RESUMEN
Methicillin resistant Staphylococcus aureus (MRSA) may be found on the skin, nose, and throats of long-term hospitalized patients. While MRSA infections are usually minor, serious infections and death may occur in immunocompromised or diabetic patients, or after exposure of MRSA to blood. This report demonstrates that the antimicrobial peptide (AMP) epinecidin-1 (Epi-1) efficiently protects against MRSA infection in a pyemia pig model. We first found that Epi-1 exhibits bactericidal activity against MRSA. Next, pharmacokinetic analysis revealed that Epi-1 was stable in serum for 4 h after injection, followed by a gradual decrease. This pharmacokinetic profile suggested Epi-1 may bind serum albumin, which was confirmed in vitro. Harmful effects were not observed for doses up to 100 mg/kg body weight in pigs. When Epi-1 was supplied as a curative agent 30 min post-infection, MRSA-induced abnormalities in blood uric acid (UA), blood urea nitrogen (BUN), creatine (CRE), GOT, and GPT levels were restored to normal levels. We further showed that the bactericidal activity of Epi-1 was higher than that of the antibiotic drug vancomycin. Epi-1 significantly decreased MRSA counts in the blood, liver, kidney, heart, and lungs of infected pigs. Elevated levels of serum C reactive protein (CRP), proinflammatory cytokine IL6, IL1ß, and TNFα were also attenuated by Epi-1 treatment. Moreover, the MRSA genes, enterotoxin (et)-A, et-B, intrinsic methicillin resistance A (mecA), and methicillin resistance factor A (femA), were significantly reduced or abolished in MRSA-infected pigs after treatment with Epi-1. Hematoxylin and eosin staining of heart, liver, lung, and kidney sections indicated that Epi-1 attenuated MRSA toxicity in infected pigs. A survival study showed that the pyemia pigs infected with MRSA alone died within a week, whereas the pigs post-treated with 2.5 mg/kg Epi-1 were completely protected against death. The present investigation, thus, demonstrates that Epi-1 effectively protects pyemia pigs against pathogenic MRSA without major toxic side effects.
Asunto(s)
Antibacterianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Proteínas de Peces/administración & dosificación , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacocinética , Péptidos Catiónicos Antimicrobianos/farmacología , Proteína C-Reactiva/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Peces/farmacocinética , Proteínas de Peces/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Porcinos , Vancomicina/farmacologíaRESUMEN
Local Treg responses are involved in Helicobacter pylori-related inflammation and clinical outcomes after infection, and H. pylori-derived HSP60 (HpHSP60) is an important virulence factor associated with gastric carcinogenesis. This study to investigate the role of HpHSP60 in immunosuppression, particularly with regard to whether it could induce the production of Treg cells. For this purpose, human peripheral blood mononuclear cells (PBMCs) were treated with or without HpHSP60 in the presence of an anti-CD3 mAb to determine the effect of HpHSP60 on cell proliferation. In this report, HpHSP60 decreased the expression of CDK4 to significantly arrest the proliferation of mitogen-stimulated T-cells, which correlated with the induction of Treg cells. Moreover, monocytic cells were essential for the induction of HpHSP60-induced Treg cells via the secretion of IL-10 and TGF-ß after treatment with HpHSP60. Blockage of HpHSP60 with specific monoclonal antibodies significantly reduced the colonization of H. pylori and the expression of Treg cells in vivo. Overall, our results suggest that HpHSP60 could act on macrophages to trigger the expression of IL-10 and TGF-ß, thereby leading to an increase in Treg cells and inhibition of T-cell proliferation.
Asunto(s)
Chaperonina 60/metabolismo , Chaperonina 60/farmacología , Helicobacter pylori/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Animales , Complejo CD3/inmunología , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Chaperonina 60/genética , Chaperonina 60/inmunología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Citocinas/metabolismo , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Regulación Bacteriana de la Expresión Génica , Infecciones por Helicobacter/inmunología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Terapia de Inmunosupresión , Inflamación , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Linfocitos T Reguladores/inmunología , Células THP-1 , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The antimicrobial peptide, epinecidin-1 (Epi), was identified from Epinephelus coioides and may have clinical application for treating sepsis. Epi has been shown to ameliorate antibiotic-resistant bacteria-induced sepsis in mice, but further evaluation in mixed-flora models and a description of the protective mechanisms are essential to establish this peptide as a potential therapeutic. Therefore, we first tested the protective effects of Epi against polymicrobial sepsis-induced bactericidal infection, inflammation and lung injury that result from cecal ligation and puncture in mice. Furthermore, since lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis, we also tested the LPS-antagonizing activity and related mechanisms of Epi-mediated protection in mice with LPS-induced endotoxemia and LPS-treated Raw264.7 mouse macrophage cells. Epi rescued mice from both polymicrobial sepsis and endotoxemia after delayed administration and suppressed both lung and systemic inflammatory responses, while attenuating lung injury and diminishing bacterial load. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species/p38/Akt/NF-κB signaling and subsequent cytokine production. Overall, our results demonstrate that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Endotoxemia/tratamiento farmacológico , Proteínas de Peces/farmacología , Sustancias Protectoras/farmacología , Animales , Antiinfecciosos/farmacología , Carga Bacteriana , Ciego/microbiología , Ciego/cirugía , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxemia/etiología , Femenino , Ligadura , Receptores de Lipopolisacáridos/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.
Asunto(s)
Lubina , Infecciones por Escherichia coli/veterinaria , Escherichia coli/fisiología , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Macrófagos/inmunología , Proteína Amiloide A Sérica/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Clonación Molecular , Citocinas , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Enfermedades de los Peces/genética , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Fagocitosis , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia/veterinaria , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMEN
The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.
Asunto(s)
Antígenos de Protozoos/inmunología , Infecciones por Cilióforos/prevención & control , Enfermedades de los Peces/prevención & control , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/administración & dosificación , Animales , Antígenos de Protozoos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Cilióforos/inmunología , Infecciones por Cilióforos/inmunología , Resistencia a la Enfermedad , Enfermedades de los Peces/inmunología , Proteínas HSP70 de Choque Térmico/administración & dosificación , Proteínas HSP70 de Choque Térmico/genética , Inmunoglobulina M/inmunología , Nanopartículas/administración & dosificación , Perciformes , Proteínas Protozoarias/administración & dosificación , Piel/inmunología , Tetrahymena thermophila/genéticaRESUMEN
Shrimp anti-lipopolysaccharide factor (SALF) is an antimicrobial peptide with reported anticancer activities, such as suppression of tumor progression. In this study, we prepared a potential cancer vaccine comprised of SALF in conjunction with the cell lysate of inactivated murine bladder carcinoma cells (MBT-2), and evaluated its efficacy in a mouse tumor model. Our study shows that SALF added to cell culture media inhibits growth progression of MBT-2, and that SALF together with inactivated MBT-2 lysate elevates the level of inflammasome activity, and modulates the levels of IL-1ß, MCP-1, IL-6, IL-12, and TNF-α in mouse macrophages. Immunization of 7, 14, and 21 day-old mice with the vaccine prevented growth of MBT-2 cell-mediated tumors. The vaccine was found to enhance expression of T-cell, cytotoxic T cells, and NK cells in the immunized mice groups. Recruitment of macrophages, T-helper cells, and NK cells was enhanced, but levels of VEGF were decreased in immunized mice. This report provides empirical evidence that our SALF as vaccine adjuvant enhances antitumor immunity in mice.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , Proteínas de Artrópodos/inmunología , Vacunas contra el Cáncer/inmunología , Lipopolisacáridos/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Animales , Antiinfecciosos/inmunología , Línea Celular Tumoral , Quimiocina CCL2/inmunología , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Antimicrobial peptides (AMPs) have recently been determined to be potential candidates for treating drug-resistant bacterial infections. Pardaxin (GE33), a marine antimicrobial peptide, has been reported to possess antimicrobial function. In this study, we investigated whether pardaxin promoted healing of contaminated wounds in mice. One square centimeter of outer skin was excised from the ventral region of mice, and a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA) was applied in the presence or absence of methicillin, vancomycin, or pardaxin. While untreated mice and mice treated with methicillin died within 3 days, mice treated with pardaxin survived infection. Pardaxin decreased MRSA bacterial counts in the wounded region and also enhanced wound closure. Reepithelialization and dermal maturation were also faster in mice treated with pardaxin than in mice treated with vancomycin. In addition, pardaxin treatment controlled excess recruitment of monocytes and macrophages and increased the expression of vascular endothelial growth factor (VEGF). In conclusion, these results suggest that pardaxin is capable of enhancing wound healing. Furthermore, this study provides an excellent platform for comparing the antimicrobial activities of peptide and nonpeptide antibiotics.
Asunto(s)
Antibacterianos/uso terapéutico , Venenos de los Peces/uso terapéutico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Animales , Carga Bacteriana , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Infecciones Cutáneas Estafilocócicas/microbiología , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/microbiologíaRESUMEN
Fish-scale collagen peptides (FSCPs) were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp.) scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature.
Asunto(s)
Estructuras Animales/química , Colágeno/farmacología , Peces/anatomía & histología , Péptidos/química , Péptidos/farmacología , Absorción Cutánea/efectos de los fármacos , Piel/efectos de los fármacos , Adulto , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Dermis/efectos de los fármacos , Dermis/fisiología , Difusión/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Ratones , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Conformación Proteica , Piel/citología , Piel/embriología , Adulto JovenRESUMEN
Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Pollos/inmunología , Sistema Inmunológico/metabolismo , Perciformes/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/análisis , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/análisis , Péptidos Catiónicos Antimicrobianos/clasificación , Péptidos Catiónicos Antimicrobianos/genética , Pollos/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Suplementos Dietéticos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Trichomoniasis is caused by infection with the protozoan parasite Trichomonas vaginalis, and prolonged persistence may lead to serious ill effects in patients. Thus, the development of new therapeutic strategies to combat drug-resistant T. vaginalis would be clinically beneficial. Antimicrobial peptides (AMPs) comprise an emerging class of molecules that may serve as effective alternatives to antibiotics. In this report, we demonstrate that the synthetic fish AMP, Epinecidin-1 (Epi-1), acts against T. vaginalis both in vitro and in vivo. Under in vitro conditions, Epi-1 disrupted the membrane of metronidazole-resistant T. vaginalis and completely killed the pathogen. To mimic human infection in vivo, estradiol-stimulated mice with vaginal Lactobacillus acidophilus colonization were infected with T. vaginalis, followed by treatment with Epi-1, Vigill, metronidazole or furazolidone. After seven days, the T. vaginalis content was effectively decreased in Epi-1 treated mice, as measured by acridine orange staining of wet smears and tissue biopsies, as well as qPCR of vaginal discharge DNA. Taken together, our results demonstrate that Epi-1 is a strong candidate for development as an alternative therapeutic for T. vaginalis infection.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/uso terapéutico , Antiprotozoarios/uso terapéutico , Proteínas de Peces/uso terapéutico , Tricomoniasis/tratamiento farmacológico , Trichomonas vaginalis/efectos de los fármacos , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Resultado del TratamientoRESUMEN
Picornavirus is a highly contagious virus that usually infects cloven hoofed animals and causes foot-and-mouth disease. This disease is a major threat to livestock breeding worldwide and may lead to huge economic losses. Because effective vaccines or antiviral drugs remain unavailable, the search for new agents to combat FMDV infections is ongoing. Antimicrobial peptides are known to possess a broad range of biological activities, including antibacterial, antiviral, antitumor and immunomodulatory effects. In this work, we used a cell culture FMDV replication assay to evaluate several antimicrobial peptides for their ability to act as antiviral agents. We found that a synthesized form of the Epinephelus coioides antimicrobial peptide, epinecidin-1 (Epi-1), was effective at combatting FMDV. Epi-1 is known to have broad spectrum antimicrobial activity and low toxicity to normal eukaryotic cells, making it a good candidate for use as a therapeutic agent.The 50% cytotoxic concentration (CC50) for BHK-21 cells was 19.5 µg/ml for synthesized Epi-1, and the 50% effective concentration (EC50) for viral inhibition was 0.6 µg/ml. The selectivity index was 31.4, as calculated by the CC50/EC50 ratio. Furthermore, Epi-1 showed virucidal activity against FMDV at high concentrations. Interestingly, our data also showed that FMDV infection was most impaired when Epi-1 was treated at the time of viral adsorption. Taken together, our data show that Epi-1 may be a promising candidate for development as an anti-FMDV agent.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Proteínas de Peces/farmacología , Virus de la Fiebre Aftosa/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Peces/genética , Virus de la Fiebre Aftosa/fisiología , Riñón/citología , Riñón/virología , Ganado/virología , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
This report shows that the antimicrobial peptide (AMP) Epinecidin-1 (Epi-1) efficiently heals MRSA-infected heat burn injuries and provides protection from infection in a pig model. The presence of an optimal level of Epi-1 induces cell proliferation by promoting cell cycle progression through an increase in S-phase cells. Epi-1 also induces proliferation to cover the wounded region in an in vitro cell proliferation assay using immortalized human epithelial HaCaT cells. Next, the in vivo wound healing efficiency of Epi-1 was tested in heat-burned pig skin infected with MRSA under in vivo conditions. Treatment of the injury with Epi-1 for 1 h at six hours post-infection completely healed the wound within 25 days. Conversely, the injury in the untreated control was not healed 25 days post-infection. Histological staining of wound sections with H&E showed that Epi-1 enhanced vascularization and increased epithelial activities in the wound region. Neutrophil recruitment to the wounded region in the Epi-1-treated sections was visualized by Giemsa staining. Additionally, Masson's trichrome staining of wound sections confirmed that Epi-1 enhanced extracellular collagen compound formation. The induction of sepsis-associated blood C-reactive protein (CRP) and the pro-inflammatory cytokine IL-6 in response to MRSA infection was also suppressed in pigs that received Epi-1. Taken together, the results demonstrate that the biomaterial Epi-1 heals wounds through increasing epithelial cell proliferation, vascularization, and the formation of collagen and controls MRSA infection-mediated sepsis in pigs.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Quemaduras/tratamiento farmacológico , Proteínas de Peces/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Piel/citología , Infecciones Estafilocócicas/tratamiento farmacológico , Infección de Heridas/tratamiento farmacológico , Animales , Quemaduras/microbiología , Células Cultivadas , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/microbiología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Piel/lesiones , Piel/microbiología , Infecciones Estafilocócicas/microbiología , Porcinos , Infección de Heridas/microbiologíaRESUMEN
Helicobacter pylori infection is marked by a strong association with various gastric diseases, including gastritis, ulcers, and gastric cancer. Antibiotic treatment regimens have low success rates due to the rapid occurrence of resistant H. pylori strains, necessitating the development of novel anti-H. pylori strategies. Here, we investigated the therapeutic potential of a novel peptide, Tilapia Piscidin 4 (TP4), against multidrug resistant gastric pathogen H. pylori, based on its in vitro and in vivo efficacy.TP4 inhibited the growth of both antibiotic-sensitive and -resistant H. pylori (CagA+, VacA+) via membrane micelle formation, which led to membrane depolarization and extravasation of cellular constituents. During colonization of gastric tissue, H. pylori infection maintains high T regulatory subsets and a low Th17/Treg ratio, and results in expression of both pro- and anti-inflammatory cytokines. Treatment with TP4 suppressed Treg subset populations and pro- and anti- inflammatory cytokines. TP4 restored the Th17/Treg balance, which resulted in early clearance of H. pylori density and recovery of gastric morphology. Toxicity studies demonstrated that TP4 treatment has no adverse effects in mice or rabbits. The results of this study indicate that TP4 may be an effective and safe monotherapeutic agent for the treatment of multidrug resistant H. pylori infections.
Asunto(s)
Antiinfecciosos/farmacología , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Timopoyetinas/farmacología , Animales , Antiinfecciosos/toxicidad , Modelos Animales de Enfermedad , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos C3H , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/toxicidad , Conejos , Distribución Aleatoria , Timopoyetinas/toxicidadRESUMEN
Helicobacter pylori (H. pylori) infection is highly prevalent, and has a strong association with various gastric diseases, including gastritis, digestive ulcers, and cancer. H. pylori strains with resistance to existing antibiotics have emerged in the past two decades. Currently, treatment of H. pylori infection (involving the use of proton pump inhibitors, followed by triple therapy with broad-spectrum antibiotics) is suboptimal, with high failure rates. As such, there is a clear need for new approaches against H. pylori. Here, we report that Epinecidin-1 (Epi-1) shows effective bactericidal activity against H. Pylori in vitro, and modulates H. Pylori-induced host immune responses in a mouse model. Epi-1 exhibited a low minimum inhibitory concentration (MIC) against antibiotic-sensitive and clinical antibiotic-resistant strains. Moreover, Epi-1 treatment caused 1-N-phenylnaphthylamine (NPN)-fluorescent probe uptake, suggesting it induced membrane lysis; transmission electron micrographs revealed that membranes were destabilized by the generation of saddle-splay membrane curvature. Oral administration of Epi-1 (quaque die dose) in a mouse infection model had strong efficacy (p < 0.00152) against H. pylori, as compared with conventional proton pump inhibitor (PPI)-triple therapeutic antibiotics. Epi-1 inhibited infection through in vivo depletion of CD4+-FOXP3+ T Regulatory and Th17 subset populations, and aided in clearance of persistent H. pylori colonization. Flow cytometry and gene expression analysis of mouse splenic and gastric tissue indicated that Epi-1 inhibits IL-10, and thereby affects FOXP3 expression levels and reduces pro-inflammatory cytokine responses. Crucially, high doses of Epi-1 did not exert toxic effects in oral, dermal, and eye irritation models. Collectively, our results suggest that Epi-1 may be a promising, effective, and safe monotherapeutic agent for the treatment of multi-drug resistant H. pylori infection.
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Péptidos Catiónicos Antimicrobianos/administración & dosificación , Proteínas de Peces/administración & dosificación , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/inmunología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/inmunología , Fluidez de la Membrana/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Infecciones por Helicobacter/patología , Helicobacter pylori/citología , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos C3H , Resultado del TratamientoRESUMEN
Antimicrobial peptides (AMPs), represent promising agents for new therapeutic approaches of infected wound treatment, on account of their antimicrobial and wound closure activities, and low potential for inducing resistance. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 23 amino-acid designer peptide TP3 possessed antimicrobial activities. Here, we analyzed the wound closure activities of TP3 both and in vivo. TP3 at doses of up to 40 µg/ml did not affect the viability of baby hamster kidney cells. Furthermore, TP3 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. TP3 treatment increased survival by 100% at 8 days after infection, and accelerated the progression of proliferation, remodeling, and maturation of infected wounds. Taken together, our results indicate that TP3 enhances the rate of survival of mice infected with the bacterial pathogen MRSA through both antimicrobial and immunomodulatory effects. Overall, these results suggest that TP3 may be suitable for development as a novel topical agent for treatment of infected wounds.
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Antiinfecciosos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Infecciones Estafilocócicas/prevención & control , Timopoyetinas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/prevención & control , Animales , Bacteriemia/tratamiento farmacológico , Bacteriemia/inmunología , Bacteriemia/microbiología , Bacteriemia/prevención & control , Cricetinae , Sinergismo Farmacológico , Femenino , Ratones , Ratones Endogámicos BALB C , Piel/lesiones , Piel/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/prevención & control , Cicatrización de Heridas/inmunología , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/inmunología , Infección de Heridas/microbiologíaRESUMEN
Pardaxin is a cationic antimicrobial peptide derived from Red Sea Moses sole. Previous studies have shown that pardaxin selectively triggers the death of cancer cells, initiating the development of a pardaxin-based cancer vaccine; however, the underlying mechanism by which pardaxin kills cancer cells has not yet been elucidated. Here, we demonstrate that this mechanism involves endoplasmic reticulum (ER) targeting and c-FOS induction. Transcriptiome analysis of pardaxin-treated HT-1080 cells revealed induction of the gene encoding c-FOS, an AP-1 transcription factor. Pardaxin mediates cell death by activating c-FOS, but not other AP-1 transcription factors. Overexpression of c-FOS caused a dramatic increase in cell death, while knockdown of c-FOS induced pardaxin resistance; such effects were observed in both an in vitro cell model and an in vivo xenograft tumor model. Treatment with pardaxin also increased the level of calcium, and blockage of cellular calcium signaling disrupted pardaxin-induced cell death. Immunocytochemistry was used to demonstrate targeting of pardaxin to the endoplasmic reticulum, but not to the Golgi apparatus or mitochondria. Importantly, pardaxin treatment or c-FOS overexpression induced cell death in diverse cancer cell lines, indicating that pardaxin and c-FOS may possess therapeutic potential for use in cancer treatment.
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Péptidos Catiónicos Antimicrobianos/farmacología , Retículo Endoplásmico/metabolismo , Venenos de los Peces/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Antineoplásicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Retículo Endoplásmico/efectos de los fármacos , Femenino , Venenos de los Peces/química , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) causes infections through open skin injuries, and its resistance makes treatment difficult. The antimicrobial peptide Epinecidin-1 (Epi-1) has been reported to possess antibacterial, antifungal, antiviral, and antitumor functions. This study investigated the antimicrobial activity of Epi-1 against skin trauma-mediated MRSA infection in mice. One square centimeter of outer skin was excised from the ventral region of mice, and a lethal dose of MRSA was applied in the presence or absence of methicillin, vancomycin, or Epi-1. While untreated mice and mice treated with methicillin died within four days, mice treated with Epi-1 survived infection. Epi-1 decreased MRSA bacterial counts in the wounded region, enhanced wound closure, and increased angiogenesis at the injury site. Treatment with Epi-1 decreased serum levels of the proinflammatory cytokines TNF-α, IL-6, and MCP-1, and regulated the recruitment of monocytes and clearance of lymphocytes around the wounded region during healing. In conclusion, Epi-1 may be effective at treating clinical MRSA, and may enhance wound recovery when combined with collagen.
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Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Proteínas de Peces/farmacología , Proteínas de Peces/uso terapéutico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Piel/lesiones , Piel/microbiología , Animales , RatonesRESUMEN
The marine antimicrobial peptide (AMP) GE33, also known as pardaxin, possesses antimicrobial and anticancer properties, and modulates host signaling. GE33 has cytotoxic effects on murine bladder carcinoma (MBT-2) cells. Here, we investigated the potential of GE33 combined with inactivated MBT-2 as a cancer vaccine. The presence of up to 12.5 µg of GE33 did not inhibit the proliferation or endogenous nitrous oxide (NO) levels of RAW264.7 cells. However, the secretion of MCP-1, IL-6, and IL-12 by RAW264.7 cells was affected by GE33. We proceeded to test the effectiveness of the vaccine by immunizing mice at 7, 14, and 21 days of age, and injecting live MBT-2 cells on the 28th day. Tumor growth by the 58th day was attenuated in mice treated with the vaccine, as compared to the control group. Induction of MBT-2 specific-tumor antigens was increased in mice immunized with our vaccine. Furthermore, activation of T-cell receptors, cytotoxic T-cells, and NK cells was enhanced, and these showed high specificity for targeting tumor cells. Finally, immunization controlled excess recruitment of monocytes, lymphocytes, T-helper cells, and NK cells, and decreased the expression of VEGF. This report provides empirical evidence that our GE33-based vaccine enhances antitumor immunity in mice.
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Vacunas contra el Cáncer/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/prevención & control , Animales , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Venenos de los Peces , Inmunohistoquímica , Interleucina-6/metabolismo , Masculino , Ratones , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Current efforts to improve the effectiveness of vaccines include incorporating antimicrobial peptides mixed with a virus. The antimicrobial peptide, epinecidin (Epi)-1, was reported to have an antiviral function, and an Epi-1-based inactivated vaccine was postulated as a model and discussed. In this report, we demonstrated modulation of immune responses by Epi-1 and an Epi-1-based Japanese encephalitis virus (JEV)-inactivated vaccine against JEV infection in mice. Under in vitro conditions, Epi-1 prevented JEV infection-mediated loss of cell viability in BHK-21 cells. When Epi-1 and JEV were co-injected into mice and mice were re-challenged with JEV after 14 days, all mice survived. In addition, Epi-1 modulated the expressions of immune-responsive genes like interleukin (IL)-6, IL-10, MCP-1, tumor necrosis factor-α, interferon-γ and IL-12, and elevated the levels of anti-JEV-neutralizing antibodies in the serum. The presence of Epi-1 suppressed the multiplication of JEV in brain sections at 4 days after an injection. Mice immunized with the developed vaccine showed complete survival against JEV infection, and it was superior to the traditional formalin-based JEV-inactivated vaccine. This study demonstrates the use of Epi-1 to develop an inactivated vaccine can provide guidelines for the future design of Epi-1-virus formulations for various in vivo applications.