Peptides 36 (2012) 100108

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Peptides

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The an ateimmun e

Shang-ChMarine Researc Rd., J

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Article history:Received 13 MReceived in reAccepted 3 ApAvailable onlin

Keywords:Epinecidin-1Immune respoBacterial infecCytokines

whice inve

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g e28 dajecte1 (40ays; idin-ction

mice elevated plasma interleukin (IL)-10 to initial peaks at 24 and 48 h, and it showed a second peak at16 days. In RAW264.7 cells, treatment with epinecidin-1 alone did not produce signicant changes intumor necrosis factor (TNF)- protein secretion at 1, 6, or 24 h after treatment with 3.75, 7.5, or 15 g/mlepinecidin-1 compared to the lipopolysaccharide group.

2012 Elsevier Inc. All rights reserved.

1. Introdu

Infectiouthreats facmultidrug-rlactamase-1given rise tocrobial peptplants, shripotency anincluding mtionarily anhypothesis tion, selectibacteria arehost cells dMany AMPbacterial plesting featu

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0196-9781/$ http://dx.doi.oction

s diseases remain some of the most serious healthing the world [1]. More distressing is the rise ofesistant pathogens such as New Delhi metallo--

(NDM-1)-producing Enterobacteriaceae [4] which has an urgent need for novel anti-infective agents. Antimi-ides (AMPs) are endogenous antibiotics identied frommp, sh, mice, humans, etc. [2,11,21] that have highd efcacy against a broad spectrum of pathogens,ultidrug-resistant ones. AMPs are described as evolu-cient weapons [28], and many studies investigated thethat AMP cationicity is important for the initial attrac-vity, and electronegative targeting of pathogens. Most

signicantly more electronegative than neighboringue to intrinsic structural and physiologic traits [27].s have hydrophobic surfaces which can permeabilizeasma membranes [8]. As described above, these inter-res of AMPs have prompted numerous scientists and

ding author. Tel.: +886 920802111; fax: +886 39871035.ress: zoocjy@gate.sinica.edu.tw (J.-Y. Chen).

biotechnological companies to begin development of various AMPsas potential therapeutics [5,10].

Recently, we identied an AMP from a marine grouper(Epinephelus coioides) named epinecidin-1, which was shown tobe active against gram-negative and -positive bacteria, viruses,Candida albicans, and Trichomonas vaginalis [1316]. In additionto direct antibacterial functions, epinecidin-1 has another impor-tant ability to regulate the innate immune system against Vibriovulnicus infection in zebrash [17]. Epinecidin-1 was reported tomodulate bacterial infection-induced cytokines and inhibit TNF-expression using an improved Tol2 transposon system to producetransgenic zebrash with epinecidin-1 which are resistant to bac-terial infection [20]. Those results suggest epinecidin-1s actionsagainst bacterial infection need to be more-clearly elucidated, andepinecidin-1 functional studies of immune-related gene modica-tions should be assessed using in vivo systems such as methods usedto study tilapia hepcidin 23s effects against V. vulnicus infectionin mice [18]. Although epinecidin-1 showed bactericidal featuresin most in vitro reports, studying the host response by produc-ing antibodies specic for antigens produced by bacteria or usingepinecidin-1 in animal (mice) systems has not been done.

To date, most patients are killed by septic shock within 48 h aftera Pseudomonas aeruginosa infection [23]. Therapy for P. aeruginosainfections greatly depends on antibiotic treatment. This may inducenumerous isolates that illustrate resistance to routinely applied

see front matter 2012 Elsevier Inc. All rights reserved.rg/10.1016/j.peptides.2012.04.002timicrobial peptide, epinecidin-1, medie response to bacterial infection in mic

un Lee, Chieh-Yu Pan, Jyh-Yih Chen

h Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen

e i n f o

arch 2012vised form 3 April 2012ril 2012e 10 April 2012

nsetion

a b s t r a c t

Epinecidin-1, an antimicrobial peptide(Epinephelus coioides). In this study, wwith Pseudomonas aeruginosa. In vivosignicant secretion of immunoglobafter injection of 40, 100, 200, or 500mice treated for 1, 2, 3, 7, 14, 21, and in IgM, IgG, or IgG2a between mice indays, and 28 days after an epinecidin-increased IgG1 to peaks at 2 and 3 dpeak at 21 days. This supports epinecproduction) against P. aeruginosa infe/ locate /pept ides

s secretion of cytokines in the

iaushi, Ilan 262, Taiwan

h encodes 21 amino acids, was isolated from a marine grouperstigated its immunomodulatory functions in mice co-injectedlts showed that the synthetic epinecidin-1 peptide induced1 (IgG1) in mice co-injected with P. aeruginosa. Moreover,

pinecidin-1/mouse, we detected IgM, IgG, IgG1, and IgG2a inys. Results showed that there were no signicant differencesd with epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7

g/mouse) injection. Injection of 500 g epinecidin-1/mouseinjection of 100 g epinecidin-1/mouse increased IgG1 to a1 being able to activate the Th2 cell response (enhance IgG1. Treatment with different concentrations of epinecidin-1 in

S.-C. Lee et al. / Peptides 36 (2012) 100108 101

antibiotics for P. aeruginosa infections, such as imipenem, lev-ooxacin, and gentamicin [9]. Grouper epinecidin-1 displayedmarked in vivo antiviral and antibacterial activities against exper-imental infections including Japanese encephalitis virus (JEV),nervous necrosis virus, V. vulnicus, and Riemerella anatipestifer inexperimental animals [7,13,16,24,25]. Furthermore, epinecidin-1 issold by Bachem (http://shop.bachem.com/ep6sf/prodH7228.html)as a product. Therefore, epinecidin-1 has emerged as a promis-ing agent, especially against antibiotic-resistant pathogens. Mostresearch results described epinecidin-1 as possessing antimicrobialactivity due to its ability to disrupt bacterial membrane integrityand cause lysis of microorganisms [15]. Those research resultsmentioned above sparked our interest in studying host immuneresponses against bacterial infection in mice using epinecidin-1 infurther clinical applications of grouper epinecidin-1 as a candidatefor an antimicrobial drug.

To conrm whether epinecidin-1 is clinically valuable as acandidate for an antimicrobial drug, we evaluated the effects ofsynthetic epinecidin-1 on mice, by comparing the antibacterialneutralization efciency, and measuring serum cytokine levels ofimmunoglobulin G (IgG), IgM, IgG1, IgG2a, interferon (IFN)-, inter-leukin (IL)-10, IL-12, and others.

2. Materials and methods

2.1. Mice and the bacterial strain

Adult Balb/C mice were purchased from BioLASCO Taiwan(Taipei, Taiwan) and housed at the Laboratory Animal House(Jiaushi, Taiwan). Mice were maintained in pathogen-free sterile

Table 1Primer sequences and Tm values listed in this paper.

Primer Sequence (53) Tm

Murine MCP-1 forward AAC TGC ATC TGC CCT AAG GTC TT 55.3Murine MCP-1 reverse TGC TTG AGG TGG TTG TGG AA 51.8Murine MCP-3 forward AAG ATC CCC AAG AGG AAT CTC AAG 55.7Murine MCP-3 reverse CAG ACATG CCC TTC TTT G 54.8Murine MIP-1 forward TCA GAC ACC AGA AGG ATA C 48.9Murine MIP-1 reverse CTG AGA AGA CTT GGT TGC 48Murine TNF- forward CAA CGG CAT GGA TCT CA 47.1Murine TNF- reverse GGA CTC CGC AAA GTC T 45.9Murine GAPDH forward TCA TCC CAG AGC TGA ACG 50.3Murine GAPDH reverse GGG AGT TGC TGT TGA AGT C 51.1

isolators according to animal house guidelines. All experimentscomplied with relevant Laboratory Animal House guidelines andinstitutional policies. Food, water, caging, and ller were steril-ized before use in the experiments. P. aeruginosa culture followeda previous publication without modication [14].

2.2. Injection of epinecidin-1 co-treated with bacteria orepinecidin-1 alone in mice for an immunological assay anddetection of antibody titers

Mice were intraperitoneally injected with 0.2 ml of P. aeruginosaalone (0.2 ml; 106 colony-forming units (cfu)/ml/per mouse),P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse) mixed with CpG(10 g/mouse), P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse) mixedwith epinecidin-1 (40 g/mouse), or medium. The day of theinjection was designated day 0. Serum was collected on days0, 1, 2, 3, 7, 14, 21, and 28. Mice were re-injected with 0.2 ml

Fig. 1. Epineci domonas aeruginosa antigen. Mice were injected with PBS alone (Medium),P. aeruginosa a AMP). Then, mice were re-challenged with P. aeruginosa alone on day 14.Serum was co bulin M (IgM), IgG, IgG1, and IgG2a antibody titers against the inactivatedP. aeruginosa a mean value from three determinations, with the standard error (SE). Data(mean SE) wdin-1 induced the production of neutralizing antibodies against an inactivated Pseulone (B), CpG with P. aeruginosa (B + CpG), or P. aeruginosa with epinecidin-1 (B +llected on days 1, 2, 3, 7, 14, 21, and 28 after the primary injection, and immunoglontigen were determined in a 96-well plate (n = 3; p < 0.05). Each bar represents theith different letters signicantly differ (p < 0.05) among treatments.

102 S.-C. Lee et al. / Peptides 36 (2012) 100108

of P. aeruginosa (0.2 ml; 106 cfu/ml/per mouse) on day 14. Theepinecidin-1 peptide was synthesized by GL Biochem (Shang-hai, China) using a solid-phase procedure of Fmoc chemistry,the detailed procedures of which were reported in our previouspublication [16].

In other trials, mice were injected with 0.2 ml of epinecidin-1 at40, 100, 200, or 500 g/mouse. The day of the injection was desig-nated day 0, and mice were re-injected with 0.2 ml of each differentconcentration of epinecidin-1 on day 14. Serum was collected ondays 0, 1, 2, 3, 7, 14, 21, and 28.

Serum was titrated using inactivated P. aeruginosa antigen-coated enzyme-linked immunosorbent assay (ELISA) plates withanti-rabbit IgG-horseradish peroxidase (HRP), IgM-HRP, IgG2a-HRP, or IgG1-HRP following our previous report with nomodications [18].

2.3. Detection of cytokine expression levels

To understand cytokine variations after injecting epinecidin-1 co-treated with bacteria or epinecidin-1 alone in mice, wemeasured the cytokines of IL-4, IL-10, IL-12, IFN-, TNF-, macrophage inammatory protein (MIP)-1, and monocytechemoattractant protein (MCP)-1 using ELISA kits (PeproTech, USA,product number: 900-K49, 900-K53, 900-K97, 900-K98, 900-K54,900-K125, and 900-K126, respectively). To detect variations inMCP-1, MIP-1, and TNF- transcription (Table 1) and translationlevels after treatment of RAW264.7 macrophages (ATCC no. TIB-71TM, American Type Culture Collection, Manassas, VA, USA) withepinecidin-1 in 24-well culture plates for 1, 6, and 24 h, we applieda real-time polymerase chain reaction (PCR). Lipopolysaccharide(LPS) (0.1 g/ml) was used to treat RAW264.7 cells. RNA was

puried using a Qiagen RNeasy Kit (Qiagen, USA). Reverse-transcription of complementary (c)DNA was performed with aniScript cDNA Synthesis Kit (Epicentre, USA) according to the manu-facturers recommendations. A real-time PCR analysis was used toanalyze gene expressions according to the manufacturers instruc-tions, and primers are shown in Table 1. SYBR Green (Toyobo,Japan) and specic primer pairs were used for selected genes, andprimer pairs for GAPDH were used as the reference gene. A quanti-tative (q)PCR was performed according to the following conditions:95 C for 180 s, followed by 45 cycles of 10 s at 95 C, 30 s at 60 C,and 12 s at 72 C. Using 0.5 l cDNA, 2 SYBR Green PCR buffer,and 500 nM of the forward and reverse primers, the threshold cyclenumber (Ct) was calculated with ABI software (Applied Biosystems,USA). Relative transcript quantities were calculated using the Ctmethod with GAPDH as the reference gene, which was ampliedfrom the same samples. Ct is the difference in the threshold cyclesof messenger (m)RNA for selected genes relative to those of GAPDHmRNA. A real-time PCR was performed in triplicate for each exper-imental group. Protein secretion were determined by ELISA kits(PeproTech).

2.4. Glutamic oxaloacetic transaminase (GOT), glutamic pyruvictransaminase (GPT), and the cell proliferation assay

To detect GOT and GPT, mice were injected with epinecidin-1co-treated with bacteria or epinecidin-1 alone as described above.Serum and urea were collected and sent to the Taiwan MouseClinic (http://tmc.sinica.edu.tw/; http://tmc.sinica.edu.tw/sopc chemistry.html; Taipei, Taiwan) for further analysis. Spleencells were collected and centrifuged with red blood cell lysisbuffer. Leukocyte pellets were collected and separated on days

Fig. 2. Effect o lone (P. aeruginosa w e on dprimary inject ted inrepresents the SE) wf Pseudomonas aeruginosa on serum cytokine levels. Mice were injected with PBS aith epinecidin-1 (B + AMP). Then, mice were re-challenged with P. aeruginosa alon

ion. Amounts of interleukin (IL)-12, interferon (IFN)-, IL-10, and IL-4 were estima mean value from three determinations, with the standard error (SE). Data (mean Medium), P. aeruginosa alone (B), CpG with P. aeruginosa (B + CpG), oray 14. Serum was collected on days 1, 2, 3, 7, 14, 21, and 28 after the

a 96-well plate using respective antibodies (see Section 2). Each barith different letters signicantly differ (p < 0.05) among treatments.

S.-C. Lee et al. / Peptides 36 (2012) 100108 103

14 and 28 from mice as described in the section of Injection ofepinecidin-1 co-treated with bacteria or epinecidin-1 alone inmice for the immunological assay and detection of antibody titers.One hundred thousand cells per milliliter was mixed with 100 lP. aeruginosa (106 cfu/ml) in 100 l of medium that contained 10%serum. To these cells was added 100 l concanavalin A (ConA;10 g/ml, Sigma, St. Louis MO, USA) as a positive control, andcells were mixed with 100 l of medium as a negative control.Cells were cultured in a 37 C incubator (5% CO2) for 72 h andassessed using a CellTiter 96 Aqueous one solution kit to ana-lyze cell proliferation (Cat. No. G3582, Promega, Madison, WI,USA).

2.5. Statistical analysis

Students t-test was used to graph and analyze the data. p valuesof

104 S.-C. Lee et al. / Peptides 36 (2012) 100108

3.2. Effects of epinecidin-1 and CpG co-treatment with P.aeruginosa on GOT and GPT in mice

Mouse plasma GOT and GPT levels were used to reect liverfunction. GOT increased to a peak at 7 days after P. aeruginosainfection (p < 0.05; Fig. 4). Compared to the P. aeruginosa group, P.aeruginosa co-treatment with epinecidin-1 increased plasma GOTat 24 h (Fig. 4). Blood GPT reached a peak at 24 h in the group co-treated with P. aeruginosa and epinecidin-1 (Fig. 4). Compared tothe P. aeruginosa group, P. aeruginosa co-treated with CpG showedincreased plasma GPT at 28 days (Fig. 4).

3.3. Effects of epinecidin-1 on plasma levels of IgM, IgG, IgG1, andIgG2a

To understand the cytokine and immune responses after inject-ing 40, 100, 200, or 500 g epinecidin-1/mouse, we detected IgM,IgG, IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28days. Results showed that there were no signicant differencesin IgM, IgG, or IgG2a between mice injected with epinecidin-1alone (Fig. 5). IgG1 increased to peaks at 24 h, 7 days, and 28 days

after the epinecidin-1 (40 g/mouse) injection. Injection of 500 gepinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days; injec-tion of 100 g epinecidin-1/mouse increased IgG1 to a peak at 21days (Fig. 5). These results support epinecidin-1 being able to acti-vate the Th2 cell response (enhance IgG1 production) against P.aeruginosa infection.

3.4. Effects of epinecidin-1 on plasma levels of TNF-, IL-4, IL-10,IL-12, and IFN-

Epinecidin-1 (200 g/mouse) elevated plasma TNF- at 1and 3 days (Fig. 6). Then, the level had returned to the basalvalue by 15 days. Epinecidin-1 (200 g/mouse) increased plasmaIL-12 at 3 and 16 days (Fig. 6). Injection of 100 g epinecidin-1/mouse increased plasma IL-12 at 3 days, and injection of 500 gepinecidin-1/mouse increased plasma IL-12 at 2 days (Fig. 6).Epinecidin-1 (200 g/mouse) signicantly increased plasma IL-4at 3, 15, 17, and 21 days (Fig. 6). The other dosage of 40 g/mouseincreased plasma IL-4 at 3, 15, and 16 days (Fig. 6). Then, thelevel had returned to the basal value by day 21. Treatment withdifferent concentrations of epinecidin-1 elevated plasma IL-10

Fig. 5. Epinec ent cocollected on d (IgM)plate (n = 3; p < ndard(p < 0.05) amoidin-1 induced the production of neutralizing antibodies after injection with differays 1, 2, 3, 7, 14, 21, and 28 after the primary injection, and immunoglobulin M

0.05). Each bar represents the mean value from three determinations, with the stang treatments.ncentrations (40, 100, 200, or 500 g/mouse) into mice. Serum was, IgG, IgG1, and IgG2a antibody titers were determined in a 96-well

error (SE). Data (mean SE) with different letters signicantly differ

S.-C. Lee et al. / Peptides 36 (2012) 100108 105

Fig. 6. Effects on days 1, 2, 3estimated in a(mean SE) w

to initial p(Fig. 6).

3.5. Effects expressions

In RAWmacrophagexpressions24 h) and Tment with (15 g/ml) TNF- mRNepinecidin-6 and 24 hand 7.5 gepinecidin-(Fig. 7b). Coproduce sigtreated witof epinecidin-1 on serum cytokine levels. Mice were injected with different concentrations, 7, 15, 16, 17, 21, and 28 after the primary injection. Amounts of interleukin (IL)-12, int

96-well plate using respective antibodies (see Section 2). Each bar represents the meanith different letters signicantly differ (p < 0.05) among treatments.

eaks at 24 and 48 h, and a second peak on day 16

of epinecidin-1 on MCP-1, MIP-1, and TNF-

264.7 cells (morphologically monocytes andes), LPS greatly elevated MCP-1 mRNA and protein

at 6 and 24 h (Fig. 7a). LPS elevated MIP-1 (6 andNF- (1 and 6 h) mRNA expressions (Fig. 7a). Treat-epinecidin-1 from a low (3.75 g/ml) to a high doseshowed dose-dependent effects on MCP-1, MIP-1, andA expressions (Fig. 7a). Compared to the LPS group,1 treatment increased MCP-1 and MIP-1 secretion at

(Fig. 7b). Compared to the group treated with 3.75/ml epinecidin-1, the group treated with 15 g/ml1 showed signicant changes in MCP-1 at 1 and 6 hmpared to the LPS group, epinecidin-1 alone did notnicant TNF- changes at 1, 6, or 24 h after being

h 3.75, 7.5, or 15 g/ml epinecidin-1.

4. Discussi

The majepinecidin-quent deteshowed thaThe inductiaeruginosa epinecidin-CpG or P. splenocytesCpG, AMP, ment was P. aeruginowith epineon establistreatment win mice indlevels [7]. Aepinecidin- of epinecidin-1 (40, 100, 200, or 500 g/mouse). Serum was collectederferon (IFN)-, IL-10, IL-4, and tumor necrosis factor (TNF)- were

value from three determinations, with the standard error (SE). Data

on

or ndings of the present study are that synthesized1 co-treated with P. aeruginosa in mice with subse-ction of antibody titers of IgG, IgM, IgG1, and IgG2at epinecidin-1 activated Th2 cells against P. aeruginosa.on level was low possibly because of the low dose of P.(0.2 ml; 106 cfu/ml/per mouse) in the treatments. Anti-1 IgG was produced in mice challenged with eitheraeruginosa. Marked cell proliferation occurred when

from bacterially infected mice were stimulated withor ConA in vitro. Cell proliferation with ConA treat-higher compared to stimulation with P. aeruginosa,sa co-treated with CpG, or P. aeruginosa co-treatedcidin-1. Thus, in agreement with our previous studyhing an epinecidin-1-based inactivated vaccine, co-ith epinecidin-1 and Japanese encephalitis virus (JEV)uced higher Th2 cytokine levels than Th1 cytokinentibody isotyping revealed that induction of IgG1 by1 was through Th2 cells, so it was a humoral response.

106 S.-C. Lee et al. / Peptides 36 (2012) 100108

Fig. 7. Changenecrosis factor15 g/ml. Thethe standard e

Antibody titaeruginosa aagainst thatincreased inat 7 days serum-neuteration fros in mRNA expressions (a) and serum secretion (b) of monocyte chemoattractant prot (TNF)- after epinecidin-1 treatment of RAW264.7 cells for 1, 6, and 24 h. Concentration

concentration of lipopolysaccharide (LPS) used to treat RAW264.7 cells was 0.1 g/ml. Error (SE). Data (mean SE) with different letters signicantly differ (p < 0.05) among trea

ers of IgG, IgM, IgG1, and IgG2a against an inactivated P.ntigen showed that epinecidin-1 can activate Th2 cells

pathogen. The Th1 cell-responsive cytokine, IL-12, also mice injected with both epinecidin-1 and P. aeruginosaafter bacterial re-challenge (Fig. 2). From results ofralizing antibody levels of IL-10, leukocyte prolif-m the spleen of MCP-1 showed slight induction,

but MCP-1iments (Fibacterial sing the epiepinecidin-against bacfunctions.ein (MCP)-1, macrophage inammatory protein (MIP)-1, and tumors of epinecidin-1 used to treat RAW264.7 cells were 0, 3.75, 7.5, andach bar represents the mean value from three determinations, withtments. *p < 0.05, **p < 0.01, and ***p < 0.001.

induction was not seen after re-challenge exper-g. 3). Recently, enhancement of resistance againsttrains was studied in transgenic sh overexpress-necidin-1 peptide [20]. Those results suggested that1 has direct antimicrobial and bacteriostatic activitiesterial infection and also possesses immunomodulatory

S.-C. Lee et al. / Peptides 36 (2012) 100108 107

Endotoxic shock is generally associated with the systemicinammatory response and causes multiple organ failure [22].Pretreatment with epinecidin-1 decreased TNF- gene expressionafter bacterial infection [14]. In this study, co-treatment with P.aeruginosa GOT and Gconcentratiicant differantibiotics (Epinecidin-after virus 1 attenuateIgG1 after organ failurels of GPT ainjection ofsignicant rthat epinecas post-treain plasma Gmean arterThus, epineliver againsseveral othe

The imming a host viruses, partant early refcacy of Ias adjunct tIgM and Igand IgG2a aresults suggtion by moepinecidin-temic immuan importantory processmight modinjection ofproduction tion. We fotreating theinfection-ining CpG- orcould strenas IL-12. Re4 and IL-10might induof both Th1epinecidin-wagandha,

In suma signicanour study epinecidin-tory effects.epinecidin-our results epinecidin-

Acknowled

Researchtion, InstituJiaushi, Ilan

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/

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Sheland epinecidin-1 attenuated the increases in plasmaPT after 7 days in mice (Fig. 4). Injection of differentons of epinecidin-1 alone in mice presented no signif-ences in GOT or GPT values compared to injection of4 and 10 mg) from days 1 to 15 (Supplementary Fig. S2).1 also alleviated histopathologic changes in the braininfection [7]. Accordingly, treatment with epinecidin-d the LPS-induced organ damage with an increase inendotoxic shock. In addition, the criteria for deninge in patients with septic shock are increased serum lev-nd GOT, which suggest liver cell injury. Interestingly,

epinecidin-1 alone in mice (this study) did not result inises in serum levels of GPT or GOT. Our ndings suggestidin-1 has a function similar to that of N-acetylcysteine,tment with N-acetylcysteine attenuated the increasesOT and GPT after an LPS injection and increased the

ial pressure and heart rate after endotoxic shock [6].cidin-1 treatment of mice might involve protecting thet LPS-induced dysfunction in and preventing damage tor organs in septic mice.une system is an important defensive system protect-from various pathogenic infections such as bacteria,asites, and so on [3]. In particular, IgM plays an impor-ole in the course of an infection. In most trials, thegM-enriched intravenous polyclonal immunoglobulinherapy in sepsis was reported [12]. We observed thatG levels were signicantly higher compared to IgG1fter injecting epinecidin-1 alone in mice (Fig. 5). Ourest that epinecidin-1 stimulates IgM and IgG produc-use splenic lymphocytes in the absence of LPS. Thus,1 might alleviate inammatory reactions in the sys-ne system of the mouse. The cytokine network playst role in immune responses and subsequent inamma-es [19]. We demonstrated that injection of epinecidin-1ulate cytokine production in splenic lymphocytes. An

epinecidin-1 (Fig. 6) stimulated IL-12, IL-4, and IL-10to a great extent, but did not affect TNF- produc-und increased cytokine production by Th2 cells afterm with epinecidin-1, even in the presence of bacterialduced inammation. These data suggest that treat-

P. aeruginosa-induced lymphocytes with epinecidin-1gthen the immune system by regulating cytokine suchgulation of Th2-related cytokine production, such as IL-, by injecting epinecidin-1 suggests that epinecidin-1ce an immune-related balance toward the production

and Th2 cytokines by splenic lymphocytes, so that1 possesses a balancing function such as Echinacea, Ash-and Brahmi [26].mary, the administration of epinecidin-1 inducedt increase in the serum IgG level. Furthermoresuggests the possibility of clinical applications of1 in mammalian systems based on its immunoregula-

Although the mechanism through which amino acids in1 modify immune indices by receptors is not yet known,point to a new possibility for a pharmacological role of1.

gment

funding was obtained from the Marine Research Sta-te of Cellular and Organismic Biology, Academia Sinica,, Taiwan.

j.pepti

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[1] BoubugAm

[2] Car b201

[3] Cha201

[4] DocPoital Esc

[5] Hanant

[6] Hsuam200

[7] Hutionand201

[8] Maphy

[9] Mikfacimi

[10] Moreg200

[11] Najcro201

[12] NorIgMin r200

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[14] Pan(PePse687

[15] PantidePro200

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[23] Traor ccot

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