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Osteosarcoma (OSA) is the most common type of cancer that originates in the bone and usually occurs in young children. OSA patients were treated with neoadjuvant chemotherapy and surgery, and the results were disappointing. Marine antimicrobial peptides (AMPs) have been the focus of antibiotic research because they are resistant to pathogen infection. Piscidin-1 is an AMP from the hybrid striped bass (Morone saxatilis × M. chrysops) and has approximately 22 amino acids. Research has shown that piscidin-1 can inhibit bacterial infections and has antinociception and anti-cancer properties; however, the regulatory effects of piscidin-1 on mitochondrial dysfunction in cancer cells are still unknown. We aimed to identify the effects of piscidin-1 on mitochondrial reactive oxygen species (mtROS) and apoptosis in OSA cells. Our analyses indicated that piscidin-1 has more cytotoxic effects against OSA cells than against lung and ovarian cancer cells; however, it has no effect on non-cancer cells. Piscidin-1 induces apoptosis in OSA cells, regulates mtROS, reduces mitochondrial antioxidant manganese superoxide dismutase and mitochondrial transmembrane potential, and decreases adenosine 5′-triphosphate production, thus leading to mitochondrial dysfunction and apoptosis. The mitochondrial antioxidant, mitoTempo, reduces the apoptosis induced by piscidin-1. Results suggest that piscidin-1 has potential for use in OSA treatment.

Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD50 &gt, 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC50) &gt, 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 &mu, m and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (&lt, 4 &mu, m) of a rat, and 58.06% (&lt, 10 &mu, m) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance, formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals.

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, IL1β, 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.

Streptococcus agalactiae infection is one of the most significant bacterial diseases in tilapia aquaculture. Identification of immune-related genes associated with Streptococcus agalactiae infection may provide a basis for breeding selection or therapeutics to augment disease resistance. Therefore, we utilized transcriptome profiling to study the host response in tilapia following Streptococcus agalactiae infection. Based on GO and KEGG enrichment analyses, we found that differentially expressed genes are widely involved in immune-related pathways, including the induction of antimicrobial peptides. Moreover, the main components of two immune-related pathways (Toll-like receptor signaling and leukocyte transendothelial migration) and four environmental information processing pathways (TNF, PI3K-Akt, Jak-STAT and MAPK) were identified. Finally, a time-course expression profile for several of the identified transcripts including tilapia piscidin 3 (TP3), tilapia piscidin 4 (TP4), TLR2, TLR5, MyD88, TRAF6, p38, and interleukin components was performed by qRT-PCR. Collectively, these results provide a starting point to study molecular mechanisms of tilapia immune response to Streptococcus agalactiae infection and may be applied as a basis for developing disease resistant strains by breeding selection.

// Han-Ning Huang 1, * , Chieh-Yu Pan 2, * , Hung-Yi Wu 3 , Jyh-Yih Chen 1 1 Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Jiaushi, Ilan, Taiwan 2 Department and Graduate Institute of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 3 Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, Taiwan * These authors have contributed equally to this work Correspondence to: Hung-Yi Wu, email: wuhy@mail.npust.edu.tw Jyh-Yih Chen, email: zoocjy@gate.sinica.edu.tw Keywords: antimicrobial peptide, Epinecidin-1, methicillin-resistant Staphylococcus aureus Abbreviations: Epi-1, Epinecidin 1; MRSA, Methicillin-resistant Staphylococcus aureus ; SA, Staphylococcus aureus ; CRP, C-reactive protein Received: October 12, 2016 Accepted: January 16, 2017 Published: February 03, 2017 ABSTRACT 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.

Osteosarcoma (OS) is the most common solid tumor of the bone that most often affects adolescents. The introduction of chemotherapy for the treatment of OS has largely improved the survival rates of patients with localized tumors. However, the 5-year survival rate of OS patients with relapsed or metastatic disease is only 10 to 20%. In this study, the antimicrobial peptide tilapia piscidin 3 (TP3), isolated from Nile tilapia (Oreochromis niloticus), was treated to OS MG63 cells. Our findings showed that TP3 concentration as low as 1 μM induced significant inhibition of cell viability and increased DNA fragmentation, as determined by the MTT and TUNEL assays, respectively. The protein expression levels of cleaved caspases 3/9 were increased. An in situ live-cell time-lapse video and cell tomographic microscopy images showed cellular blebbing, shrinkage, nuclear fragmentation, and chromatin condensation, with the formation of beaded apoptopodia. Moreover, there were significant increase in the production of TP3-induced mitochondrial and cellular reactive oxygen species (ROS), as well as down-regulated mitochondrial oxygen consumption and extracellular acidification rates. Additionally, TP3 enhanced mitochondrial fission, whereas fusion was attenuated. Furthermore, after administration of the mitochondria targeted antioxidant mitoTempo, TP3-induced ROS oxidant levels and alterations in cleaved caspases 3/9 expression were rescued. TP3 promoted mitochondria-modulated intrinsic apoptosis through the induction of ROS production, activation of caspases 3/9, and the down-regulation of mitochondrial oxygen consumption and extracellular acidification rates, suggesting that TP3 has potential as an innovative alternative for OS treatment.

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.

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.

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-1β, 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.

This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 μg/mouse) or TP4 (50 μg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.

Pardaxin is an antimicrobial peptide of 33 amino acids, originally isolated from marine fish. We previously demonstrated that pardaxin has anti-tumor activity against murine fibrosarcoma, both in vitro and in vivo. In this study, we examined the anti-tumor activity, toxicity profile, and maximally-tolerated dose of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resection of canine histiocytomas revealed large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. Pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our findings indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data justify the veterinary application of pardaxin, and also provide invaluable information for veterinary medicine and future human clinical trials.

To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from Oreochromis niloticus, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (Oreochromis spp.) with Vibrio vulnificus and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. In vivo experiments showed that co-treatment with V. vulnificus and TP3 (20 μg/fish) or TP4 (20 μg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and V. vulnificus into tilapia and then re-challenged the fish with V. vulnificus after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 μg/fish) or TP4 (20 μg/fish) for 30 minutes prior to V. vulnificus infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 μg/fish or 30 μg/fish) or TP4 (20 μg/fish) 30 minutes after V. vulnificus infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of V. vulnificus bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with V. vulnificus were 7.8 and 62.5 μg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 μg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed that V. vulnificus disrupted the outer membranes of cells, resulting in the loss of cell shape and integrity. We examined whether TP3 and TP4 increased the membrane permeability of V. vulnificus by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). Treating fish with TP3 and TP4 under different pH and temperature conditions did not significantly increase MIC values, suggesting that temperature and the acid-base environment do not affect AMP function. In addition, the qPCR results showed that TP3 and TP4 influence the expression of immune-responsive genes, including interleukin (IL)-1β, IL-6, and IL-8. In this study, we demonstrate that TP3 and TP4 show potential for development as drugs to combat fish bacterial infections in aquaculture.

Epinecidin-1, an antimicrobial peptide which encodes 21 amino acids, was isolated from a marine grouper (Epinephelus coioides). In this study, we investigated its immunomodulatory functions in mice co-injected with Pseudomonas aeruginosa. In vivo results showed that the synthetic epinecidin-1 peptide induced significant secretion of immunoglobulin G1 (IgG1) in mice co-injected with P. aeruginosa. Moreover, after injection of 40, 100, 200, or 500 μg epinecidin-1/mouse, we detected IgM, IgG, IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28 days. Results showed that there were no significant differences in IgM, IgG, or IgG2a between mice injected with epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7 days, and 28 days after an epinecidin-1 (40 μg/mouse) injection. Injection of 500 μg epinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days; injection of 100 μg epinecidin-1/mouse increased IgG1 to a peak at 21 days. This supports epinecidin-1 being able to activate the Th2 cell response (enhance IgG1 production) against P. aeruginosa infection. Treatment with different concentrations of epinecidin-1 in mice elevated plasma interleukin (IL)-10 to initial peaks at 24 and 48 h, and it showed a second peak at 16 days. In RAW264.7 cells, treatment with epinecidin-1 alone did not produce significant changes in tumor necrosis factor (TNF)-α protein secretion at 1, 6, or 24h after treatment with 3.75, 7.5, or 15 μg/ml epinecidin-1 compared to the lipopolysaccharide group.

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V . vulnificus (204) or S . agalactiae (SA48) had decreased at 96 h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1–24 h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.

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.

The antimicrobial peptide, tilapia hepcidin (TH) 2-3, belongs to the hepcidin family, and its antibacterial function has been reported. Here, we examined the TH2-3-mediated regulation of proinflammatory cytokines in bacterial endotoxin lipopolysaccharide (LPS)-stimulated mouse macrophages. The presence of TH2-3 in LPS-stimulated cells reduced the amount of tumor necrosis factor (TNF)-α secretion. From a microarray, real-time polymerase chain reaction (PCR), and cytokine array studies, we showed down-regulation of the proinflammatory cytokines TNF-α, interleukin (IL)-1α, IL-1β, IL-6, and the prostaglandin synthesis gene, cyclooxygenase (COX)-2, by TH2-3. Studies with the COX-2-specific inhibitor, melaxicam, and with COX-2-overexpressing cells demonstrated the positive regulation of TNF-α and negative regulation of cAMP degradation-specific phosphodiesterase (PDE) 4D by COX-2. In LPS-stimulated cells, TH2-3 acts like melaxicam and down-regulates COX-2 and up-regulates PDE4D. The reduction in intracellular cAMP by TH2-3 or melaxicam in LPS-stimulated cells supports the negative regulation of PDE4D by COX-2 and TH2-3. This demonstrates that the inhibition of COX-2 is among the mechanisms through which TH2-3 controls TNF-α release. At 1 h after treatment, the presence of TH2-3 in LPS-stimulated cells had suppressed the induction of pERK1/2 and prevented the LPS-stimulated nuclear accumulation of NF-κB family proteins of p65, NF-κB2, and c-Rel. In conclusion, TH2-3 inhibits TNF-α and other proinflammatory cytokines through COX-2-, PDE4D-, and pERK1/2-dependent mechanisms.

We tested the activity of epinecidin-1, a novel antimicrobial peptide structurally related to pleurocidin, in commercial cleaning solutions stored at 4 and 25 degrees C for 7 and 14 days. The peptide's activities against Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans were measured in a minimum inhibitory concentration (MIC) determination, minimal bactericidal concentration (MBC) determination, disk diffusion test, and a count of the bacterial numbers. Exposure to epinecidn-1 in a cleaning solution following MIC value comparisons in the disk diffusion test and counts of bacterial numbers after 16, 24, 48, and 72 h suggested that bacterial numbers were much lower than those treated with only commercial cleaning solutions for all bacteria. The efficacy of the antimicrobial activities of inhibiting bacterial numbers by epinecidin-1 in cleaning solutions at a low pH and a low temperature was not affected. Given its simple structure and antimicrobial activity, epinecidin-1 may be a useful component of microbicides designed to prevent pathogen infections and/or remediate abnormal vaginal or skin flora.

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

Antimicrobial peptides (AMPs) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of epinecidin-1 against a multidrug-resistant clinical isolate of P. aeruginosa ( P. aeruginosa R) and a P. aeruginosa strain from ATCC ( P. aeruginosa ATCC 19660) in vivo . The MICs of epinecidin-1 against P. aeruginosa R and P. aeruginosa ATCC 19660 were determined and compared with those of imipenem. Epinecidin-1 was found to be highly effective at combating peritonitis infection caused by P. aeruginosa R or P. aeruginosa ATCC 19660 in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that epinecidin-1 enhances the rate of survival of mice infected with the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory effects.

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.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (10(4), 10(6), 10(8), and 10(10) colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1β, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1β expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1β promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.

In the present study, we used Vibrio vulnificus and a zebrafish model system to investigate the inhibitory effect of epinecidin-1 on acute bacterial infection and studied the impacts of pretreatment, co-treatment, and post-treatment with epinecidin-1 on its protective efficacy. In vivo experiments showed that co-treatment with epinecidin-1 and V. vulnificus achieved 78%-97% survival rates after 30 days. When epinecidin-1 and V. vulnificus were co-injected into zebrafish and zebrafish were re-challenged with V. vulnificus after 30 days, zebrafish had survival rates of 22%-47%. Pretreatment and post-treatment with epinecidin-1 obtained respective survival rates of 57% and 60%. In addition, epinecidin-1 modulated the expressions of immune-responsive genes like interleukin (IL)-10, IL-1b, tumor necrosis factor-α, and interferon-γ as analyzed by a microarray and qPCR approach. This study demonstrates the use of epinecidin-1 to develop inactivated material for fish bacterial infections which can provide guidelines for the future design of epinecidin-1-bacterial formulations for various in vivo applications.

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, against a bacterial endotoxin under in vitro conditions was previously reported. In this study, we investigated the antibacterial and immunomodulatory functions of TH2-3 in mice infected with the pathogen, Vibrio vulnificus. A TH2-3 injection in V. vulnificus-infected mice produced an increased survival rate compared to mice injected with V. vulnificus only. In addition, a TH2-3 injection increased the bacteriostatic property against V. vulnificus in mice. Gene expressions examined using a microarray demonstrated that TH2-3 modulated several V. vulnificus-responsive genes in the host. A neutralizing antibody assay of mice serum against inactivated V. vulnificus antigen-coated plates demonstrated the induction of an immune response by TH2-3 against the pathogen. Taken together, TH2-3 enhanced the survival rate of mice against the bacterial pathogen V. vulnificus through both antimicrobial and immunomodulatory functions. These properties make the TH2-3 peptide a good candidate for development as a new antimicrobial drug and suggest that TH2-3 can underpin the design of adjuvants for further development of vaccines.

The inhibitory function of tilapia hepcidin (TH)1-5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1-5 peptide and tested TH1-5's antitumor activity against several tumor cell lines. We show that TH1-5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1-5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1-5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1-5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1-5 peptide. However, real-time qualitative RT-PCR indicated that TH1-5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-γ, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1-5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1-5 possesses the potential to be a novel peptide for cancer therapy.

Hepcidin is an antimicrobial peptide (AMP) secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. But the function of hepcidin in fish is still not completely understood. We recently described three different hepcidins (named tilapia hepcidin (TH)1-5, TH2-2, and TH2-3) from tilapia Oreochromis mossambicus, the cDNA sequences were determined, the predicted peptides were synthesized, and the TH2-3 peptide showed antimicrobial activity against several bacteria. We hypothesized that TH2-3 may have a biological function like an AMP in fishes and can be used as a transgene to boost resistance against bacterial infection. To examine the antimicrobial effects of TH2-3, we produced and engineered the overexpression of TH2-3 in zebrafish (Danio rerio) and the convict cichlid (Archocentrus nigrofasciatus). The microinjected plasmid also contained a green fluorescent protein (GFP) which was used as an indicator to trace germline transmission. In vivo, transgenic TH2-3 fish (of the F3 generation) were challenged with Vibrio vulnificus (204) and Streptococcus agalactiae (SA). Results showed significant clearance of bacterial numbers of V. vulnificus (204) but not of S. agalactiae in transgenic TH2-3 fish. A gene expression study using a real-time RT-PCR revealed that transgenic TH2-3 zebrafish showed increased endogenous expressions of Myd88, tumor necrosis factor-alpha, and TRAM1 in vivo. After transgenic TH2-3 zebrafish were infected with V. vulnificus (204), interleukin (IL)-10, IL-26, lysozyme, toll-like receptor (TLR)-4a, and Myd88 were upregulated, but IL-1beta (at 12-24 h) and IL-15 (at 1-12 h) were downregulated post-infection. After transgenic TH2-3 zebrafish were infected with S. agalactiae, IL-1beta (at 1-24 h), IL-15 (at 6 h), IL-22 (at 1-6 h), and TLR3 (at 1-24 h) were downregulated, but TLR4a (at 6-12 h) and c3b (at 12 h) were upregulated post-infection. Our findings identify the TH2-3 transgene in transgenic fish as an active component of the host response to bacterial pathogens. These results suggest that using TH2-3 as a transgene in zebrafish can effectively inhibit bacterial growth, specifically the V. vulnificus (204) strain for up to 24 h.

In order to advance the application of antimicrobial peptides in aquaculture, transgenic zebrafish expressing the antimicrobial peptide, epinecidin-1, were developed and are reported on here. First, we cloned the zebrafish mylz2 promoter for this purpose. To characterize the activity of the mylz2 promoter, various fragments of it were analyzed using a firefly luciferase transient expression assay, in which maximum promoter activity was found with a 2.5-kb fragment. In addition, the 2.5-kb fragment also expressed considerable red fluorescent proteins in skeletal muscles of transgenic zebrafish. Second, in order to improve the translation efficiency of the Tol2 transposase, we constructed untranslated regions (UTRs) of zebrafish ba1 globin flanked by a transposase. A transient embryonic excision assay (TEEA) and in vivo fluorescent observations showed high transposition efficiency during embryonic development. After optimization of the promoter and transgene efficiencies, transgenic zebrafish with the Epi-1/DsRed plasmid (pTLR-m2.5 K-K.Epinecidin-1/DsRed vector) were developed, and expressions of Epi-1/DsRed in muscles and blood were demonstrated by immunohistochemical staining techniques. Moreover, we also found that the Epi-1/DsRed gene was efficiently and significantly expressed in vivo against Vibrio vulnificus and Streptococcus agalactiae after injecting the bacteria and determining bacterial counts. A gene expression study using real-time RT-PCR revealed that Epi-1/DsRed itself induced endogenous MyD88 expression in vivo. After Epi-1/DsRed transgenic zebrafish were infected with V. vulnificus 204, interleukin (IL)-10, IL-22, IL-26, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, MyD88, and nuclear factor (NF)-kappaB activating protein-like were upregulated, but IL-1beta and tumor necrosis factor-alpha were downregulated at 12h post-infection; IL-21, complement component c3b, and NF-kappaB activating protein-like were downregulated, but MyD88 was upregulated at 24h post-infection. These results suggest that using epinecidin-1 as a transgene in zebrafish can effectively inhibit bacterial growth for up to 24h after infection.

The synthetic epinecidin-1(22-42) peptide was derived from positions 22-42 of Epinephelus coioides epinecidin-1. The synthetic SALF(55-76) cyclic peptide (csSALF(55-76)) and SALF(55-76) linear peptide (lsSALF(55-76)) contained sequences from positions 55 to 76 of the Penaeus monodon anti-lipopolysaccharide factor (ALF), respectively. We studied the in vitro activities of epinecidin-1(22-42), csSALF(55-76), and lsSALF(55-76) against Propionibacterium acnes, Candida albicans, and Trichomonas vaginalis. The minimum inhibitory concentrations (MICs) of epinecidin-1(22-42) for the test pathogen strains ranged 12.5-200 microg/ml, those of csSALF(55-76) ranged 100-200 microg/ml, and those of lsSALF(55-76) ranged 25-200 microg/ml. epinecidin-1(22-42) exhibited cytotoxicity towards P. acnes, C. albicans, and T. vaginalis (one strain of which was a metronidazole-resistant strain, while the other strain was not), suggesting that epinecidin-1 functions like a lytic peptide. Similar cytotoxicity was identified against T. vaginalis treated with the csSALF(55-76) and lsSALF(55-76) peptides. The antimicrobial activities of these peptides were confirmed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), a viable cell count assay, and flow cytometric analysis. TEM and SEM examinations of T. vaginalis treated with these three peptides showed that severe swelling preceded cell death and breakage of the outer membrane, and the intracellular inclusion was found to have effluxed extracellularly. This phenomenon was also found with epinecidin-1(22-42) treatment of P. acnes and C. albicans. Our results suggest that the epinecidin-1(22-42), csSALF(55-76), and lsSALF(55-76) peptides may be good candidates for treating trichomoniasis, and epinecidin-1(22-42) may have potential as a drug supporting therapy for acne and candidiasis.