines; Disease Models, Animal; Female; Humans; Inflammation; Macrophages; Melitten; Mice; Nanoparticles; Pancreatitis; Phospholipases A2; THP-1 Cells

Inflammation is an essential biological response that eliminates pathogenic bacteria and repairs tissue after injury. Acute kidney injury (AKI) is associated with systemic and intrarenal inflammation as the inflammatory process decreases renal function and promotes progression to advanced chronic kidney disease. Macrophages are key mediators of the inflammatory response; their activation influences the immune system and may have various effects. Classically activated type I macrophages (M1) produce a variety of pro-inflammatory cytokines at the lesion site. However, anti-inflammatory type II macrophages (M2) are alternatively activated upon exposure to anti-inflammatory cytokines and are associated with wound healing and tissue repair following AKI. Here, we used melittin from bee venom to enhance the polarization of M2 macrophages and promote renal recovery after AKI. Melittin was administered to mice intraperitoneally for 5 days at various concentrations (10, 50, and 100 µg/kg); serum creatinine and blood urea nitrogen (BUN) levels were analyzed 72 h after cisplatin administration to confirm renal dysfunction. Melittin inhibited the cisplatin-induced increase in creatinine and BUN, an indicator of renal dysfunction. The expression of M1 markers (CD16/32) decreased significantly, whereas that of M2 markers (CD206, Arg1nase I) increased after melittin administration. Consistently, tubular necrosis was substantially reduced in melittin-treated mice. Thus, melittin alleviates cisplatin-induced AKI by regulating M2 macrophage expression.

Topics: Acute Kidney Injury; Animals; Anti-Inflammatory Agents; Bee Venoms; Cisplatin; Cytokines; Disease Models, Animal; Inflammation Mediators; Kidney; Macrophage Activation; Macrophages; Male; Melitten; Mice; Phenotype

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.

Topics: Adult; Animals; Apoptosis; Biomarkers; CADASIL; Cell Proliferation; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Genetic Predisposition to Disease; Humans; Male; Melitten; Mice; Mice, Transgenic; Middle Aged; Muscle, Smooth, Vascular; Mutation; Myocytes, Smooth Muscle; Receptor, Notch3; rho-Associated Kinases; Signal Transduction; Vascular Diseases

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.

Topics: Acinetobacter; Acinetobacter Infections; Animals; Anti-Infective Agents; Bacteriolysis; Burns; Cell Membrane; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Melitten; Mice; Microbial Viability; Treatment Outcome; Wound Infection

To explore the effect of combination therapy of epirubicin (Epi) and melittin (Mel) to cancer cells, calcium carbonate nanoparticles (CCN), as carriers, were developed which were modified with MUC1-Dimer aptamers as targeting agents. Both Epi and Mel were delivered at the same time to cancer cells overexpressing the target of MUC1 aptamer, mucin 1 glycoproteins (MCF7 and C26 cells). CCN were prepared with a water-in-oil emulsion method. Epi and Mel were separately encapsulated in CCN and the nanoparticles were modified with MUC1-Dimer aptamers. In vitro studies, including MTT assay, flow cytometry analysis and fluorescence imaging were applied to investigate the targeting and cell proliferation inhibition capabilities of MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex in the target (MCF-7 and C26 cells) and nontarget (HepG2) cells. Also, the function of the developed complexes was analyzed using in vivo tumor growth inhibition. The release of Epi from MUC1-Dimer aptamer-CCN-Epi complex was pH-sensitive. Cellular uptake studies showed more internalization of the MUC1-Dimer aptamer-CCN-Epi complex into MCF-7 and C26 cells (target) compared to HepG2 cells (nontarget). Interestingly, the MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex indicated very low toxicity as compared to target cells. Moreover, co-delivery of Epi and Mel using the mixture of MUC1-Dimer aptamer-CCN-Mel complex and MUC1-Dimer aptamer-CCN-Epi complex exhibited strong synergistic cytotoxicity in MCF-7 and C26 cells. Furthermore, the presented complexes had a better function to control tumor growth in vivo compared to free Epi.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aptamers, Peptide; Calcium Carbonate; Cell Line, Tumor; Delayed-Action Preparations; Disease Models, Animal; Drug Carriers; Drug Screening Assays, Antitumor; Drug Synergism; Epirubicin; Female; Humans; Melitten; Mice; Mice, Inbred BALB C; Mucin-1; Nanoparticles; Neoplasms; Treatment Outcome

Melittin (MEL) is a 26-amino acid peptide with numerous biological activities. Paraquat (PQ) is one of the most widely used herbicides, although it is extremely toxic to humans. To date, PQ poisoning has no effective treatment, and therefore the current study aimed to assess for the first time the possible effects of MEL on PQ-induced lung injuries in mice. Mice received a single intraperitoneal (IP) injection of PQ (30 mg/kg), followed by IP treatment with MEL (0.1 and 0.5 mg/kg) twice per week for four consecutive weeks. Histological alterations, oxidative stress, and apoptosis in the lungs were studied. Hematoxylin and eosin (H&E) staining indicated that MEL markedly reduced lung injuries induced by PQ. Furthermore, treatment with MEL increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity, and decreased malonaldehyde (MDA) and nitric oxide (NO) levels in lung tissue homogenates. Moreover, immunohistochemical staining showed that B-cell lymphoma-2 (Bcl-2) and survivin expressions were upregulated after MEL treatment, while Ki-67 expression was downregulated. The high dose of MEL was more effective than the low dose in all experiments. In summary, MEL efficiently reduced PQ-induced lung injuries in mice. Specific pharmacological examinations are required to determine the effectiveness of MEL in cases of human PQ poisoning.

Topics: Animals; Apoptosis; Biopsy; Catalase; Disease Models, Animal; Glutathione Peroxidase; Histocytochemistry; Lung Injury; Malondialdehyde; Melitten; Mice; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Paraquat; Proto-Oncogene Proteins c-bcl-2; Superoxide Dismutase

Melittin is a major peptide component of sweet bee venom that possesses anti-allergic, anti-inflammatory, anti-arthritis, anti-cancer, and neuroprotective properties. However, the therapeutic effects of melittin on muscle injury have not been elucidated. We investigated the therapeutic effects of melittin on muscle injury in a mouse model of muscle contusion. The biceps femoris muscle of the mice was injured using drop mass method, and the animals were treated with melittin (4, 20, or 100 μg/kg) for 7 days. Melittin significantly increased: locomotor activity in open field test, and treadmill running activity in a dose-dependent manner to level comparable to the positive control, diclofenac (30 mg/kg). Melittin treatment attenuated the pro-inflammatory cytokine MCP-1, TNF-α and IL-6. The expression of muscle regeneration biomarkers, including MyoD (muscle differentiation marker), myogenin, smooth muscle actin, and myosin heavy chain was markedly increased in the injured muscle tissue of melittin-treated mice, as determined by western blotting and quantitative real-time polymerase chain reaction. These results demonstrate that melittin inhibits inflammatory response and improves muscle damage by regenerating muscles in a mouse model of muscle contusion. Taken together, the results of present study suggest that melittin is a promising candidate for the muscle injury treatment.

Topics: Actins; Animals; Anti-Inflammatory Agents; Bee Venoms; Chemokine CCL2; Contusions; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression; Inflammation Mediators; Interleukin-6; Male; Melitten; Mice, Inbred C57BL; Muscle, Skeletal; MyoD Protein; Myogenin; Myosin Heavy Chains; Regeneration; Tumor Necrosis Factor-alpha

Bee venom contains a number of pharmacologically active components, including enzymes and polypeptides such as phospholipase A

Topics: Animals; Bee Venoms; Disease Models, Animal; Dopaminergic Neurons; Male; Melitten; Mice, Inbred C57BL; Neuroprotective Agents; Parkinsonian Disorders; Phospholipases A2; T-Lymphocytes, Regulatory

Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.

Topics: Acinetobacter baumannii; Acinetobacter Infections; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Disease Models, Animal; Hydrogel, Polyethylene Glycol Dimethacrylate; Magainins; Melitten; Mice; Microscopy, Confocal; Microscopy, Electron; Recombinant Fusion Proteins; Time Factors; Treatment Outcome; Wound Healing; Wound Infection; Xenopus Proteins

Atopic dermatitis (AD) is a multifactorial skin condition with complex interactions of innate and adaptive immune responses. There are several existing therapies for AD, including topical glucocorticosteroids, emollients, phototherapies, calcineurin inhibitors and immunosuppressants, such as cyclosporine A. Although these therapies reduce inflammation, they also cause serious side effects. Therefore, it is necessary to develop new therapeutic approaches for AD treatment without side effects. There are several studies on natural materials or toxins, such as herbs, ginseng extract and snake venom, for AD treatment. However, treatment of AD with bee venom and its major component, melittin has rarely been studied.. Effects of bee venom and melittin were studied in a model of AD in vivo induced by 1-chloro-2,4-dinitrobenzene (DNCB) in female Balb/c mice and in cultures of human keratinocytes, stimulated by TNF-α/IFN-γ. The potential pharmacological effects of bee venom and melittin on these in vivo and in vitro AD-like skin disease models were studied.. Bee venom and melittin exhibited potent anti-atopic activities, shown by decreased AD-like skin lesions, induced by DNCB in mice. In vitro studies using TNF-α/IFN-γ-stimulated human keratinocytes showed that bee venom and melittin inhibited the increased expression of chemokines, such as CCL17 and CCL22, and pro-inflammatory cytokines, including IL-1β, IL-6 and IFN-γ, through the blockade of the NF-κB and STAT signalling pathways.. Our results suggest that bee venom and melittin would be suitable for epicutaneous application, as topical administration is often appropriate for the treatment of AD.

Topics: Animals; Bee Venoms; Cell Differentiation; Cell Survival; Cells, Cultured; Dermatitis, Atopic; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Melitten; Mice; Mice, Inbred BALB C; Structure-Activity Relationship

The present study was aimed to establish a model of chronic prostatitis in rat with the use of intraprostatic injection of Complete Freund's Adjuvant, and to examine the anti-inflammatory and analgesic effects of melittin on the newly-developed chronic prostatic pain model.. Adult male Sprague-Dawley rats were injected with Complete Freund's Adjuvant (CFA) into the prostate. Twelve days after model rats of the treatment group were injected melittin into the prostate, while those of the control group received sterile saline injection. The nociceptive effects of CFA were evaluated by using a behavior approach (i.e. mechanical pain threshold measurement) on the day of CFA injection and 6, 12, and 18days after CFA injection. After the in-live study was done, the prostate was collected for histological examination of inflammatory cell infiltration. Levels of cyclooxygenase (COX)-2 in prostate and glial fibrillary acidic protein (GFAP) in spinal cord were determined using immunohistochemistry. Rats of the sham control group received intraprostatic injection of sterile saline and were studied using the same methods RESULTS: Intraprostatic CFA injection induced local allodynia that lasted over at least 2 weeks. The pain behavior of rat was associated with increases in inflammatory cell infiltration into the prostate. Levels of COX-2 in prostate and GFAP in spinal cord were also elevated. Treatment with melittin significantly raised pain threshold, decreased inflammatory infiltrates, and suppressed COX-2 and GFAP expression.. Intraprostatic injection of CFA induced neurogenic prostatitis and prostatic pain. The established model will be useful to the study of CP/CPPS pathogenesis. Melittin demonstrated profound anti-inflammatory and analgesic effects on the chronic prostatic pain model, suggesting melittin may hold promise as a novel therapeutic for treatment of CP/CPPS.

Topics: Animals; Chronic Disease; Cyclooxygenase 2; Disease Models, Animal; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Hyperalgesia; Inflammation; Male; Melitten; Pain; Pain Measurement; Pain Threshold; Prostatitis; Rats; Rats, Sprague-Dawley; Spinal Cord

Atopic dermatitis (AD) is an inflammatory skin disease characterized by intense pruritus and relapsable eczematous lesions. The hallmarks of AD are defects in the epidermal barrier and immunoglobulin E (IgE)-mediated sensitization to several environmental allergens, as well as an immune disorder mediated by an imbalance toward T-helper-2 response. Melittin, a major component of bee venom, has been studied in various inflammatory diseases. However, the beneficial effects of melittin on mouse with AD-like symptoms have not been explored. Therefore, we investigated the anti-allergic effects of melittin. AD was induced by ovalbumin (OVA) patch. After agent treatment, skin tissues and sera were extracted from the sacrificed mice were used to demonstrate the effects of melittin through various molecular biological methods. The results showed that OVA-induced skin thickening and inflammatory infiltration were decreased in the melittin-treated group. Melittin prevented OVA-induced filaggrin deficiency and imbalanced inflammatory mediators. Furthermore, melittin inhibited IL-4/IL-13-induced filaggrin downregulation through the blockade of STAT3 activation in human keratinocytes. In summary, this study has shown that melittin ameliorated OVA-induced AD-like symptoms from various perspectives. The findings of this study may be the first evidence of the anti-inflammatory effects of melittin on OVA-induced AD.

Topics: Allergens; Animals; Cells, Cultured; Dermatitis, Atopic; Disease Models, Animal; Female; Filaggrin Proteins; Immunoglobulin E; Interleukin-13; Keratinocytes; Melitten; Mice; Mice, Inbred BALB C; Ovalbumin; Skin; STAT3 Transcription Factor

Endothelial progenitor cells (EPCs) are important in tumor angiogenesis. Stromal cell-derived factor-1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) are key in stem cell homing. Melittin, a component of bee venom, exerts antitumor activity, however, the underlying mechanisms remain to be elucidated. The present study aimed to assess the effects of melittin on EPCs and angiogenesis in a mouse model of osteosarcoma. UMR‑106 cells and EPCs were treated with various concentrations of melittin and cell viability was determined using the MTT assay. EPC adherence, migration and tube forming ability were assessed. Furthermore, SDF‑1α, AKT and extracellular signal‑regulated kinase (ERK)1/2 expression levels were detected by western blotting. Nude mice were inoculated with UMR‑106 cells to establish an osteosarcoma mouse model. The tumors were injected with melittin, and its effects were assessed by immunohistochemistry and immunofluorescence. Melittin decreased the viability of UMR‑106 cells and EPCs. In addition, it decreased EPC adhesion, migration and tube formation when compared with control and SDF‑1α‑treated cells. Melittin decreased the expression of phosphorylated (p)‑AKT, p‑ERK1/2, SDF‑1α and CXCR4 in UMR‑106 cells and EPCs when compared with the control. The proportions of cluster of differentiation (CD)34/CD133 double‑positive cells were 16.4±10.4% in the control, and 7.0±4.4, 2.9±1.2 and 1.3±0.3% in tumors treated with 160, 320 and 640 µg/kg melittin per day, respectively (P<0.05). At 11 days, melittin reduced the tumor size when compared with that of the control (control, 4.8±1.3 cm3; melittin, 3.2±0.6, 2.6±0.5, and 2.0±0.2 cm3 for 160, 320 and 640 µg/kg, respectively; all P<0.05). Melittin decreased the microvessel density, and SDF‑1α and CXCR4 protein expression levels in the tumors. Melittin may decrease the effect of osteosarcoma on EPC‑mediated angiogenesis, possibly via inhibition of the SDF-1α/CXCR4 signaling pathway.

Topics: Animals; Biomarkers; Bone Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Chemokine CXCL12; Disease Models, Animal; Endothelial Progenitor Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Melitten; Mice; Neovascularization, Pathologic; Osteosarcoma; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Cytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.. The specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h (51)Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.. The melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.. This study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cellular Microenvironment; Cytotoxicity, Immunologic; Disease Models, Animal; Female; Humans; Immunomodulation; Immunotherapy; Interferon-gamma; Lung Neoplasms; Lymphocyte Activation; Melitten; Mice, Inbred BALB C; Mutant Proteins; Neoplasms; Recombinant Fusion Proteins

Renal fibrosis is the principal pathological process underlying the progression of chronic kidney disease that leads to end-stage renal disease. Melittin is a major component of bee venom, and it has anti-bacterial, anti-viral, and anti-inflammatory properties in various cell types. Thus, this study examined the therapeutic effects of melittin on the progression of renal fibrosis using the unilateral ureteral obstruction (UUO) model. In addition, the effects of melittin on inflammation and fibrosis in renal fibroblast cells were explored using transforming growth factor-β1 (TGF-β1). Histological observation revealed that UUO induced a considerable increase in the number of infiltrated inflammatory cells. However, melittin treatment markedly reduced these reactions compared with untreated UUO mice. The expression levels of inflammatory cytokines and pro-fibrotic genes were significantly reduced in melittin-treated mice compared with UUO mice. Melittin also effectively inhibited fibrosis-related gene expression in renal fibroblasts NRK-49F cells. These findings suggest that melittin attenuates renal fibrosis and reduces inflammatory responses by the suppression of multiple growth factor-mediated pro-fibrotic genes. In conclusion, melittin may be a useful therapeutic agent for the prevention of fibrosis that characterizes the progression of chronic kidney disease.

Topics: Animals; Cell Line; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Male; Melitten; Mice; Mice, Inbred BALB C; Renal Insufficiency, Chronic; Transforming Growth Factor beta1; Ureteral Obstruction

Melittin is the main component in the venom of the honey bee (Apis mellifera). It has multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-inflammatory mechanisms of melittin have not been elucidated in Propionibactierium acnes (P. acnes)-induced keratinocyte or inflammatory skin disease animal models. In this study, we examined the effects of melittin on the production of inflammatory cytokines in heat-killed P. acnes-induced HaCaT cells. Heat-killed P. acnes-treated keratinocytes increased the expression of pro-inflammatory cytokines and Toll-like receptor 2. However, melittin treatment significantly suppressed the expression of these cytokines through regulation of the NF-κB and MAPK signaling pathways. Subsequently, the living P. acnes (1 × 10(7) CFU) were intradermally injected into the ear of mice. Living P. acnes-injected ears showed cutaneous erythema, swelling, and granulomatous response at 24 hours after injection. However, melittin-treated ears showed markedly reduced swelling and granulomatous responses compared with ears injected with only living P. acnes. These results demonstrate the feasibility of applying melittin for the prevention of inflammatory skin diseases induced by P. acnes.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Disease Models, Animal; Gene Expression; Gram-Positive Bacterial Infections; Humans; Interleukin-1beta; Keratinocytes; MAP Kinase Signaling System; Melitten; Mice; Mice, Inbred ICR; NF-kappa B; Propionibacterium acnes; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

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.. 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.. 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.. 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.

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Goats; Kidney; Kidney Function Tests; Male; Melitten; Renal Replacement Therapy

Acute hepatic failure remains an extremely poor prognosis and still results in high mortality. Therefore, better treatment is urgently needed. Melittin, a major component of bee venom, is known to inhibit inflammatory reactions induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α in various cell types. However, there is no evidence of the anti-inflammatory and anti-apoptotic effect of melittin on liver cells. In the present study, we investigated the effects of melittin on D: -galactosamine (GalN)/lipopolysaccharide (LPS)-induced acute hepatic failure. Acute liver injury was induced with GalN/LPS to determine in vivo efficacy of melittin. Mice were randomly divided into four groups: sterile saline treated group (NC), melittin only treated group (NM), GalN/LPS-treated group (GalN/LPS), and GalN/LPS treated with melittin group (M+GalN/LPS). Mice were given intraperitoneal GalN/LPS with or without melittin treatment. Liver injury was assessed biochemically and histologically. Inflammatory cytokines in the serum, apoptosis of hepatocytes, and cleavage of caspase-3 in the liver were determined. The expression of TNF-α and interleukin (IL)-1β were increased in the GalN/LPS group. However, treatment of melittin attenuated the increase of inflammatory cytokines. The M+GalN/LPS group showed significantly fewer apoptotic cells compared to the GalN/LPS group. Melittin significantly inhibited the expression of caspase and bax protein levels as well as cytochrome c release in vivo. In addition, melittin prevented the activation of the transcription factor nuclear factor-kappa B (NF-κB) induced by GalN/LPS. These results clearly indicate that melittin provided protection against GalN/LPS-induced acute hepatic failure through the inhibition of inflammatory cytokines and apoptosis.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cytokines; Disease Models, Animal; Hepatocytes; Humans; Liver Failure, Acute; Male; Melitten; Mice; Mice, Inbred C57BL; Protective Agents

Melittin is the main peptide in bee venom and causes both persistent spontaneous nociception and pain hypersensitivity. Our recent studies indicated that both transient receptor potential (TRP) vanilloid receptor 1 (TRPV1) and canonical TRPs (TRPCs) are involved in mediating the melittin-induced activation of different subpopulations of primary nociceptive cells. Here, we further determined whether TRPC channels are involved in melittin-induced inflammatory nociceptive responses in behavioral assays.. The anti-nociceptive and anti-hyperalgesic effects of localized peripheral administration of three doses of the non-selective TRPC antagonist, SKF-96365 (1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl}-1H-imidazole hydrochloride), were evaluated in melittin tests. Pain-related behaviors were rated by counting the number of paw flinches, and measuring paw withdrawal thermal latency (s) and paw withdrawl mechanical threshold (g), over a 1-h time-course.. Localized peripheral SKF-96365 given before melittin prevented, and given after melittin significantly suppressed, the melittin-evoked persistent spontaneous nociception. Pre-blockade and post-suppression of activation of primary nociceptive activity resulted in decreased hypersensitivity to both thermal and mechanical stimuli applied to the primary injury site of the ipsilateral hindpaw, despite dose-effect differences between thermal and mechanical hyperalgesia. However, local administration of SKF-96365 into the contralateral hindpaw had no significant effect on any pain-associated behaviors. In addition, SKF-96365 had no effect on baseline threshold for either thermal or mechanical sensitivity under normal conditions.. Besides TRPV1, SKF-96365-sensitive TRPC channels might also be involved in the pathophysiological processing of melittin-induced inflammatory pain and hypersensitivity. Therapeutically, SKF-96365 is equally effective in preventing primary thermal and mechanical hyperalgesia as well as persistent spontaneous nociception. However, this drug is likely to be more effective in the relief of thermal hyperalgesia than mechanical hyperalgesia when applied 5 min after establishment of primary afferent activation.

Topics: Animals; Calcium Channel Blockers; Chronic Pain; Disease Models, Animal; Ganglia, Spinal; Imidazoles; Inflammation; Inflammation Mediators; Male; Melitten; Nociceptors; Rats; Rats, Sprague-Dawley; TRPC Cation Channels

Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder characterized by the progressive degeneration and death of motor neurons and occurs both as a sporadic and familial disease. Mutant SOD1 (mtSOD1) in motor neurons induces vulnerability to the disease through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport- and growth factor signaling, excitotoxicity, and neuro-inflammation.Melittin is a 26 amino acid protein and is one of the components of bee venom which is used in traditional Chinese medicine to inhibit of cancer cell proliferation and is known to have anti-inflammatory and anti-arthritic effects.The purpose of the present study was to determine if melittin could suppress motor neuron loss and protein misfolding in the hSOD1G93A mouse, which is commonly used as a model for inherited ALS. Meltittin was injected at the 'ZuSanLi' (ST36) acupuncture point in the hSOD1G93A animal model. Melittin-treated animals showed a decrease in the number of microglia and in the expression level of phospho-p38 in the spinal cord and brainstem. Interestingly, melittin treatment in symptomatic ALS animals improved motor function and reduced the level of neuron death in the spinal cord when compared to the control group. Furthermore, we found increased of α-synuclein modifications, such as phosphorylation or nitration, in both the brainstem and spinal cord in hSOD1G93A mice. However, melittin treatment reduced α-synuclein misfolding and restored the proteasomal activity in the brainstem and spinal cord of symptomatic hSOD1G93A transgenic mice.Our research suggests a potential functional link between melittin and the inhibition of neuroinflammation in an ALS animal model.

Topics: alpha-Synuclein; Amyotrophic Lateral Sclerosis; Animals; Behavior, Animal; Brain Stem; Cell Death; Disease Models, Animal; Humans; Male; Melitten; Mice; Mice, Transgenic; Motor Activity; Nerve Degeneration; Proteasome Endopeptidase Complex; Protein Folding; Rotarod Performance Test; Spinal Cord; Superoxide Dismutase; Survival Rate

Bee stings are a health concern in the Americas, where fatal envenomings due to massive attacks by Africanized honeybees have been documented in the last decades. Most studies on the toxic effects of honeybee venom in experimental animals have been performed using the intravenous or intraperitoneal injection routes. The aim of this study was to develop a mouse model that would better resemble a massive honeybee attack by using the subcutaneous (s.c.) route to induce a severe, sublethal systemic envenoming. An array of acute venom effects were characterized, including biochemical, hematological, histological, and inflammatory alterations, after the s.c. injection of 0.5 median lethal dose of venom. Rapid increases in serum alanine (ALT) and aspartate (AST) transaminases, creatinine, urea nitrogen, uric acid, sodium and chloride electrolytes, and creatine kinase (CK) were recorded, indicating damage to liver, kidneys, and skeletal muscle. Also, coagulation disturbances (fibrinogen decrease, and moderate delay in prothrombin and partial thromboplastin times) were demonstrated. Circulating platelet and leukocyte numbers remained unaltered, but a hemoconcentration effect (hematocrit and hemoglobin increase) was observed. This effect might be related to the marked edema induced by the venom. In addition, this inflammatory response included a systemic increase in cytokines (IL-1 beta, IL-6, TNF-alpha), together with an elevation of serum malondialdehyde and nitric oxide. The myotoxic effects of venom, melittin, and phospholipase A(2) were demonstrated after injection by s.c. route. No synergistic myotoxicity between melittin and PLA(2) was observed. Moreover, these two components, when injected at equivalent concentrations to those present in venom, induced a lower increase in serum CK than venom, suggesting that other components also contribute to its strong systemic toxicity towards skeletal muscle. The model here presented may be useful in preclinical studies to assess therapeutic antivenoms developed to cope with the problem of massive bee attacks.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bee Venoms; Bees; Biomarkers; Blood Coagulation; Cytokines; Disease Models, Animal; Drug Synergism; Female; Injections, Subcutaneous; Insect Bites and Stings; Lethal Dose 50; Male; Melitten; Mice; Mice, Inbred Strains; Myositis; Oxidative Stress; Phospholipases A2

Bee venom (BV) has been used for millennia in Chinese traditional medicine to treat rheumatoid arthritis (RA). However, its components and mechanism remain unclear, which has hampered its development and application for the treatment of RA. In this study, we examined the anti-arthritis effects of melittin, which composes nearly 50% of the dry weight of whole BV, on the complete Freund's adjuvant-induced (CFA-induced) RA model in rats. The RA animal models were treated with solutions of BV, melittin, and saline by injection into a specific acupoint (Zusanli). The BV and melittin treatments statistically diminished the thickness of the arthroses in the injected side of the paw, compared to the saline treatment. Melittin therapy also significantly reduced arthritis-induced nociceptive behaviors, as assessed by the thermal hyperalgesia test. In addition, CFA-induced Fos expression in the superficial layer of the lumbar spinal cord was significantly suppressed by the BV and melittin treatments, compared to the saline treatment. These results indicate that melittin is an effective anti-arthritis component of whole bee venom, making it a promising candidate as an anti-arthritis drug.

Topics: Acupuncture Points; Animals; Antirheumatic Agents; Apitherapy; Arthritis, Experimental; Arthritis, Rheumatoid; Bee Venoms; Disease Models, Animal; Freund's Adjuvant; Hot Temperature; Hyperalgesia; Lower Extremity; Male; Melitten; Pain; Rats; Rats, Sprague-Dawley; Spinal Cord

Surfactant protein (SP)-D plays an important role in host defense and pulmonary surfactant homeostasis. In SP-D-deficient (Sftpd(-/-)) mice, the abnormal large surfactant forms seen at the ultrastructural level are taken up inefficiently by type II cells, resulting in an over threefold increase in the surfactant pool size. The mechanisms by which SP-D influences surfactant ultrastructure are unknown. We hypothesized that SP-D binds to surfactant immediately after being secreted and influences surfactant ultrastructure conversion. In newborn and adult sheep lungs, immunogold-labeled SP-D was associated with both lamellated membranous lipid structures of newly secreted surfactant and with small aggregate surfactant but not with tubular myelin. Since SP-D preferentially binds to phosphatidylinositol (PI) in vitro, the postnatal changes in PI were assessed. PI content in the bronchoalveolar lavage fluid increased after birth and peaked at 2-5 days of age, a time of rapid conversion of surfactant forms that is associated with the peak of surfactant lipid pool size. SP-D selectively interacted with PI-rich liposomes in vitro, causing their lysis. Similarly, the abnormal surfactant ultrastructure in Sftpd(-/-) mice was corrected by the addition of SP-D or melittin, and both peptides caused lysis of lipid vesicles. The normal conversion of surfactant ultrastructure requires SP-D that preferentially interacts with PI-rich, newly secreted surfactant, causing lysis of surfactant lipid membranes, converting the lipid forms into smaller surfactant lamellated structures that are critical for surfactant uptake by type II cells and normal surfactant homeostasis. SP-D regulates the dramatic decreases in the surfactant pool size that occurs in the newborn period.

Topics: Age Factors; Animals; Animals, Newborn; Bronchoalveolar Lavage Fluid; Cytoplasmic Granules; Disease Models, Animal; Humans; Immunohistochemistry; Lung; Melitten; Membrane Lipids; Mice; Mice, Knockout; Phosphatidylcholines; Pulmonary Surfactant-Associated Protein D; Pulmonary Surfactants; Recombinant Proteins; Sheep

Melittin, a water-soluble toxic peptide derived from bee venom of Apis mellifera was reported to have inhibitory effects on hepatocellular carcinoma (HCC). However, its role in antimetastasis and the underlying mechanism remains elusive. By utilizing both HCC cell lines and an animal model based assay system, we found that Rac1, which has been shown to be involved in cancer cell metastasis, is highly expressed in aggressive HCC cell lines and its activity correlated with cell motility and cytoskeleton polymerization. In addition, Rac1-dependent activity and metastatic potential of aggressive HCC cells are remarkably high in both cellular and nude mouse models. We provide evidence here that melittin inhibits the viability and motility of HCC cells in vitro, which correlates with its suppression of Rac1-dependent activity, cell motility, and microfilament depolymerization. Furthermore, melittin suppresses both HCC metastasis and Rac1-dependent activity in nude mouse models. The specificity of the effect of melittin on Rac1 was confirmed in HCC cells both in vitro and in vivo.. Melittin inhibits tumor cell metastasis by reducing cell motility and migration via the suppression of Rac1-dependent pathway, suggesting that melittin is a potential therapeutic agent for HCC.

Topics: Actin Cytoskeleton; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MAP Kinase Kinase 4; Melitten; Mice; Mice, Nude; Neoplasm Metastasis; rac1 GTP-Binding Protein; Signal Transduction; Xenograft Model Antitumor Assays

Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistant to chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid (81-95) fragment of the human chorionic gonadotropin beta (CGbeta) chain, which would selectively kill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To prove the principle that Hecate-CGbeta conjugate may eradicate tumors ectopically expressing plasma membrane receptors, transgenic (TG) inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimental model. Wild-type control littermates and TG mice with adrenal tumors were treated with either Hecate or Hecate-CGbeta conjugate at the age of 6.5 months for 3 weeks and killed 7 days after the last treatment. The Hecate-CGbeta conjugate reduced the adrenal tumor burden significantly in TG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGbeta conjugate treatment did not affect normal adrenocortical function as the serum corticosterone level between Hecate and Hecate-CGbeta conjugate groups were similar. The mRNA and protein expressions of GATA-4 and LHR colocalized only in tumor area, and a significant downregulation of gene expression was found after the Hecate-CGbeta conjugate in comparison with Hecate- and/or non-treated adrenal tumors by western blotting. This finding provides evidence for a selective destruction of the tumor cells by the Hecate-CGbeta conjugate. Hereby, our findings support the principle that Hecate-CGbeta conjugate is able to specifically destroy tumor cells that ectopically express LHR.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Adenoma; Animals; Antineoplastic Agents; Chorionic Gonadotropin, beta Subunit, Human; Disease Models, Animal; Female; GATA4 Transcription Factor; Gene Expression Regulation, Neoplastic; Luteinizing Hormone; Male; Melitten; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptors, LH; RNA, Messenger

To investigate whether phospholipase A2 (PLA2) plays a role in the pathogenesis of spinal cord injury (SCI).. Biochemical, Western blot, histological, immunohistochemical, electron microscopic, electrophysiological, and behavior assessments were performed to investigate (1) SCI-induced PLA2 activity, expression, and cellular localization after a contusive SCI; and (2) the effects of exogenous PLA2 on spinal cord neuronal death in vitro and tissue damage, inflammation, and function in vivo.. After SCI, both PLA2 activity and cytosolic PLA2 expression increased significantly, with cytosolic PLA2 expression being localized mainly in neurons and oligodendrocytes. Both PLA2 and melittin, an activator of endogenous PLA2, induced spinal neuronal death in vitro, which was substantially reversed by mepacrine, a PLA2 inhibitor. When PLA2 or melittin was microinjected into the normal spinal cord, the former induced confined demyelination and latter diffuse tissue necrosis. Both injections induced inflammation, oxidation, and tissue damage, resulting in corresponding electrophysiological and behavioral impairments. Importantly, the PLA2-induced demyelination was significantly reversed by mepacrine.. PLA2, increased significantly after SCI, may play a key role in mediating neuronal death and oligodendrocyte demyelination following SCI. Blocking PLA2 action may represent a novel repair strategy to reduce tissue damage and increase function after SCI.

Topics: Aldehydes; Animals; Apoptosis; Blotting, Western; CD11b Antigen; Cell Count; Cells, Cultured; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Female; Gene Expression; Glial Fibrillary Acidic Protein; Hydro-Lyases; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Melitten; Microscopy, Electron, Transmission; Motor Activity; Neurons; Oligodendroglia; Phospholipases A; Phospholipases A2; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Time Factors

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Topics: alpha-Defensins; Animals; Anti-Bacterial Agents; Bacterial Proteins; Burkholderia; Burkholderia Infections; Colony Count, Microbial; Disease Models, Animal; Drug Resistance, Bacterial; Gene Deletion; Genetic Complementation Test; Lipopolysaccharides; Lung; Melitten; O Antigens; Oligosaccharides; Peptides; Polymyxin B; Rats; Virulence

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Northern; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cell Separation; Chorionic Gonadotropin, beta Subunit, Human; Disease Models, Animal; Enzyme Activation; Female; Flow Cytometry; Granulosa Cell Tumor; Humans; Leydig Cell Tumor; Male; Melitten; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Necrosis; Ovarian Neoplasms; Progesterone; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, LH; Recombinant Fusion Proteins; Testicular Neoplasms; Time Factors

Synthetic peptides, ranging from 12 to 18 residues, containing partial sequences from natural cecropin A and melittin were tested for activity in an experimental pseudomonas keratitis model in rabbits. In separate experiments, two Pseudomonas aeruginosa strains: (a) a clinical isolated strain, and (b) an American Type Culture Collection (ATCC) strain, were inoculated into the stroma of one cornea of each rabbit. Peptides were topically applied at 0.1% in phosphate-buffered saline (PBS) and compared with PBS alone and 0.3% gentamicin eye drops. Clinical evaluation, based on the McDonald-Shadduck scale, was performed during a > 48-h period after the bacterial inoculation. The peptide-treated animals showed significantly lower (p < 0.05) inflammatory signs and lower anterior-segment bacterial damage compared with PBS-treated animals, after the first 6 h. The antiinflammatory/antimicrobial activity was non significantly differnt (p > 0.05) from that in animals treated with gentamicin. We conclude that peptides keeping the sequence KWKLFKK from cecropin A and at least the sequence VLKVL from melittin show promise as novel agents in topical ocular therapy of bacterial keratitis.

Topics: Administration, Topical; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Chromatography, High Pressure Liquid; Cornea; Corneal Ulcer; Disease Models, Animal; Eye Infections, Bacterial; Male; Melitten; Ophthalmic Solutions; Peptide Fragments; Peptides; Pseudomonas Infections; Rabbits; Random Allocation

Melittin (MLT) (10 micrograms/paw) and D49 (0.4 micrograms/paw) were injected into the hind paw of male CD-1 mice and elicited 70-80% of maximal paw edema responses at 60 and 30 min after injection, respectively. D49 paw edema was significantly inhibited by anti-histamine/serotonin agents, a PAF antagonist, a PLA2 inhibitor, and some but not all 5-LO and CO inhibitors, indicating that this edema is produced by several classes of inflammatory mediators with mast cell degranulation apparently playing a major role. In contrast, MLT paw edema was not inhibited effectively using the same pharmacological agents except theophylline, suggesting it was elicited via a different sequence of inflammatory events. In summary, D49 and MLT paw edema models were found to be ineffective models to identify experimental PLA2 compounds in our laboratory.

Topics: Animals; Anti-Inflammatory Agents; Cytoplasmic Granules; Disease Models, Animal; Edema; Foot; Histamine Antagonists; Male; Mast Cells; Melitten; Mice; Phospholipases A; Phospholipases A2

Injection into the rat paw of either melittin, its C-terminal dekapeptide, tetralysine or triarginine causes an inflammatory response with melittin having the highest and the last mentioned peptides the lowest activity. The inflammatory action of melittin and the dekapeptide has the following characteristics: 1. it is mainly due to the liberation of histamine, since on both compounds the anti-histaminic diphenhydramine has a pronounced inhibitory effect; 2. di-sodium-cromoglycate inhibits only the inflammatory action of the dekapeptide but has no effect on the activity of melittin. Melittin and its fragment thus liberate histamine via different routes.

Topics: Animals; Arginine; Cromolyn Sodium; Disease Models, Animal; Hindlimb; Histamine Release; Inflammation; Irritants; Lysine; Male; Melitten; Oligopeptides; Peptide Fragments; Rats; Venoms