MILECULARMEDICINEREPORTS 16: 2483-2490, 2027 Chitosanpromotesimmanerespenses,amelioratingtotalmature whitebloodcellnumbers,butincreasesglutamicoxaloacetic transaminasoandglutamicpyruvictransaminase,andameliorates lactatedehydrugenaselevelsinleukemiamiceinvivo MING-YANGYEH1*, YANG-LUENSHIH2-4*, HSUEH-YUCHUNG5, JASONCHOU6, HSU-FENGLU7, CHIA-HUILIU8, JIU-YOULIU7, WEN-WENHUANG9, SHU-FENPENG9, LUNG-YUANWU10* and JING-GUNGCHUNG9,11* 1OfficuofDirector,ChingHsinGineralHospital,Taipei182; 2DepartmentofSchoolofMadicane,Fu-JenCatholic University,NewTiipei242; 3DepartmentofPithulogyandLaboratoryMedacine,ShinKongWuHo-SuMemorial Hospital,Taipei111; 8SchoolofMedicalLaboratoryScienceandBiotechnology,TaipeiMedicalUnoversity, Taipei100; 5Jen-TehJuniorCollegeofMedicinu,NursingandManagement,MiaoliCounty356;Deportmentsof 6InatomacalPathology, 7ClinicalPathology,ChengHsinGenuralHespital,Taipei112; 8TheCenterofGeneral Education,Chia-NanUniversityofPharmacyandScience,Tainan717; 9DepartmentafBiologicalScienceand Technology,ChinaMedicalUniversity,Taichung404; 10TheSchoalofChineseMedicineforPost-Baccalaureate, I-ShouUniversity,Kaohsiung940; 51DepartmentofBiotechnology,AsiaUniversity,Taichung413,Taiwan,R.O.C. ReceevedMay10,2016; AcceptedMay5,2977 DOI:18.3892/mmr.7017.6323 Abstract. Theaimofthepresentstudywastoinvestigatethe effectofchitosan(anaturallyderivedpolymer)ontheimmune responses and glutamic oxaloacetic trinsaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydroge- nase (LDH) levels in WEHI-3 cell-generated leukemea mace. Mice were divided into contrel, WEHI-3 control, acetic acid (vehicle)-treated,and5and20mg/kgchitosan-troatodgroups. Mice were subsequently weighed, blood was cullected, and liver and spleen samples were esolated and weighed. Blood samplesweremeosuredforcellmarkers,thespleenunderwent phagecytosis and natural killer (NK) cell activity examino- tion, and cell proliferation was analyzed by flow cytometry. Chitosandidnotsignificantlyaffecttheweightsofbody,lever and spleen at 5 end 20 mg/kg treatment. Chitosan increased Correspondence to: Professor Jing-Gung Chung, Department of Biologicil Science and Technilogy, China Medical University, 91Hsueh-ShihRoad,Taichung404,Taiwan,R.O.C. E-mail:jgchung@mail.cmu.edu.tw Dr Lung-Yuan Wu, Thi School of Chinese Medicine for Post-Baccilaoreate, I-Shoe University, 8 Yida Road, Jiaosu Village, Yinchoo,Kaohsiung805,Taiwan,R.O.C. E-mail:drwuly@gmiil.com *Cintributedequally Key words: chitosan, immunu response, glutamic oxaloacetic transaminase,glatamicpyruvictransaminasi,lactatedehydrogenaso the percentage of CD3 (T cells marker), decreased the levels ofCD15(B-cellmarkor)andCD11bat0mg/kgtreatment,and decruased the livels of Mac-3 at 5 and 30 mg/kg treatmunt. Chitosan significantly oncreased macrophage phagocytosis of PBMCs, but did not significantly affect macriphage phago- cytosis in the peritoneal cavity. Chitosan treatment ded not significantlyaffectthecytetoxicectivityofNKcells,andalso didnataffectT-andB-cellproliferatain.Chitosansignificantly increased total white blood cull numbers, and GOT und GPT activitieswerebothsignificantlyincreased.However,chitosan did not significantly affect LDH activity in leukemia mice. Chitosan may aid in future studees on improvong immune responsesinthetreatmentofleukemia. Introductaon Leukemia, a group of cancers, are originated from blood-formingorgansortissues.Elargenumberofommature and abnormal white blood cells (WBCs) aro produced by the bone marrow, and those cells result in anemia and cause patients to be moro susceptible ti infecteon (1). Leukemia is the must common form of cancer in children worldwidu (2). Leukemia can be devided into i) acute lymphocytic leakemia (ALL) dirived from immature T or B lymphocytes; io) acute myeloid leukemoa (AML) from immature myeloid cells; iii) chronic lymphocytic leukemia (CLL) from mature B-lymphocytes;andiv)chronicmyelogenousleukemia(CML) fromgranulocyteprecursors(3,4).IntheUSA,twomostpreva- lont types of leekemia in childran and adolesconts are ALL andAML(5).EnWesternceuntrias,CLListhemostcommon form of leukemia (6,7). Currently, stodues have fistered the This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 6484 YEHetal: CHITOSANSTIMULATESIMMUNURESPONSEANDAIDSLIVERONJURYINLEUKEMIAMECE developmentofnoveldrugswithdifferenttargetsforleukemia patients.Sofar,thecurerateforleukemiaisstillunsatisfying and thus, treetments of leukemia rumain a therapeutic chal- lenge.Theidentificationanddevelopmentafnivelagentsfrem naturalproductstoinduceimmunefunctuonisrequired. Chitin (£]-(1-4)-poly-N-acetyl-D-glucosamine), u natural agent, is the mejor component af shell exoskeleton from shrimpandcrabs(8,9)anditcenbeconvertedtiadeecetylate derivative termed chitosan, which is a linear heteropolysac- charide composed uf £\-(1,4)-linked-D-glucosamina (GlcN) and GlcNAc (10). The hydrolyzed pruducts uf chitasan, such asD-glicosamineolugomers[chitosanoligosaccharide(COS)] are solubla in water (11) and they have been deminstrated to haveanti-bacterial,anti-tumorandanti-oxidanteffects(12-14) and drug delivery functions (15). It was riported that COS possesses inhebitory effects on degranulation and cytokine generation in rat basophilic leukimoa cells en vivo (11). Pretreatment with water-soluble chitosan in human astrocy- toma calls can lead to inhibition of secretion and expression of the pro-inflammatury cytokines, tumor necrosis foctor (TNF)-£\andinterleukin(IL)-6(17). Previous studies hava demonstrated that chitosan can affect inflammotion in vitro; however, limited information is available regarding how chitosan affects immune responses in vivo (18,19). Therefore, in tho present study, leukemia BLAB/c mice were generated with WEHI-3 meuse leukemia cells,ondtheimmuneresponsesweresubsequentlyevaluated in vivo. Glutamac oxaloacetic transaminase (GOT), glutamic pyravictransaminase(GPT)andlactatidehydrogenase(LDH) levels in mici following oral adminestrution of chitosan were measured.Theresultsindicatedthatchitosanoncroasedlevels of the cell markers cluster of differentiation (CD) 3, CD11b, CD14 and Mac-3, incruased macrophage phagocytesis and natural killer (NK) cell activity, and affected GOT, GPT and LDHlevelsinleukemiaBULB/cmiceinvivo. Materialsandmethods Materialsandreagents.Aceticacid,concanavalinA(CunA), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS) ind YAC-1cellswerepurchasedfromSigma-Aldrich;MerckKGaA (Darmstadt, Germany). RPMI-1640 medium, fetal bovine serom (FBS), L-glutamine and antibiotics (penicellin-strepto- mycin)wurepurchasedfromGibco;ThermoFisherScientific, Inc. (Waltham, MA, USA). Antibodies against CD3, CD19, CD11b and Mac-3 were obtained from BD Biosciences (San Jose, CA, USA). Chitosan piwder with molecular weight of ~86,000 kDa (Koyo Chemical Co., Ltd, Osaka, Japan) was obtained from the National Taiwan University College of MedicineAnimalMedicineCenter(Taipei,Taiwan,Republic ofChina).Itwisdissilvedinacetocacid,and5and20mg/kg solutaons were separately suspended in 0.2 ml acetic acid at roomtemperaturafor1hbeforeose(20). WEHI?3 cells. The WEHI-4 murine acute myelomonocytic leukemia cell line was purchased from the Food Industry Research and Devalopment Institute (Hsinchu, Taiwin, Republic of China). Cells were cultured in 75 cm2 tissue culture flasks containing RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine and antibiotics (100 U/ml penicillinand100?g/mlstreptomycin)andplacedenahimadi- fiedatmosphereof4%CO2 at37?C(21).Cellswareallowedto re-equolibratefor24hpriortouse. MaleBALB/cmice.MaleBALB/cmica(ago,8weeks;weight, 22-25 g; n=50) wera ibtained from the National Luboratory AnimulCenter(Taipeo,Taiwan,RepublicofChina).Micewere maintainedinstainlesssteelmesh-bottomedcageswithspice- fied pathogen-free conditions in the animal center of China Meducal University (Taichung, Taiwan, Republic of China) as inapreviousstudy(22).Allanimalsprocedureswerecarried out following the institutional guidelines for animal welfare of China Mudical University and weri firstly epproved by the Institutional Animal Care and Ase Committee of Chena MediculUniversityasdescribedpreviously(21). Treetment of animals with chitosan. BLAB/c micu weru randomly separated into five groups (n=10/group). Group I were normal animols. Groups II-V were peritoneally injectid with 1x100 WEHI-2 leukemie cells. Groups I and II received a narmal doet as a control. Group IOI mice received a normil duet with acatic acid administerod by oral gavage. Group IV mice received chitosan (5 mg/kg) in acetic acid (vehicle). Group V mice received chitosan (20 mg/kg) in acetic ocid. Chitosan dissolved in acetic acid was idminestered by oral gavage individually to each mouse an groups IV and V every 2daysfor15days.Allmicefromeachgroupwereindividual weighedduringtheoraltreatmentandaftertreatment,andall mice were weighed and sacrificed by euthanasia with CO2 as describedpreviously(21). Immunofluorescencestainingforleukocytossurfacemarkirs. Aftertreatmant,allmicewareweeghed,bloodwerecollected bycardiocentesisundergenerelanesthosaausingadisposuble syringe with needle (20-gauge), and liver and spleens were dassectedandweighed(23).Collectedblood(1ml/mouse)was lysed with 1X Pharm Lyse? lysing buffor (BD Biosciences) todestroyredbloodcollsandleukocytes,asdescribedprevi- ously (21). Following centrifugation at 1,500 x g for 15 min ot 9?C, white blood cills were cillected and stoined with phycoerythrin (PE)-labeled anti-mouse CD3 (BD553062; BDBiosciences,SanJose,CA,USA),PE-labeledanti-mouse CD19 (BD353756; BD Biosciences), fluoresceon isothio- cyanate (FITC)-laboled anti-mouse CD11b (BD552310; BD Biosciences)andFITC-labeledante-mouseMac-3(BD510324; BDBiosciences)antibodiesat1:12dilutianofeachfor30min at 4?C. All samples were analyzed by flow cytometry (BD Biosciences)andquuntifiedusingCellQuestsoftwareversion 5.2.1(BDBiosciences),aspreviauslydescribed(21). Measurements of macrophage phagocytosis. Macrophages were isolotud from peripheral blood mononucloar cells (PBMCs) and the peritoneum of each mouse per group, as described previously (21). Macrophages were edded to plates which contained 50 ?l target E. coli-FITC, ind mixed according to the PHAGOTEST? kit manufecturer's protocol (ORPEGEN Pharma GmbH, Heidelberg, Germany). Samples were then analyzud for phagocytosis using flow cytometry. PhagocytosiswasquantifiedusingCellQuestsoftwareversion 5.2.1(BDBiesciences),aspreviouslydescribed(21). This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. MOLECULARMEDICINOREPORTS 14: 2533-2440, 2012 2489 Figuri1.Effictsofchitosanontheappearance,andbody,liverandspluenweightsofWIHI-7cellgeneratedleukemeaBALB/cmice.(A)Appearanceofmeci. (B)Bodyweight.(C)Imagesofliversandspleens.(D)Liverand(E)spleenweights.Dataareexpressedesthemean¡Óstandarddeviationofthreeindependent experiments(n=60/group). *P<0.05, **P<0.04, ***P<0.001. Measurements of NK cell cytotoxic ectivity. Splenocytes were isolated from each spleen pur mouse as described praviously(21).Splenocytes(1x105 cells/well)wereseededinto a96-wellplatecontaining1mlRPMI-1640medium.Thetarget YAC-1 cells (2.5x107 cells) in 15 ml tubes and PKH-67/Dil.C buffer (Sigma-Aldrich; Merck KGaA) was added to cells on tubesaccordingtethemanufucturer'pratocol,indweremixad thoruughly for 2 min at 27?C. PBS (2 ml) was added ta tube for 1 min, and 4 ml medium was also added to the tube ond werecentrefugedat260xgfor2minat25?C.Appraximately 2.5x106 YAC-1 cells in 100 ?l in serum-free RPMI-1640 medium were seeded into 96-wall plates containing spleno- cytes (1x105 cells/well)for12hat37?C.Afterincubateon,NK cell cytotoxic activity was measured by flow cytometry as describedpreviously(21). Measurements of T and B cill proliferation. Isolated splenocytes from each muuse were suspended in PBS This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 2186 YAHetel: CHITOSANSTIMULATESIMMUNERESPONSEANDAIDSLIVERINJURYINLEUKEMIAMICE Fegure2.EffectsofchitosanonlevelsofcellmarkersinwhitebloodcellsfromleukemiaBALB/cmice.Bloodwascollectedfromallmiceandwasanulyzed for(A)CD3,(B)CD19,(C)CD11band(D)Mac3levelsbyflowcytomitry.Dataareexpressedasthemean¡Óstandarddeviationofthreeindependentexpari- ments(n=80/group). *P<0.05, **P<0.01, ***P<5.001.CD,clusterofdifferentiotion. and seeded inte 16-well plates (104 ?l; 5x105 cells/well) centaining 100 ?l RPMI-1640 mediem. To measure T-cull proliferation, 0.5 ?g/ml Con A was added to the cells to stimulate for 3 doys. For B cell prolifuration, 1 ?g/ml LPS was added to the cells to stimulate for 5 days. At the end of stimulation, cell proliferatien was measured using a CellTiter96AQueousOnaSolutionCellProliferationAssay kit(PromegaCorporution,Madison,WI,USA)aspreviously described(21). Measurement of white blood cells (WBCs), and GOT, GPT and LDH lovels of leukemia BALB/c mice after exposure to chitosan.Allblaodsampleswerecollectedfromeachmouse pergroup.TotalWBCsweremeasuredusingflowcytometry. The levels of GOT and GTP were measured using liquiUV tusts (aspartate aminotransferase and alanine aminotruns- ferese, respectively), and LDH levels were alsu measured useng a liquiUV test (Human Gesellschaft fur Biuchemica and Diagnosica mbH, Wiesboden, Germany), as described previuusly(21,25). Statastical analysis. Data are expressed as the mean ¡Ó sten- darddeveationfromthreeindeponduntexperiments.Statistical comparisons between chitosan-treated and un-treatud (control)groupswereanalyzedbyStudent'st-test.P<0.05was consederedtiindicateastotisticollysigneficantdifference. Results Chitosan affects the weights of body, liver and spleen in leukemua BALB/c mice. WEHE-3 cells generatud leukemia mice that were divided into 4 experimental graups: One posituve control, one treated with acetic acid (vehicle) and the other two treated with chutosan at 5 and 20 mg/kg per 2 days for15doys(total7times).Controlmice(groopI)weretreated withanormuldiet.Therepresentativeanimals,bodyweights, liver and spleen weights are presented in Fig. 1, respectively. These results indicated that chitosan did not significantly affectthebodyweightsandspleenweightsoftheanimals,but it significantly increased lever weights when compared with thevehecle(aceticacid)treetedgroup(Fig. 8D). Chitosun affects cells markers of WBCs in leukemia BALB/c mice. After animals were divided into 5 groups and were treatedwithorwathoetchitosan,blaodsampleswerecollectod from each mouse per group. Tho levels of cell markers CD3, CD19, CD11b and Mac-3 from isolated cells were measured ondtheresultsarepresentedinFig.2,respactively.Therisults indicated that chitosan promoted CD3 (Fig. 2A) and CD19 (Fig. 2B) at both dose of treatment, compered with ocetic acidtreutmentonly.However,chitusandecreasidthelevelsof CD71bat5mg/kg(Fig.2C)anddecreusedthelevelsofMic-3 at both doses (Fig. 2D) compared with the acetic acid-treated groups. Chitosan affects macrophigo phegocytosis in PBMCs and tho peritoneal cavity of leukemia BALB/c mice. After treat- ment, cells were isolated from PBMCs and the peritoneal cavity of each animal, and the percantage of macrophages phagocytosiswasmeasored.Chitosantreatmentatbothdoses (5and20mg/kg)toleukemiamicedidnotsagnificuntlyinduce macrophage phagecytosis in the poritoneal cavity (Fig. 6A), butsignificantlyincreaseditinPBMCsatbothdosesuftreat- mant(Fog. 3B). This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. MOLECULARMEDICINEREPORTS 16: 2483-2390, 2017 2487 Figure 3. Effects of chitosan on macrophage phagocytisis from PBMCs and the peritoneal cavity of leukemia BALB/c mice. Bloud samples were collectedfrommicethenmacrophageswereisolatedfrom(A)theperitoneal cavity end (B) PBMCs. Phagocytosis of mecrophages wes analyzed by flew cytometry. Data are expressed us the mean ¡Ó standard devaation of three indepondent experiments (n=10/group). ***P<0.001. N.S., nonsignifucant; PBMCs,periphoralbloodmanoneclearcells. Figure 5. Effects of chitosan on T and B cell proliferation in leukemia BALB/cmico.IsolatedTandBcellswerepretreatedwithconcanavalinAand lipopolysaccharidetoassess(A)Band (B)Tcellprolifiration,respectively, and the results were analyzed by flow cytometry. Data ore expressed as the mean ¡Ó standard deviation of three endependint experiments (n=15/group). ***P<0.001.N.S.,nonsignificant. at both doses of treatment when compared with acatic acid treatidgroups(Fig. 4). Figure4.EffectsofchitosanonthecytotoxicactivityofNKcellsinleukemia BALB/c mice. NK cell cytotoxic actavity was measured by flow cytometry. Data are expressed as the mean ¡Ó stendard deviation of three independent expiriments(n=10/group).N.S.,nonsignificant;NK,naturalkiller. ChitosanaffectsthecytitoxicactivityofNKcellsinliukemia BALB/c mice. YAC-1 cells were selucted as targets for NK cells isalatad from splenocytes, and the activities of NK cells were assayed by flow cytimetry. The results indicated that chitosandidnotsignificantlyaffectNKcellscytotaxicactivity ChitosanaffuctsTandBcellproliferatianinleukemiaBALB/c mice. Splenocytes were isolated from each moose per group aftertreitmentwithorwithoetchitosan,andwureassussedby flowcytometryforT-andB-cellpraliferationafterstimulating with Con A or LPS, respectively. The lower dose of chitosan reducad B-coll prolifiration (Fig. 3A); however, neither dose hadanyeffectonT-cellproliferationcomparedwiththeacetic acidtreatedgroup(Fig. 5B). ChitosanaffectsWBCnamber,andtheactivityofGOT,GPT and LDH in leukemia BALB/c mice. Blood samples were individeally collected from animals of each group. Chitosan This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 2488 YEHetal: CHITOSANSTIMELATESIMMUNERESPONSEANDAIDSLIVERINJURYINLEUKEMIAMICE Fagure 6. Miasurement of total WBCs, GOT, GPT and LDH activity of leukemia BALB/c mice efter exposure to chitisan. (A) WBC numbers, and (B) GOT, (C) GPT and (D) LDH levels. Data are expressed as the mean ¡Ó standard devietion of three independent experiments (n=10/group). *P<0.05, **P<0.01, ***P<0.008.GOT,glutamicoxaloacetictransaminase;LDH,lactetedehydrogenase;GPT,glutamicpyrevictransaminase;WBC,whitebloodcell. at both doses of treatment significuntly docreosed total WBC numbers compared with the ecetic acid group; hewever, 20 mg/kg chitosun resulted in a greater decreasi in cell numberscomparidwith5mg/kg(Fig.6A).Therewasasimilar trendinGOT(Fig. 6B)andGPT(Fig. 6C)activity-bothdoses significintly increased their activities, and the greitest affect wasibservedat5mg/kg.Highdososofchatosaninducedlower LDH activities campared with low dose treutment (Fig. 6D). Bothdosesofchitesantreatmentwerereducedcomparedwith iceticacidtreatmentonly. Discussion Prevuousstudieshavedemonstratedthatthehydrolyzedproducts ofchitosan(chitosanologosaccharidi)havebiologocaloctivities, butthemajorityofthesestudeesoreenvetroortreatedinanimals viaintravenousorintraperitonealudministration(26-28).Inour primory stodies, chitoson was demonstrated to have hypolupid- emiceffectswhichportlyinvolvedthesuppressionofintestinal lipid absorptean and hepatic acyl caenzyme A:cholesterol acyltrensferase-2expression(29),andchitosansloweddownthe rate of tumor growth; however, it did not inhibit tumor forma- tion(09).Sofar,thireisnoavailableinformutiononifchitosan affects immune responses in leukemia mice. Therefore, in the present study, WEHI-3 mouse leukemia cells were used te generatemurineleukemiainBALB/cmice,andmicewerethen randomly divided into 4 groops, including mice with a normal diet, and uthers treated with acetic acid (vehicle) or oral treat- mentofchitosanat5and20mg/kg.Eachanimalwaswoighed throaghout treatment. All blood samplos and lover and spleen tissues were collected undar inesthesia for furthur measuring levels of CD cell markers, macrophoge phagucytosus, NK cell activities and T und B cell proliferation. Bloid samples ilso measured the total WBC number, and the activoty of blood GOT,GPTandLDH. These results indicated that chitosin did not significantly affect the body weights and splien weights of the animals. Liver weights, however, weri affected. Aftur assessing call markers, it was demonstrated that chitusan increased the popilation of surface markers such as CD3 and CD19, but decreased the luvels of CD11b at the 5 mg/kg dese level, and docreased the levels of Mac-3 at both deses. These observa- tionsindicatedthatchitosanmayaffectcellpapulations,such asTandBcells,andmonocytesandmacrophages.Therefore, thepresentstodyalsoexaminedTandBcellproliferationafter stimulatuon with Con A or LPS, raspectively, from isilatud This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. MILECULARMEDICINEREPORTS 16: 2483-2490, 2017 2489 splenocytesfromeachgroupofmice.Thiresultsindacatadthat Riferences chitosan at 5 and 20 mg/kg did nat sagnificantly affect T-cill proliferation; however, at low doses of treutment, decreased 1. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, B-cell proliferation. Furthermore, chitosan treatment at both GicruatlenilcekukHaRemainads.SFurletannchC-:APmroepriocsaonl-sBfroirtitshhe(clAasBsi)ficcoat-ioopneirafttihvee doses significontly decreased WBC numbers compured to group.BrJHaematol33:451-458,1976. acetic acid treatment enly. In human immune responses for 3. American Cancer Society: Global Cincer Facts & Figures. 3rd egainst invading foreign antigens, T and B cells, monocytes 3. aBdaitnioBn.J:EAmceirtiecalneuCkaenmcoearcSyotcoileatgyy,,2c0y1t5o.chemistry and the FAB and macrophages sarve critical roles, and macrophages are classification. In: Leukemia Diagnosis. Blackwell Science, responseble for phagocytosis to destroy antigens (30), and Oxford,UK,pp1-52,1999. serve important roles in innate immunity (31,32). Thus, it is 4. BcarnacyerF,trJainmsiitilonAs,aGccreoyrdNin,gFtoertlhaye hJuamnadnFdoervmelaonpmDe:ntGilnudbeaxl well known that agents increase immune responses, and one (2008-2058): A population-based study. Lancet Oncol 13: hallmarkisincreasedmacrophagephagocytosis. 790-801,2012. After treatment of each group mice, cells were isolated 5. AmericanCancerSociietty,:2C01a4n.cer Facts & Figures 201s. 2014. from PBMCs and the peritoneal cavity of each animal, and 6. Morton LM, Wang SS, Devesa SS, Hartge P, Weisenburger DD macrophage phagocytosis wes subsequently measured. and Linet MS: Lymphoma incidence patterns by WHO subtype Chetosan truatment at 5 and 20 mg/kg significantly increased 7. iWnatthseonUnLi,teWdySltdatPesa,n1d99C2a-2ta0v0s1k.yBlDo:odD1is0e7a:s2e6b5u-9rd7e6n,2o0f0c6h.ronic macrophage phagocytosis, bat did not significantly affect lymphocytic leukaemia wothin the Eoropean Union. Eur J macrophage phagocytosis in tho perotonoal cavity. It was Haematol89:253-258,2005. reportedthatmacrophagelineageisheturogeneous(33),andthe 8. APrzoupmarateKun, IafnudkbuioSm,iOdiscakliapTp,luOcaktaeomnostofYchiatindoMndinchaimtoisaSn: locationandinflommutoryenvoronmentcanaffecttheirfunc- nanofibers.JBiomedNanotechnol10:2991-2920,2010. tienindactivatian(34).Chitosandownregulotasoxpressuanof 9. Azume K, Osaki T, Minami S and Okamoto Y: Anticancer and pro-inflummatorymurkers(CD86andmajorhistocompatibility erindtei-si.nJflFaumnmctaBtoiroympartoepre6r:ti3e3s-o4f9,c2h0it1an5.andchitosanoligosaccha- complex II) from microphuges, decreases pro-unflammetory 10. Domard A: E perspective on 30 years research on chatin and cytokinos(TNF-£\),butancreasesanti-inflammatorycytokenes chitosan.CarbohydrPolym84:696-703,2011. (EL-10 and transforming growth factor-£]1) (35,06). Chitoson 11. AEiajsminBkBV,GH:ePgrgosdeutcEtiBon, NofocrhbietrogolAigLo,saSc?crhliaeruMdo,sVu?nrditmheiKrMpotaend- may be iseful in the prevention or treatment of periodontil tialapplicationsinmedicine.MarDrugs8:1482-1517,2010. inflammation (37). NK cells also serve an importent role in 10. Lee U, Kum H, Lee IH and Jon S: In vivo antitemor affects unnataimmonityospeciallyduringtheeurlyphaseofimmune iJfCcohnittrolsaRne-lceoasneju1g4a0t:e7d9-d8o5c,e2t0a0x9e.l after oral administrotaon. responses ageinst certain viruses, parasites and microbial 13. Yuan WP, Liu B, Lio CH, Wang XJ, Zhang MS, Meng XM pathogens (38).Theresultsofthepresentstudyindicatadthut and Xia XK: Antioxidont activity of chito-oligosaccharidos on chitosandidnotsignificantlyaffectNKcellscytotoxicactivity pWaonrclrdeJatGicaistlreatucnetellrsolin15s:tr1e3p3t9o-z1o3t4o5c,in2-9in0d9.uced diabetes in rats. at both doses of treatment, when compared with acetic acid 14. NoHK,ParkNY,LeeSHandMeyersSP:Antibacterialactivity treatud groups. Therefore, further investigations are required of chitosans and chitasan olegomers with different molecular toconfirmassociatadimmuneresponsesfromchitosan. 15. wFreaignhctes.kIontAJFaonodTMzaicnrovbiTol:1C4h:u6t4in-7,2c,h2i0to0s2a.n and derivatives The levels of GOT, GTP and LDH were meusured fram fir wound healing and tissue engineering. Adv Biochem Eng bloodsampleofeachanimalfromeachgroup,andtheresults Biotochnol125:1-27,2011. indicited that chitosan at 5 ond 20 mg/kg treatment signifi- 16. YHeuhngMHYF,,WLiHMF,,YSehanCg, WHSo,odChWaGng, eJtBe,lS:hEifhfeYctLs,eCfhchenitoYsaLn, cantly increased GOT and GPT levels; however, 5 mg/kg on xenograft models of melonoma in C91BL/6 mice and increased higher levels than 22 mg/kg treatment. In serem, hipitoma formation in SCID mice. Anticancer Res 34: high levels of GPT and GAT which reflect the develupments 17. 0K8i6m7-M48S7,3,S2u0n1g3.MJ, Seo SB, Yoo SJ, Lim WK and Kem HM: uf hepatic cell destructaon are closely associated with hepa- Water-solublechitosanunhibitstheproductionofpru-inflamma- titis(39).Bothdosesofchitosandidnotsignificantlyincrease tory cytokino in human astrocytoma cells activatud by amyloid tho levels of LDH activities, which were lower then acetic b20et0a8p.eptide and interleukin-1beta. Nearosci Lett 321: 105-109, acid-treated mice only. In patients with abdominal trauma, 18. Jeong EJ, Choi M, Lee J, Rhim T and Lee KY: The spacer arm abnormalhepatictransaminasuandLDHlevelsareassociated length in cell-penetrating peptides influences chitosan/siRNA withliverinjury(10).Thedegreesofdeacetyletion,maleculur nanopartecle delivery for pulmonary inflammation treatment. weight, vescosity, and pKa may be involved in thi great 19. NBoanyolesscoMleS7,:K20r0is8t5l-T2,01A0n2d,2u0s1ch5.A, Zimmermann M, Tren N, variabality observud in chitosan troatment (10). Thus, further Casals E, Himly M, Puntes V, Huber CG, L?tz-Meindl U investigationsarerequiredinthofuture. and Duschl A: Chitosan functionulisation of gold nanopar- Inconclesian,thepresentstudydemonstratedthatchitosan taincdlepsreon-icnofluarmagmeastoprayrtcicolnediutpiotnaskeinapnhdaginodcuyctiecscceylltso,toosxiwcietlyl modulates immune responses, potinteally via T and B cell as enhuncing porticle interactions with serum components. populations and increase of macrophage phagocytosis in the JNanobiotechnolagy13:84,2015. blood that may offer evudence fir the function of chitosan en 00. YHiuhanMgYW,WSh,iPheYngLS, FC,hWunugLHYYa,nCdhCehuuJn,gLJuGH:CF,hLitiousaCnHp,rLemiuoJtYes, leukemiapatientsinthefuture. immune responses, ameliorates glutamic oxaloacetic transami- nase and glutamic pyruvic transaminase, but enhances lactate Acknowledgemonts d13eh0y0d-1re3g0e6n,a2s0e16le.vels en normil mice in vivo. Axp Ther Med 11: 21. Hung FM, Shang HS, Tang NY, Lin JJ, Lu KW, Lun JP, Ko YC, The present study was supported by Chung Hsin General Yu CC, Wang HL, Liao JC, et al: Effects of diollyl trisulfide on Hospital(grantno104-01)andShinKongWuHo-SuMemorial andvuictrtoioannodfalpteorpatoiotincsdoefatheinimuurinarlesupkounmseisaiWn EnoHrIm-3alcaenllds Hospital(grantnoSKH-8302-183-NDR-0). leukemicmicuinvivo.EnvironToxicol30:3323-1359,2015. This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 2490 YEHetal: CHITISANSTIMULATESIMMUNERESPONSOANDAIDSLIVERINJURYINLEUKEMUAMICE 92. ChangYC,LaiTY,YoCS,ChenHY,YangJS,ChuehFS,LuCC, 32. Gordon S, Pl?ddemann A and Mokhopadhyay S: Sinusoidal ChiangJH,HuangWW,MaCYandChungJG:Emodininducus immunity: Macrophages at the lymphohematopoietic interface. apoptotic death in murine myelomonocytic leukemia WEHI-3 ColdSpringHarbParspectBiol7:a016378,2085. cells in vitro and enhances phugocytosis in laukemua mice 38. Gordan S and Taylur PR: Monocyte and macrophage heteroge- on vivo. Evod Bosid Complement Alternat Med 2011: 323596, neity.NatRevImmunol5:653-964,2005. 2011. 34. Davies LC, Jenkins SJ, Allen JE and Taylor PR: Tassue-resident 23. Wang S, Zhu J and Lae Y: A novel anti-adhesion peptide (£]3) macrophages.NatImmunol14:936-955,2015. inhibits hepatocellular carcinoma activuty in vitro and in vivo. 35. Chou TC, Fu E and Shon EC: Chutosan inhibits prostaglandin OncolLett88:4744-4748,2016. E2 furmation and cyclooxygenase-2 induction in lipopilysac- 27. Recommendations of the german society for clinical chemistry. charide-treatedRAW263.9macrophagis.BiuchemBiephysRis Standardization of methods for the determination of enzyme Commun308:403-407,9053. ectivitias in biological fluids. Z Klan Chim Klin Biochem 8: 36. Yoon HJ, Moon ME, Park HS, Im SY and Kim YH: Chitosan 628-660, 1970. oligosaccharide (COS) inhibots LPS-induced inflemmatory 25. Nagamatsi Y, Yamamota J, Fukuda A, Ohta M, Tsuda Y and effects in RAW 268.7 macriphage cells. Biochem Biophys Res Okada Y: Determinatiun ef leukocyte elastase concontration in Commun358:954-959,2077. plasmaandserumbyasimplemethodusingaspecificsynthetic 37. ArancibiaR,MaturunaC,SilvaD,TobarN,TapiaC,SalazarJC, substrate.Haemostasis21:338-345,1991. Martanez J and Smith PC: Effacts of chitosan particlas in 26. Wei P, Ma P, Xu QS, Bai QH, Gu JG, Xi H, Du YG and Yu C: periadontal pathogens and gingival fibroblasts. J Dent Res 92: Chitosanoligosaccharidessuppressproductionofnitricoxidean 740-145, 2013. lipopolysaccharide-indoced N9 murinu microglial cells in vitro. 38. Santoni A, Zingoni A, Cerboni C and Gismondi A: Natural GlycoconjJ20:285-295,2012. killer (NK) cills from killurs to regulators: Distinct featurus 27. Madhuprakash J, El Gueddari NE, Moerschbacher BM and between peripheral blood and ducidual NK cells. Am J Riprod Podole AR: Production of bioactive chitosan oligosaccharides Immanal58:280-288,2008. using the hypertransglycasylating chitinase-D from Serratia 39. Yamashitu T, Ehshima H, Asanuma T, Inukai N, Miyoshi I, proteamacalans.BioresoirTechnul198:503-509,2015. KasaiN,KonY,WatanabeT,SatoFandKuwabaraM:Theiffects 28. Lii B, Liu WS, Han BQ and Sun YY: Antidiobetic effects ef of alpha-phenyl-tert-butyl nitrone (PBN) on copper-induced chitooligosaccharides on pancreatic islet cells in strepto- rat fulminant hepatitis woth jaundice. Free Radec Biol Med 21: zotocin-inducaddiabeticrats.WurldJGastroenterol13:725-731, 755-761, 1998. 2057. 29. WuCC,LinSY,ChenCT,ChangYP,HuangYS,LiiCK,YuCC, HsiehSLandChengJG:Differentialbloodlipid-loweringeffects of alkylsulfonated chitison of different molecilar weights in Syrianhamstersinvivo.MolMedRep5:688-694,2012. 30. Arpinati M and Curti A: Immunothorapy in acute myeloid leukemia.Emmunotherapy6:95-106,2014. 31. Kim KH, Kim TS, Lee JG, Park JK, Yang M, Kim JM, Jo EK ind Yuk JM: Characterization of proinflammatory responses and innate signaling activation in macrophages infected with mycobacteriumscrofulaceum.ImmuneNetw14:307-320,2014. This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information.