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Expression and inhibition of il 23 by colon cancer cells a promising approach in prevention of ibd.

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  • 1. Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.4, 2013Expression and InhibitionPromising ApproachDr. Vishal Bhargava1. Department of Biochemistry, G.R. Medical College2. Department of Biotechnology DRDO Gwalior* E-mail of the corresponding author:The research is financed by G.R. Medical College Autonomous SocietyAbstractInflammatory bowel disease is chronic uncontrolled inflammaactivated leukocytes. Here we have developed anepithelial cell line HT-29 expressed elevated ILstrains. In this in-vitro model of IBD, ILanti-inflammatory drug Sulfasalizine was studied and was found to block the up regulation of ILKeywords: IBD (Inflammatory Bowel Disease), HT(Lipopolysaccharide).1. IntroductionInflammation can be defined as a series of nonmediators respond to tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterizedin the acute phase by increased blood flow and vascular permeability along with the accumulation of fluid,leukocytes, and inflammatory mediators such as cydevelopment of specific humoral and cellular immune responses to the pathogen(s) present at the site of tissue injury(Carol A. 1997).Most cytokines involved in the inflammation processestheir effects locally or systemically in an autocrine or paracrine manner (Carol A. 1997). Cytokines are involved inextensive networks that involve synergistic (proinflammatory cytokines) as(anti-inflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cells(Adams G 2003).Sometimes immune cells fail to distinguish among the foreign and its native cells, as a resultinflammation is targeted towards its own cells, the condition is termed as Auto inflammation.One such condition is Inflammatory Bowel Diseaseinteracted with its own underlying immunthere by leading to the destruction of tissue as suchInflammatory bowel disease (IBD)Ulcerative colitis (UC) and Crohn’s disease (CD).uncontrolled inflammation characterized by intense mucosal recruitment of activated leukocytes (Hanauer. S 2006).Although the diseases have some features in common, there arOver the past 15 years wide variety of candidate genes have been studied for IBD. Significant linkages have beenreported on chromosomes 1,3,6,7,12,14,16 and 19. Detailed mapping of chromosome 16 resulted in identification ofthe gene responsible, at least in part for this linkage (Podolsky D.K. 2002).This gene encodes a cytoplasmic proteindesignated nucleotide-binding oligomerization domaindomain CARD15 (Hanauer B 2006)immune system. It is the first gene to be clearly associated with IBD, and >60 mutations have been recognized, 3 ofwhich have been linked to development of CD. The mechanism wheredevelopment of IBD remains unclear. The NOD2 gene is expressed mainly in monocyte/macrophage cell lines,where it has a role in host signaling pathway. Reasonable data suggest thCrohn’s disease is dominated by CD4+ lymphocytes with a type 1 helperJournal of Biology, Agriculture and Healthcare3208 (Paper) ISSN 2225-093X (Online)25nd Inhibition of Il-23 by Colon Cancer Cells: APromising Approach in Prevention of Ibd.Dr. Vishal Bhargava1*Dr. Neelima Singh1, Dr.Richa SijoriaDepartment of Biochemistry, G.R. Medical College, Gwalior (M.P.) India. (474009)Department of Biotechnology DRDO Gwalior (M.P) Indiamail of the corresponding author: bhargavavishal6@gmail.comThe research is financed by G.R. Medical College Autonomous SocietyInflammatory bowel disease is chronic uncontrolled inflammation characterized by intense mucosal recruitment ofactivated leukocytes. Here we have developed an in vitro model for IBD using colon cancer cell line HT29 expressed elevated IL-23 levels when induced with LPS isolatedmodel of IBD, IL-23 was playing a key role in inflammation cascade. Action ofinflammatory drug Sulfasalizine was studied and was found to block the up regulation of ILIBD (Inflammatory Bowel Disease), HT-29, IL-23 (Interleukin-Inflammation can be defined as a series of non-specific defense mechanism of body, in which cells and differento tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterizedin the acute phase by increased blood flow and vascular permeability along with the accumulation of fluid,leukocytes, and inflammatory mediators such as cytokines. In the sub acute/chronic phase it is characterized by thedevelopment of specific humoral and cellular immune responses to the pathogen(s) present at the site of tissue injuryMost cytokines involved in the inflammation processes are multifunctional. They are pleiotropic molecules that elicittheir effects locally or systemically in an autocrine or paracrine manner (Carol A. 1997). Cytokines are involved inextensive networks that involve synergistic (proinflammatory cytokines) as well as antagonistic interactionsinflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cellsSometimes immune cells fail to distinguish among the foreign and its native cells, as a resultinflammation is targeted towards its own cells, the condition is termed as Auto inflammation.Inflammatory Bowel Disease in which the gut micro biota if through any pathway getsinteracted with its own underlying immune cells; the process of inflammation towards these microorganisms beginsthere by leading to the destruction of tissue as suchInflammatory bowel disease (IBD) refers to two chronic diseases that cause inflammation of the intestines:Crohn’s disease (CD). The hallmark of inflammatory bowel disease is chronicuncontrolled inflammation characterized by intense mucosal recruitment of activated leukocytes (Hanauer. S 2006).Although the diseases have some features in common, there are some important differences.Over the past 15 years wide variety of candidate genes have been studied for IBD. Significant linkages have beenreported on chromosomes 1,3,6,7,12,14,16 and 19. Detailed mapping of chromosome 16 resulted in identification ofthe gene responsible, at least in part for this linkage (Podolsky D.K. 2002).This gene encodes a cytoplasmic proteinbinding oligomerization domain 2 NOD2 also known as caspase activation and recruitmentdomain CARD15 (Hanauer B 2006). This is a polymorphic gene the product of which is involved in the innateimmune system. It is the first gene to be clearly associated with IBD, and >60 mutations have been recognized, 3 ofwhich have been linked to development of CD. The mechanism whereby defects in the NOD2 gene lead to thedevelopment of IBD remains unclear. The NOD2 gene is expressed mainly in monocyte/macrophage cell lines,where it has a role in host signaling pathway. Reasonable data suggest that mucosa of the patients with establiCrohn’s disease is dominated by CD4+ lymphocytes with a type 1 helper-T-cell (Th1) phenotype, characterized bywww.iiste.orgy Colon Cancer Cells: Af Ibd., Dr.Richa Sijoria2Gwalior (M.P.) India. (474009)Indiabhargavavishal6@gmail.comtion characterized by intense mucosal recruitment ofmodel for IBD using colon cancer cell line HT-29. Colon23 levels when induced with LPS isolated from various bacterial23 was playing a key role in inflammation cascade. Action ofinflammatory drug Sulfasalizine was studied and was found to block the up regulation of IL-23.-23), Sulfasalizine, LPSspecific defense mechanism of body, in which cells and differento tissue injury (Elenkov IJ 2005). Inflammation, the response of tissue to injury, is characterizedin the acute phase by increased blood flow and vascular permeability along with the accumulation of fluid,tokines. In the sub acute/chronic phase it is characterized by thedevelopment of specific humoral and cellular immune responses to the pathogen(s) present at the site of tissue injuryare multifunctional. They are pleiotropic molecules that elicittheir effects locally or systemically in an autocrine or paracrine manner (Carol A. 1997). Cytokines are involved inwell as antagonistic interactionsinflammatory cytokines) and exhibit both negative and positive regulatory effects on various target cellsSometimes immune cells fail to distinguish among the foreign and its native cells, as a result the process ofinflammation is targeted towards its own cells, the condition is termed as Auto inflammation.in which the gut micro biota if through any pathway getse cells; the process of inflammation towards these microorganisms beginsrefers to two chronic diseases that cause inflammation of the intestines:The hallmark of inflammatory bowel disease is chronicuncontrolled inflammation characterized by intense mucosal recruitment of activated leukocytes (Hanauer. S 2006).e some important differences.Over the past 15 years wide variety of candidate genes have been studied for IBD. Significant linkages have beenreported on chromosomes 1,3,6,7,12,14,16 and 19. Detailed mapping of chromosome 16 resulted in identification ofthe gene responsible, at least in part for this linkage (Podolsky D.K. 2002).This gene encodes a cytoplasmic proteinalso known as caspase activation and recruitment. This is a polymorphic gene the product of which is involved in the innateimmune system. It is the first gene to be clearly associated with IBD, and >60 mutations have been recognized, 3 ofby defects in the NOD2 gene lead to thedevelopment of IBD remains unclear. The NOD2 gene is expressed mainly in monocyte/macrophage cell lines,at mucosa of the patients with establishedcell (Th1) phenotype, characterized by
  • 2. Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.4, 2013production of IFN-γ, IL-2 IL-8 and ILby CD4+ lymphocytes with an atypical type 2 helpertransforming growth factor β (TGF-2. Materials and Methods2.1 HT-29These cells are also adherent colonic epithelial cells obtainewith different cellular products mainly carcinoembryonic antigen (CEA); transforming growth factor beta bindingprotein; mucin. The cell line used in the present study was purchased from American Type Cu(ATCC), Manassas VA 20108, USA.2.2 ReagentsDMEM Powder, Bovine serum albumin (BSA), DMSO, Sodium bi carbonate, Trizma, EDTA and others wereobtained from Sigma (Sigma Aldrich co. St. Louis MO USA).2.3 ELISA kitKit for both IL-23 was quantitated from the cell supernatants using DuoSet ELISA Development KITS.ELISAs are sensitive enzyme immunoassays that measure soluble levels of proteins in biological samples. R&DSYSTEM provides Complete ELISA kits that offer accurate andDuoSet kits contain the basic components required to develop an immunoassay to measure natural or recombinantproteins. All these were obtained from3. MethodologyIn our study HT-29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)and 1% penicillin –streptomycin antibiotic solution .The cells were incubated at 37of 5% CO2 in air.3.1 SeedingAround 80% confluency stage, the cells were sub cultured. Medium contained in the flask was completely discardedusing a sterile disposable pipette. Cells were washed with 1X PBS so as to remove the dead cells and the cell debris.Cells were treated with trypsin-EDTA solution (0.25%W/V) and kept at37cells were detached from the substratum, as monitored under the phase contrast microscope. Once the cells werecompletely detached, immediately excess amount ofprolonged exposure of cells with the trypsinthe complete medium acts as an inhibitor of trypsin. Flushing gently with a pipette tFrom this cell suspension, a sample was subjected to counting using a hemocytometer. The desired no. of cells wasselected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the respplate. (12, 24, or 96 well plate) according to the requirement of the experiment. Incubate the respective plates overnight at 37°C in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.3.2Treatment with LPS and the drug additionSpecific cell line was selected based on the experiment and the cells were allowed to get confluent. Post confluentcells were seeded in 96 well plate as described above, with a concentration of 30,000then left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to bepre-incubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in differentconcentrations. Based on the solubility of the drug if soluble in water was directly diluted with medium and appliedto the cells or if insoluble was the diluted in DMSO and the applied. 3 hours later of drug incubation cells weretreated with LPS to get inflamed. LPS treatment was optimizefficient strain to cause inflammation was chosen for the experiment. Based on the volume of the cell suspension inthe well LPS was introduced respectively. Followed this plate containing cells were incubovernight. Next day supernatant from each well was collected by centrifuging plate at 1500 rpm for 15 min.Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA atplate with the adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess themaximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISAper the user manual provided with the RObservations of ELISA are depicted graphically as follows with interpretations.Journal of Biology, Agriculture and Healthcare3208 (Paper) ISSN 2225-093X (Online)268 and IL-23. In contrast the mucosa in patients with ulcerative colitis may be dominatedth an atypical type 2 helper-T-cell (Th2) phenotype, characterized by the production of-β) and IL-5 (Podolsky. D.K 2002).are also adherent colonic epithelial cells obtained from the patients of colorectal adenocarcinoma butwith different cellular products mainly carcinoembryonic antigen (CEA); transforming growth factor beta bindingprotein; mucin. The cell line used in the present study was purchased from American Type Cu(ATCC), Manassas VA 20108, USA.DMEM Powder, Bovine serum albumin (BSA), DMSO, Sodium bi carbonate, Trizma, EDTA and others were(Sigma Aldrich co. St. Louis MO USA).quantitated from the cell supernatants using DuoSet ELISA Development KITS.ELISAs are sensitive enzyme immunoassays that measure soluble levels of proteins in biological samples. R&DSYSTEM provides Complete ELISA kits that offer accurate and reproducible results with no development timeDuoSet kits contain the basic components required to develop an immunoassay to measure natural or recombinantproteins. All these were obtained from R&D Systems, Inc 614 McKinley Place NE, Minneapolis, USA.29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)streptomycin antibiotic solution .The cells were incubated at 37°C under a humidified atmosphereAround 80% confluency stage, the cells were sub cultured. Medium contained in the flask was completely discardedusing a sterile disposable pipette. Cells were washed with 1X PBS so as to remove the dead cells and the cell debris.EDTA solution (0.25%W/V) and kept at37°C in the CO2 incubator for 2cells were detached from the substratum, as monitored under the phase contrast microscope. Once the cells werecompletely detached, immediately excess amount of complete medium was added to the cells. This is because aprolonged exposure of cells with the trypsin -EDTA solution is known to cause cell death. The serum contained inthe complete medium acts as an inhibitor of trypsin. Flushing gently with a pipette tip mixed the contents of the flask.From this cell suspension, a sample was subjected to counting using a hemocytometer. The desired no. of cells wasselected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the respplate. (12, 24, or 96 well plate) according to the requirement of the experiment. Incubate the respective plates overC in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.and the drug additionSpecific cell line was selected based on the experiment and the cells were allowed to get confluent. Post confluentcells were seeded in 96 well plate as described above, with a concentration of 30,000-40,000 cells/well. Plate washen left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to beincubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in differentility of the drug if soluble in water was directly diluted with medium and appliedto the cells or if insoluble was the diluted in DMSO and the applied. 3 hours later of drug incubation cells weretreated with LPS to get inflamed. LPS treatment was optimized using different strains of bacteria and maximumefficient strain to cause inflammation was chosen for the experiment. Based on the volume of the cell suspension inthe well LPS was introduced respectively. Followed this plate containing cells were incubovernight. Next day supernatant from each well was collected by centrifuging plate at 1500 rpm for 15 min.Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA atthe adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess themaximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISAper the user manual provided with the R&D Duo kit system.Observations of ELISA are depicted graphically as follows with interpretations.www.iiste.org23. In contrast the mucosa in patients with ulcerative colitis may be dominatedcell (Th2) phenotype, characterized by the production ofd from the patients of colorectal adenocarcinoma butwith different cellular products mainly carcinoembryonic antigen (CEA); transforming growth factor beta bindingprotein; mucin. The cell line used in the present study was purchased from American Type Culture collectionDMEM Powder, Bovine serum albumin (BSA), DMSO, Sodium bi carbonate, Trizma, EDTA and others werequantitated from the cell supernatants using DuoSet ELISA Development KITS. SandwichELISAs are sensitive enzyme immunoassays that measure soluble levels of proteins in biological samples. R&Dreproducible results with no development timeDuoSet kits contain the basic components required to develop an immunoassay to measure natural or recombinant, Inc 614 McKinley Place NE, Minneapolis, USA.29 cells were cultured with a medium consisting of DMEM, 10%FETAL BOVINE SERUM (FBS)C under a humidified atmosphereAround 80% confluency stage, the cells were sub cultured. Medium contained in the flask was completely discardedusing a sterile disposable pipette. Cells were washed with 1X PBS so as to remove the dead cells and the cell debris.C in the CO2 incubator for 2-5 mins. Thecells were detached from the substratum, as monitored under the phase contrast microscope. Once the cells werecomplete medium was added to the cells. This is because aEDTA solution is known to cause cell death. The serum contained inip mixed the contents of the flask.From this cell suspension, a sample was subjected to counting using a hemocytometer. The desired no. of cells wasselected by diluting it with complete medium. Seed the cell suspension diluted with the medium in the respectiveplate. (12, 24, or 96 well plate) according to the requirement of the experiment. Incubate the respective plates overC in the CO2 incubator prior to any treatment, for ensuring proper growth and spreading of cells.Specific cell line was selected based on the experiment and the cells were allowed to get confluent. Post confluent40,000 cells/well. Plate washen left at CO2 incubator for over night incubation. As designed for the experiment 1) if the cells are to beincubated with drug followed by LPS treatment, the cells in the plate were incubated with drug in differentility of the drug if soluble in water was directly diluted with medium and appliedto the cells or if insoluble was the diluted in DMSO and the applied. 3 hours later of drug incubation cells wereed using different strains of bacteria and maximumefficient strain to cause inflammation was chosen for the experiment. Based on the volume of the cell suspension inthe well LPS was introduced respectively. Followed this plate containing cells were incubated in CO2 incubator forovernight. Next day supernatant from each well was collected by centrifuging plate at 1500 rpm for 15 min.Supernatant from the plate was collected in sterile eppendorfs and was stored as samples for ELISA at -80°C. Thethe adherent cells was then undertaken for the MTT assay for measuring the cell viability. To assess themaximum tolerance of cells for the given concentration of drug MTT assay was performed. ELISA was performed as
  • 3. Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.4, 20134. Observations and Results.The Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disease where intestinal epithelial cells (IEC)gets differentiated as cancer cells when induced by some inflammatory agents. Normally inremain separated from underlying immune cells by a membrane lining. In most of the cases of IBD the underlyingimmune cells comes in direct contact(Hendrickson BA 2002), thereby leading to uncontrolled tissue destruction (Weber CR 2007).The process of inflammation could be studied with the help of certain tumour markers. In prepamodel for IBD several cytokines were used as prominent markers whose upregulation determined the stages ofinflammation (Carol A 1997).In-vitro model for IBD could be developed by using colon cancer cell line like HTwhich show some inflammatory responses. In our experiment we have used HTproteins indicating inflammatory responses (Haller D 2004).Development of inflammatory conditions in HTstudies the bacterial DNA or bacteria as a whole were confirmed to express inflammatory responses (Haller D 2000& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell walas an inflammatory agent is a new approach of our research laboratory for this study since no study in this regard hasbeen made so far.In normal intestinal flora (in-vivo) numbers of bacterial strains exist but few of them have been found to bepathogenic in nature. LPS was isolated from different bacterial strains and were introduced in HTselection of LPS we used strains of E.coli BE.coli B-4 did not induce much inflammatory conditions in HTof bacteria like Salmonella Minnesota (S.Minn.), Salmonella Enteritidis (S.Ent.) and Pseudomonas to study theinflammatory reactions. It was observed that S.Minn andAfter developing inflammatory conditions in colon cancer cell line HTS.Minn and S.Ent we then checked inflammatory responses with cytokines as a marker (Neurath MF 2003).Cytokines are categorized under proInterleukin-23 (IL-23) cytokine for observing the effect of LPS. LPS induction expressed different levels of ILwith respect to each bacterial strain (Puleston J 2005). Upreguthe inflammatory conditions in given cell line.Further in our study to check the inflammation process developed, the effect of certain antistudied in the same (Botoman VA 1991).concentration was introduced to inhibit the upregulated cytokine ILinhibition of upregulated IL-23 with minimum concentration of Sulfasalizine wasmimic the in-vivo model of IBD (Mulder J 1988) (Fig.1).Dose response curve (DRC) was set up for each given concentration of drug with respect to bacterial strain and cellline to study the cytotoxic effect and most effect4.1Figure.14.2 Figure.25. ConclusionHT-29 cells expressed different levels of inflammatory processes for inLipopolysaccharide (LPS) isolated from strains ofgram-negative bacteria induced inflammation in the monolayer and cowere highly expressed in the process of inflammation. Sulfasalizine at different concentration was found effective intreatment of inflammatory bowel disease.6. ReferencesAraki, Y. Sugihara, H. & Hattori T. (2006) In vitro effects of dextran sulfate sodium on a Cacoplausible mechanisms for dextran sulfate sodiumBisping, G. Lügering, N. Lütke-Brintrup, S. Pauels, H.G. Schürmann, G, Domschke, W. & Kucharzik, T. (2001)Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellularinterferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes coJournal of Biology, Agriculture and Healthcare3208 (Paper) ISSN 2225-093X (Online)27The Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disease where intestinal epithelial cells (IEC)rentiated as cancer cells when induced by some inflammatory agents. Normally inremain separated from underlying immune cells by a membrane lining. In most of the cases of IBD the underlyingimmune cells comes in direct contact with micro-biota of intestine resulting in the process of inflammation(Hendrickson BA 2002), thereby leading to uncontrolled tissue destruction (Weber CR 2007).The process of inflammation could be studied with the help of certain tumour markers. In prepamodel for IBD several cytokines were used as prominent markers whose upregulation determined the stages ofvitro model for IBD could be developed by using colon cancer cell line like HT-29, CaCowhich show some inflammatory responses. In our experiment we have used HT-29 cell which expressed variousproteins indicating inflammatory responses (Haller D 2004).Development of inflammatory conditions in HT-29 cell line could be achieved through various ways. In severalstudies the bacterial DNA or bacteria as a whole were confirmed to express inflammatory responses (Haller D 2000& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell walas an inflammatory agent is a new approach of our research laboratory for this study since no study in this regard hasvivo) numbers of bacterial strains exist but few of them have been found to benic in nature. LPS was isolated from different bacterial strains and were introduced in HTselection of LPS we used strains of E.coli B-4 (present in abundance in intestine) to elicit infection. It was found thatmuch inflammatory conditions in HT-29 cells. Then LPS was isolated from different strainsof bacteria like Salmonella Minnesota (S.Minn.), Salmonella Enteritidis (S.Ent.) and Pseudomonas to study theinflammatory reactions. It was observed that S.Minn and S.Ent were most potent inflammatory agents.After developing inflammatory conditions in colon cancer cell line HT-29 with LPS isolated from bacterial strains ofS.Minn and S.Ent we then checked inflammatory responses with cytokines as a marker (Neurath MF 2003).Cytokines are categorized under pro-inflammatory and anti-inflammatory in nature. In our present study we selected23) cytokine for observing the effect of LPS. LPS induction expressed different levels of ILwith respect to each bacterial strain (Puleston J 2005). Upregulation of IL-23 cytokine (proinflammatory) confirmedthe inflammatory conditions in given cell line.Further in our study to check the inflammation process developed, the effect of certain antistudied in the same (Botoman VA 1991). The popularly used drug for IBD like Sulfasalizine at differentconcentration was introduced to inhibit the upregulated cytokine IL-23 (Bonner GB 1996). Complete or above 70%23 with minimum concentration of Sulfasalizine was regarded as efficiency of drug tovivo model of IBD (Mulder J 1988) (Fig.1).Dose response curve (DRC) was set up for each given concentration of drug with respect to bacterial strain and cellline to study the cytotoxic effect and most effective concentration with least cytotoxic effect was analyzed (Fig 2).29 cells expressed different levels of inflammatory processes for in-vitro model of inflammatory bowel disease.ed from strains of Salmonella Enteritidis and Salmonella Minnesotanegative bacteria induced inflammation in the monolayer and co-culture system of inflammation. ILwere highly expressed in the process of inflammation. Sulfasalizine at different concentration was found effective intory bowel disease.Araki, Y. Sugihara, H. & Hattori T. (2006) In vitro effects of dextran sulfate sodium on a Cacoplausible mechanisms for dextran sulfate sodium-induced colitis. Oncol Rep, 16,1357-62.Brintrup, S. Pauels, H.G. Schürmann, G, Domschke, W. & Kucharzik, T. (2001)Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellulargamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells.www.iiste.orgThe Inflammatory Bowel Disease (IBD) is a chronic autoinflammatory disease where intestinal epithelial cells (IEC)rentiated as cancer cells when induced by some inflammatory agents. Normally in-vivo these epithelial cellsremain separated from underlying immune cells by a membrane lining. In most of the cases of IBD the underlyingbiota of intestine resulting in the process of inflammation(Hendrickson BA 2002), thereby leading to uncontrolled tissue destruction (Weber CR 2007).The process of inflammation could be studied with the help of certain tumour markers. In preparing the in-vitromodel for IBD several cytokines were used as prominent markers whose upregulation determined the stages of29, CaCo-2, SW-620 and others29 cell which expressed variousough various ways. In severalstudies the bacterial DNA or bacteria as a whole were confirmed to express inflammatory responses (Haller D 2000& Mahmood A 2003). The idea to introduce Lipopolysaccharide (LPS), a product of gram negative bacterial cell wallas an inflammatory agent is a new approach of our research laboratory for this study since no study in this regard hasvivo) numbers of bacterial strains exist but few of them have been found to benic in nature. LPS was isolated from different bacterial strains and were introduced in HT-29 cells. In the4 (present in abundance in intestine) to elicit infection. It was found that29 cells. Then LPS was isolated from different strainsof bacteria like Salmonella Minnesota (S.Minn.), Salmonella Enteritidis (S.Ent.) and Pseudomonas to study theS.Ent were most potent inflammatory agents.29 with LPS isolated from bacterial strains ofS.Minn and S.Ent we then checked inflammatory responses with cytokines as a marker (Neurath MF 2003).inflammatory in nature. In our present study we selected23) cytokine for observing the effect of LPS. LPS induction expressed different levels of IL-2323 cytokine (proinflammatory) confirmedFurther in our study to check the inflammation process developed, the effect of certain anti-inflammatory drugs wasThe popularly used drug for IBD like Sulfasalizine at different23 (Bonner GB 1996). Complete or above 70%regarded as efficiency of drug toDose response curve (DRC) was set up for each given concentration of drug with respect to bacterial strain and cellive concentration with least cytotoxic effect was analyzed (Fig 2).vitro model of inflammatory bowel disease.Salmonella Minnesota ofculture system of inflammation. IL-23 levelswere highly expressed in the process of inflammation. Sulfasalizine at different concentration was found effective inAraki, Y. Sugihara, H. & Hattori T. (2006) In vitro effects of dextran sulfate sodium on a Caco-2 cell line and.Brintrup, S. Pauels, H.G. Schürmann, G, Domschke, W. & Kucharzik, T. (2001)Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellulared with intestinal epithelial cells. Clin
  • 4. Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.4, 2013Exp Immunol.123, 15-22.Bonner, G.B. & Ruderman,W.B., (1993) 5disease. In: Inflammopharmacology. Norwell, Mass.: Kluwer Academic, 247Bonner, G.F., (1996) Current medical therapy for inflammatory bowel disease.Botoman, V.A, Kozarek, R.A. & Taylor RB, (1991). Inflammatory bowel disease difficult medical management.Philadelphia: Saunders, 374-86Carol, A. Feghali & Timothy M. Wright. (1997) Cytokines in acute and chronic inflammation Frontiers in Bioscience2, d12-26, 12Farmer, R.G, Whelan G & Fazio, V.W, (1985) Longbetween the clinical pattern and prognosis.Griffiths, A.M., Ohlsson, A. Sherman, P.M., & Sutherland LR. (1995) Metaprimary treatment of active Crohns disease.Haller, D. Bode, C. Hammes, W.P., Pfeifer, A.M., Schiffrin, E.J &Blum, S. 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McKproduction by a MAPK-dependent, NFκΒ10.1096/fj.02-0950fje.Markus F Neurath (2003) Inflammatory bowel disease: Cytokines and cytokine therapies4075–4082.Mulder CJ, Tytgat GN, Weterman IT, Dekker W, Blok P. & Schrijver M. (1988) Doubleslow-release 5-aminosalicylate and sulfasalazine in remission maintenance in ulcerative colitis.95,1449-53.Nugent FW & Roy MA. (1989) Duodenal Crohns disease: an analysis of 89 cases.Owen D. Bayless TM (1989) Endoscopic biopsy Current management of inflammatory bowel disease. Philadelphia:Decker, 1989,13-6.Podolsky DK. (2002) Inflammatory bowel diseasePuleston J, Cooper M, Murch S, Bid K, Makh S, Ashwood P, Bingham AH, Green H, Moss P, Dhillon A, Morris R,Strobel S, Gelinas R, Pounder RE, Platt A. (2005) DistAliment Pharmacol Ther. 21, 109-120Rampton D.S & Phil D. 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  • 5. Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.4, 2013Figure 1.Legend 1.The above graph shows induction of ILand Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of ILby LPS strain. From third plot onwards different concentration of drug is provided to down regulate the ILproduction and almost 100% inhibitionFigure 2.Legend 2.Cell viability for the above drug concentration was asdrug can be safely used till a concentration of around 1mM and there after number of cell death was found to bemore.0100200300400500600700800900HT conIL-23(pg/ml)Inhibition of IL020406080100120PercentageviabilityCell viability upon treatment of HT29 cells with SulfasalizineJournal of Biology, Agriculture and Healthcare3208 (Paper) ISSN 2225-093X (Online)29The above graph shows induction of IL-23 in HT-29 cells by LPS isolated from the strains of Salmonella Minnesotaand Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of ILFrom third plot onwards different concentration of drug is provided to down regulate the ILinhibition is seen around 1mM – 2.5 mM drug concentrationCell viability for the above drug concentration was assayed simultaneously using MTT assay and it was found thatdrug can be safely used till a concentration of around 1mM and there after number of cell death was found to beNo drug 25uM 50uM 100uM 500uM 1mMInhibition of IL-23 in HT 29 cells by SulfasalizineS.minnSal.entconcentration of drugCell viability upon treatment of HT29 cells with Sulfasalizinesal.minn LPSSal.ent LPSwww.iiste.org29 cells by LPS isolated from the strains of Salmonella Minnesotaand Salmonella Enteritidis. First plot in the graph shows the control group. Second plot shows the induction of IL-23From third plot onwards different concentration of drug is provided to down regulate the IL-82.5 mM drug concentrationsayed simultaneously using MTT assay and it was found thatdrug can be safely used till a concentration of around 1mM and there after number of cell death was found to be1mM 2.5mMS.minnSal.entsal.minn LPSSal.ent LPS
  • 6. This academic article was published by The International Institute for Science,Technology and Education (IISTE). The IISTE is a pioneer in the Open AccessPublishing service based in the U.S. and Europe. The aim of the institute isAccelerating Global Knowledge Sharing.More information about the publisher can be found in the IISTE’s homepage:http://www.iiste.orgCALL FOR PAPERSThe IISTE is currently hosting more than 30 peer-reviewed academic journals andcollaborating with academic institutions around the world. There’s no deadline forsubmission. Prospective authors of IISTE journals can find the submissioninstruction on the following page: http://www.iiste.org/Journals/The IISTE editorial team promises to the review and publish all the qualifiedsubmissions in a fast manner. All the journals articles are available online to thereaders all over the world without financial, legal, or technical barriers other thanthose inseparable from gaining access to the internet itself. Printed version of thejournals is also available upon request of readers and authors.IISTE Knowledge Sharing PartnersEBSCO, Index Copernicus, Ulrichs Periodicals Directory, JournalTOCS, PKP OpenArchives Harvester, Bielefeld Academic Search Engine, ElektronischeZeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe DigtialLibrary , NewJour, Google Scholar