The GI system is under changeless antigenic challenge from immense Numberss of bugs. The GI mucosal surface is a portal entry to a big Numberss of foreign antigens including those from bacteriums. The uninterrupted exposures of the GI mucous membrane to big Numberss of bacteriums have made the mucosal immune system evolve mechanisms to arouse different responses to pathogenic bacteriums and commensal bacteriums. Both commensal and infective bacteriums are recognized by the host ‘s pattern acknowledgment molecules, including TLR and NLR. Although commensal and infective bacteriums have common conserved microbic motives that can trip the form acknowledgment molecules, the different result of pathogen-host interaction from non-pathogen-host interaction is the activation of strong innate and adaptative immune responses directed at eliminating the pathogens ( as reviewed by ( Magalhaes et al. , 2007 ) and ( Salzman et al. , 2007 )
Mucosal epithelial cells run alonging the GI piece of land provide more than a mere physical barrier to infection. Mucin glycoproteins produced by mucus-producing cells in the epithelial tissue or submucosal secretory organs are an built-in portion of the mucosal barrier to pathogens ( as reviewed by ( Linden et al. , 2008b ) ) . MUC1 and MUC13 belong to cell surface mucin household, and have an of import function in modulating immune responses against bacteriums in the tummy and bowel, severally ( Williams et al. , 2001, McAuley et al. , 2007, McGuckin et al. , 2007 ) . Following the invasion of the mucous secretion bed by bacteriums, the big extracellular sphere of cell surface mucins can move as releasable steerer ligands for bacterial adhesions, thereby forestalling subsequent bacterial fond regard to and invasion of epithelial cells ( McAuley et al. , 2007, Every et al. , 2008, Linden et al. , 2009 ) . Furthermore, cell surface mucins initiate intracellular signalling that may be of import as detector mechanisms in response to invasion or epithelial harm ( as reviewed by ( Carraway et al. , 2003 ) ) .
It was shown that mice missing MUC1 are predisposed to more terrible pathology following Helicobacter pylori infection ( McGuckin et al. , 2007 ) and Campylobacter jejuni infection ( McAuley et al. , 2007 ) . However, crosstalk between mucins and TLR and NOD signalling in the context of anti-inflammatory activity of mucins in response to pathogens is of possible importance in finding the nature of the inflammatory response to infection.
The Gastrointestinal Immune System against Pathogenic Bacteria
Distinguishing between Commensal and Pathogenic Bacteria
The mucosal surfaces of the GI piece of land are vulnerable to infection due to their anatomical and physiological belongingss. They are thin and permeable which allow nutrient soaking up. This permeableness creates exposure to infection. However, even though the GI surroundings is colonized by a dynamic microbic ecosystem, the huge bulk of these bugs do non do infection and disease. The mucosal immune system has evolved mechanisms to arouse different responses to pathogenic bacteriums and commensal bacteriums. On one manus, the GI immune system is required to avoid a vigorous immune response to nutrient antigens or commensal bacteriums, whilst it is obliged to observe and kill pathogens making the GI mucosal surface ( as reviewed by ( Magalhaes et al. , 2007 ) and ( Blum and Schiffrin, 2003 ) ) .
Despite the uninterrupted exposure of bacteriums in close propinquity to the enteric surface epithelial tissue, it is really unusual that the commensal bacterium cause disease. The host ‘s immune system has several mechanisms to command the immune response towards the commensal bacterium. Bing recognized by TLRs and NODs, so the bacteriums are sampled by dendritic cells. The sampling by dendritic cells occurs in M cells that overlay the enteric Peyer ‘s spots and lymphoid follicles or in the epithelial junctional composites. Particular subsets of the dendritic cell population ( CD11c+CD11b+CD8i??- ) are responsible for keeping tolerance to harmless bacteriums by bring oning Th2-induced cytokines and IL-10, taking to T cell-dependent IgA production. Other dendritic subsets ( CD8+ cytoid plasma ) besides induce IL-10 bring forthing Treg cells. The commanding influence of Treg cells can forestall the Th1 response to commensal bacteriums which can be responsible for redness and tissue harm. Furthermore, the commensal bacteriums can rarefy TLR signalling and NF-i?«B activation through suppression of Ii?«B-i?? ubiquination, so that NF-i?«B-mediated response can be prevented. Additionally, the mesenteric lymph nodes can restrict the mucosal immune initiation procedure to the mucosal immune system itself. ( as reviewed by ( Blum and Schiffrin, 2003 ) , ( Kelly et al. , 2004 ) , and ( Magalhaes et al. , 2007 ) ) . In decision, the normal intestine immune system can interact with commensal bacteriums in a good mode, non to kill them but to utilize them as a trigger factor to modulate the GI immune system to be ever in a homeostatic state of affairs.
Interactions between the Innate and Adaptive Immune System against Pathogenic Bacteria
The of import different characteristic between commensal and infective bacterium is the presence of virulency factors. Virulence factors determine the pathogenicity of the bacteriums and are encoded by organized cistrons called Pathogenicity Islands. The virulency factors can ease bacteriums to adhere, to occupy, to colonize and to do infection and disease in a susceptible host. The same virulency factors may non do infection in an immunized host, so virulence factors may non merely be seen independently as infective bacterial feature but besides as a consequence of host-pathogen interaction. By and large, the virulency factors can be adherent molecules, invasion systems, enzymes and toxins. Bacterias have different mechanisms which enable to bring forth toxin in the external environment or to shoot the virulency factors straight into the cytosol of the mark cell utilizing a secernment system. The secreted virulency factors allow bacteriums to traverse the epithelial barriers ( as reviewed by ( Magalhaes et al. , 2007 ) ) . Several mechanisms can be utilised by bacteriums to traverse the physical barrier of the epithelial tissue viz. invasion of M cells, invasion of epithelial cells or interrupting the epithelial liner of the bowel ( Janeway, 2005 ) .
The epithelial cells that line the bowel sense the bacteriums through the innate immune system. In the intestine, there are two categories of pattern acknowledgment receptors ( PRRs ) called Toll-like receptors and NOD-like receptors which are expressed at the cell surface or associated with intracellular cell organs and localise to the cell cytosol, severally. The host PRRs can separate bacterial constituents which are called pathogen associated molecule forms ( PAMPs ) from host constituents. Twelve different members of TLRs detect different microbial-derived ligands, for illustration TLR2 detects lipoproteins and lipoteichoic acid, TLR4 detect lipopolysaccharide from Gram negative bacteriums, and TLR5 detects flagellin ( reviewed in ( Kumar et al. , 2009 ) and ( Takeuchi and Akira, 2010 ) ) . The PAMP acknowledgment by PRR leads to signal bringing to host sing the presence of infection and trigger proinflammatory and anti microbic response ( as reviewed by ( Akira et al. , 2003 ) and ( Takeuchi and Akira, 2010 ) ) .
Following antigen acknowledgment by TLRs, different cistrons are transcriptionally regulated depending on the TLRs and cell types involved. TLR signalling relies on the use of the distinguishable adapter molecules such as MyD88 and TRIF. By and large, there are two distinguishable tracts of TLR signalling, viz. MyD88-dependent and TRIF-dependent tracts. In the early stage of NF-i?«B and MAPK activation, MyD88-dependent tract is activated whereas in the late stage, TRIF-dependent tract is stimulated. Furthermore, the latest is besides responsible for IRF-3 activation which leads to the initiation of IFN-i?? and IFN-inducible cistrons. In the terminal, TLR signalling contributes to the inflammatory response ( reviewed in ( Mogensen, 2009 ) , ( Kawai and Akira, 2010 ) and ( Takeuchi and Akira, 2010 ) ) .
Other pattern acknowledgment receptors are NOD1 and NOD2 which belong to a household of NLRs. These two receptors can function as intracellular detectors of muramyl dipeptide, a constituent of peptidoglycan, from bacteriums or other pathogens ( as reviewed by ( Bourhis and Werts, 2007 ) and ( Benko et al. , 2008 ) ) . The look of NOD protein is alone. There can be an autocrine stimulation but TLR-induced NF-i?«B activation can take straight to paracrine stimulation, for illustration, via IL-8 ( Takahashi, 2006, Werts et al. , 2007 ) . Therefore, NODs synergize with TLRs to bring on redness in response to pathogens.
Both NODs and TLRs can bring on the ripening of human dendritic cells which links between innate and adaptative immune responses. Being activated, the dendritic cells migrate to regional lymph nodes and show the antigenic peptide to relevant MHC molecules. Simultaneously, phagocytosis and upregulation of costimulary molecules happen. Finally, the procedure switches in chemokine receptor look and cytokine secernment. Additionally, the stimulated dendritic cells are besides able to excite naA?ve CD4+ T lymphocytes into different Th subsets, which is, in portion, controlled by TLR-induced cytokines ( as reviewed by ( Mogensen, 2009 ) ) . The innate immune response can besides originate further adaptative immune response by bring oning Th1 and Th17 response. NOD1 besides synergize with TLRs to trip Th1 and Th17 response, which both are of import in protection against bacterial infections ( as reviewed by ( Fritz et al. , 2007 ) and ( Mogensen, 2009 ) ) . In decision, both innate and adaptative immune responses work together to counter the pathogens synergistically.
Pathogenic bacteriums differ from non-pathogenic or commensal bacteriums due to the presence of specific pathogenicity cistrons. The cistrons, which can be organized in pathogenicity islands, are responsible for look of virulency factors such as secernment system proteins and toxin. Helicobacter pylori and Campylobacter jejuni are pathogens which infect the GI mucous membrane and cause serious sequelae ( Lecuit et al. , 2004 ) . The pathogenesis and immune response triggered by the pathogens will be discussed briefly in the undermentioned subdivisions.
1.2.1. Pathogenesis of Helicobacter pylori Infection and Host Immune Response
Helicobacter pylori infection still presents a major public wellness job worldwide. It is estimated that about half of the universe ‘s population are colonized with H. pylori ( Zhang et al. , 2009 ) . Although many septic persons remain mostly symptomless, H. pylori infection is associated with peptic ulcer disease, stomachic ulcers, stomachic MALT lymphoma and stomachic glandular cancer ( Uemura et al. , 2002, Ferreccio et al. , 2007 ) and as reviewed by ( Konturek et al. , 2009 ) . Gastric malignant neoplastic disease is the 4th most common malignant neoplastic disease in the universe ( as reviewed by ( Crew and Neugut, 2006 ) ) .
H. pylori infect stomachic epithelial cells. The bacteriums have several mechanisms to last in a hostile environment and to colonize the mucous membrane and interact with the host ‘s epithelial cells to do chronic redness. H. pylori have adapted to populate in extremely acidic environment of the tummy due to their urease enzyme which can hydrolyze urea into ammonium hydroxide so that the sourness of the tummy can be neutralised ( as reviewed by ( Kusters et al. , 2006 ) ) . Furthermore, H. pylori have several virulency factors which facilitate the bacteriums to infect host and arouse an inflammatory response. Certain virulency factors are harboured by certain strains and are associated with specific pathogenesis of the infection. For illustration, cagPAI cistrons encode CagA, a protein associated with the increased hazard of developing peptic ulcer or stomachic malignant neoplastic disease. The protein has indirect oncogenic belongingss and is believed to do mutant of the tumor suppresser cistrons ( Deguchi et al. , 2001 ) and as reviewed by ( Nguyen et al. , 2008 ) . Furthermore, cagPAI cistrons besides encode a Type IV Secretion System ( TFSS ) which can reassign CagA protein and peptidoglycan into host cells and finally, bring on secernment of pro-inflammatory cytokines including IL-8. The secernment setup stimulates intracellular signaling including NF-i?«B and MAPK which leads to the written text of cistrons responsible for redness and programmed cell death ( Shibata et al. , 2005, Ferrero et al. , 2008 ) . Another virulency factor viz. vacA, which harboured by 50 % of all H. pylori can bring on programmed cell death and modulate the immune response ( as reviewed by ( Kusters et al. , 2006 ) ) .
Both excess cellular and intracellular form acknowledgment molecules are of import in the acknowledgment of H. pylori. The bacterial constituent, peptidoglycan, is transferred into the cytol by cag-PAI mediated contact between the epithelial cells and bacteria, and so is recognized by NOD1 ( Viala et al. , 2004 ) . The peptidoglycan is besides recognised by extracellular receptor NOD2 ( reviewed in ( Kusters et al. , 2006 ) ) . The function of TLRs in acknowledgment of bacterial constituents is controversial. Some TLRs such as TLR2, TLR4 and TLR9 can bring on NF-i?«B activation and chemokine look in the stomachic cells ( Rad et al. , 2009 ) , although TLRs do non look to be prevailing in the initiation of innate immune responses against H. pylori. TLR5 is less powerful in triping immune response because the flagellin of H. pylori is non recognised good by TLR5, so that H. pylori can hedge TLR5 immunosurveillance ( Gewirtz et al. , 2004 ) .
The development of H. pylori disease is determined by many factors sing the host-pathogen relationship during the infection. Both innate and adaptative immune responses are of importance to eliminate the pathogen, but most persons get chronic infection likely because the immune system does non unclutter the infection. The failure of one of the constituent can take to the patterned advance of the disease. Naturally, the innate immune response take few yearss after pathogen invasion so is followed by induction of adaptative immune response. H. pylori are able to excite ripening of dendritic cells which leads to the stimulation of Th1 cells, which is TLR-dependent ( Algood et al. , 2007, Rad et al. , 2007 ) .
Another innate immune constituent which has an of import function in the redness procedure caused by H. pylori is mucin. This molecule is of involvement to be investigated sing the fact that mucin is the first molecule that bacteriums have to get by with during incursion to gastric epithelial cells. A really recent survey really relevant to my undertaking has showed that MUC1 can rarefy epithelial redness in response to H. pylori infection. This fading is caused by suppression of Ii?«Bi?? phosphorylation, NF-i?«B inactivation which eventually prevent farther redness ( Guang et al. , 2010 ) . However, the XT between MUC1 and TLR and NOD signalling in modulating epithelial immune response demands to be investigated further.
1.2.2. Pathogenesis of Campylobacter jejuni Infection and Host Immune Response
Campylobacter jejuni is a major cause of diarrheal disease in worlds and is by and large regarded as one of the most common bacterial causes of gastro-enterocolitis in many states including Australia. The campylobacteriosis eruption in Australia during 2001 to 2006 showed that contaminated nutrient and domestic fowl is the of import vehicles to convey the disease ( Unicomb et al. , 2009 ) . The high incidence of diarrhea caused by Campylobacter jejuni and its possible sequelae, makes this infection extremely of import in footings of socio-economic impact ( Ivanova, 2010 ) . Additionally, Campylobacter jejuni infection can do post-infection complications such as MALT lymphoma ( Schmidt-Ott et al. , 2006 ) ( Lecuit et al. , 2004 ) and Guillain-Barre syndrome ( Schmidt-Ott, 2006 ) .
C. jejuni infects the little and big bowel. After consumption, the pathogenesis of C. jejuni infection is commenced when the bacterium penetrate the enteric mucous secretion bed and the production of bacterial protein toxins called cytolethal dilating toxin ( CDT ) . It has been shown that C. jejuni utilize outer membrane cysts to transport all the sub unit of cytolethal dilating toxin. The cell rhythms can highly be arrested by the toxin ensuing in cell decease ( Lindmark et al. , 2009 ) . The release of the toxin so can do epithelial harm because of the toxin itself and because of the inflammatory response to the bacterium. The subsequent infection can so trip host ‘s immune response both mucosal and systemic antigen-specific IgA and IgG both in homo and in ferret theoretical account ( Cawthraw et al. , 2002, Nemelka et al. , 2009 ) .
However, C. jejuni has evolved mechanism to get away from host ‘s innate immune system by hedging from TLR 5- and TLR 9-mediated immunosurveillance. Normally, flagellin, the structural constituent of scourge, can trip enteric epithelial cells to show pro-inflammatory cistrons but C. jejuni have amino acids alterations in their flagellin at the TLR5 acknowledgment site. Therefore, C. jejuni flagellin is a hapless agonist of TLR to bring on pro-inflammatory cytokine production ( Andersen-Nissen et al. , 2005, Watson and Galan, 2005 ) . Such immune equivocation is aimed to do the infection persist. However, upon C. jejuni infection, the bacterial constituent can be recognised by host internal form acknowledgment receptor viz. NOD1 and so NF-i?«B is activated taking to IL-8 production ( Johanesen and Dwinell, 2006, Zilbauer et al. , 2007 ) . Therefore, C. jejuni can bring on IL-8 production independently of TLR5 activation. It has been studied that C. jejuni infection can bring on IL-8 secernment due to bacterial invasion and the toxin/CDT ( Hickey et al. , 1999, Hickey et al. , 2000 ) . The signalling tract responsible for IL-8 production is Mitogen-activated Protein Kinase including extracellular signal-regulated kinase ( ERK ) and p38 MAP kinase with the ERK tract being critical for IL-8 initiation ( Watson and Galan, 2005 ) .
The first measure of pathogenesis in C.jejuni infection is the incursion of enteric mucosal bed by C. jejuni. It is a critical measure to guarantee the farther invasion by the pathogen. The presence of mucin in the epithelial surface has a great benefit to forestall farther harm triggered by the pathogen. It was shown that mucin ( MUC1 ) can protect the epithelial cells from the effects of cytolethal dilating toxin and restrict the C. jejuni infection ( McAuley et al. , 2007 ) . Another epithelial surface mucin is MUC13 which is expressed in GI epithelial cells. Although MUC13 is the same household as MUC1 ( cell surface mucin ) , they may hold differences in the transition of immune responses as respects intracellular signalling.
Mucins are Part of The Immune System
The Characteristics of Mucins
The liner of full GI piece of land is protected by a mucous secretion bed. The chief constituents of the mucous secretion bed are mucin glycoproteins which are produced by mucus-producing cells in the epithelial tissue or submucosal secretory organs. The physical and biochemical feature of the mucin is closely related to their map, for illustration, high viscoelasticity is necessary for protection from mechanical harm and as a trap for bugs. The viscoelasticity is produced by the high molecular weight of mucin macro-molecular composites and the high hydrous nature of mucous secretion ( as reviewed by ( Linden et al. , 2008b ) and ( Byrd and Bresalier, 2000 ) ) .
Mucins are big glycoproteins with O-linked oligosaccharides which are the chief constituent comprising over 70 % of the entire mass. The O-glycosylated glycoprotein province will act upon the biochemical belongingss and the map of mucin. The types of saccharide construction on mucin or glycosylation depends on tissue-specific enzyme look, host and environmental factors such as can be altered during bacterial infection or in the presence of mucosal disease ( as reviewed by ( Linden et al. , 2008b ) and ( Dharmani et al. , 2009 ) )
The look and distribution of mucins in different tissues are determined by 21 different cistrons. The different type of mucin is dependent on the construction and location which can be subdivided into secretory and cell-surface mucin ( reviewed in ( Dharmani et al. , 2009 ) ) . The undermentioned treatment is chiefly about the cell surface mucins which will be studied in my research.
The Cell Surface Mucins
Cell surface or membrane edge mucins are transmembrane glycoproteins that belong to household consisting MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17 and MUC20. Among them, MUC1 and MUC4 are well-characterised. Membrane bound mucins are present on the apical membrane of all mucosal epithelial cells and incorporate big extracellular spheres predicted to organize stiff elongated constructions. With regard to their high look, these molecules are likely to be a dominant component of the glycolayx ( as reviewed by ( Linden et al. , 2008a ) and ( Jonckheere and Van Seuningen, 2009 ) ) .
Cell surface mucins have two spheres viz. the extracellular and cytoplasmatic spheres. The extracellular sphere signifiers an highly big thread-like construction covered by complex of O-linked oligosaccharide and can be shed from the cell surface. The cytoplasmatic spheres of the single mucins are extremely conserved across species and have an of import function in signal transduction due to the ability to interact with kinases and adapter molecules and undergo tyrosine phosphorylation ( Meerzaman et al. , 2001, Thompson et al. , 2006 ) . The MUC1 cytoplasmatic sphere can be cleaved and so the cleaved sphere translocates to chondriosomes and together with p53 written text factor, translocate to the karyon to modulate cell rhythms and prevent programmed cell death caused by genotoxic emphasis ( as reviewed by ( Linden et al. , 2008b ) ) . Most cell-surface mucins appear to be cleaved into two sub units during synthesis. The cleavage procedure occurs via car proteolysis. Additionally, the extracellular i??-subunit can be shed from the cell surface either mediated via a 2nd distinguishable cleavage event or by physical shear ( as reviewed by ( Linden et al. , 2008a ) ) .
MUC1 is expressed by most mucosal tissues including tummy and enteric tissue. MUC1 has been estimated to be 200-500 nanometer in length, so that it will transcend other molecules attached to the plasma membrane. The molecule surface contains complex array of O-linked oligosaccharides that have been shown to adhere to microbic molecules. For illustration, distinguishable set of saccharides play an of import function in adhesion of H. pylori to epithelial cells, fading of H. pylori colonization and facilitation of inflammatory response following H. pylori infection ( as reviewed by ( Linden et al. , 2008b ) and ( Kobayashi et al. , 2009 ) ) .
Much research has been done to look into the ordinance of mucin secernment and their function in signalling tracts both in normal cells and malignant cells. MUC1 is produced by most normal epithelial cells but MUC1 is besides produced by mammary, pancreatic and colon malignant neoplastic disease cells. The different mucin look in normal, preneoplastic and neoplastic cells is determined by different cistrons taking to different phenotypes such as the O-linked saccharides on MUC1 mucin differ between malignant and normal epithelial cells. Such difference can be detected utilizing different monoclonal antibodies ( reviewed in ( Ho et al. , 1995 ) and ( Byrd and Bresalier, 2000 ) ) .
Sing the function of MUC1 in signalling tracts, the intent appears to be to protect the epithelial cells from farther harm and enhance cell endurance. MUC1 can modulate signalling pathway in response to oxidative emphasis in malignant neoplastic disease cells. Additionally, MUC1 is besides involved in NF-i?«B signalling ( Yin et al. , 2004, Ahmad et al. , 2007 ) . Consequently, the activated NF-i?«B tract will trip written text of proinflammatory and anti-apoptotic cistrons ( reviewed in ( Thompson et al. , 2006 ) ) .
MUC13 is extremely expressed in the GI piece of land which has somewhat different GI look form from MUC1 as in tummy ; MUC1 is more uttered than MUC13 ; whereas in bowel, MUC13 is more dominant than MUC1. Like MUC1, MUC13 has similar construction consisting extracellular fractional monetary unit and cytoplasmatic sphere. MUC13 is besides expressed in windpipe, kidney, appendix, in-between ear epithelial tissue and hematopoietic cells ( ( Williams et al. , 2001 ) and as reviewed by ( Linden et al. , 2008b ) ) . MUC13 is besides expressed in malignant cells such as colorectal, oesophageal, gastric, pancreatic and lung malignant neoplastic disease. It has been studied that MUC13 has an of import function in the development of ovarian malignant neoplastic disease due to involvement in signalling tract ( Chauhan et al. , 2009 ) .
The Role of Mucins against Pathogenic Bacteria
With respect to pathogenic bacteriums, there are of import interactions between mucins and bacteriums. These interactions will find the destiny of the bugs and the diseases they cause. The construction and physical belongings of mucins allow these molecules to organize a physical barrier to pathogen invasion. For illustration, MUC1 can screen the epithelial surface from exposure to H. pylori virulency factors, forestalling adhesion-dependent synthesis of proinflammatory cytokines. However, the saccharide construction of mucins favour bacteriums to colonize and to utilize it as an energy beginning and finally bacteriums can work underlying signalling tracts. Such stimulations will typically heighten the mucous secretion secernment in response to bacteriums ( reviewed in ( Byrd and Bresalier, 2000 ) and ( Dharmani et al. , 2009 ) ) .
The secernment of mucin can be altered during infection and redness where infective bacteriums can utilize assorted tracts to bring on or cut down mucin secernment. LPS from H. pylori can trip caspase-3 and bring on programmed cell death which is dependent on p38 MAPK. It was shown that LPS of H. pylori can suppress mucin glycosylation which may hold hurtful effects on mucin assembly. The reduced mucin synthesis farther additions H. pylori adhesion taking to disease ( reviewed in ( Byrd and Bresalier, 2000 ) and ( Dharmani et al. , 2009 ) ) .
However, McGuckin et Al has shown that MUC1 can forestall H. pylori colonization due to the map of MUC1 as a “ releasable steerer ligand ” . The extracellular sphere can be cleaved by host peptidase and later shed the bacterium from epithelial surface ( McGuckin et al. , 2007 ) . This consequence is besides supported by McAuley who found that MUC1 besides can besides restrict the fond regard of C. jejuni to epithelial surface and prevent farther invasion. Furthermore, following release of the extracellular sphere, the cytoplasmatic sphere together with p53 play an of import function in cell signalling related to the transition of cell growing and programmed cell death in response to bacterial toxin ( McAuley et al. , 2007 ) . In decision, the presence of MUC1 is of import to forestall the more terrible pathology following bacterial infection.
As mentioned earlier that bacterial infection is a trigger for proinflammatory responses. The chief intent of redness in the context of bacterial infection is to decide the infection quickly and expeditiously. However, the redness reaction may do tissue harm. Therefore, the host ‘s immune system needs a counter mechanism to avoid inordinate tissue harm but still efficaciously neutralise danger effects caused by pathogens. Such mechanism will assist to forestall farther redness and to cut down the badness of the diseased effects. Mucin glycoproteins have been shown to play an of import function to forestall the development of chronic redness. It was shown by Ueno et Al ( Ueno et al. , 2008 ) that the decreased proinflammatory response in cells bearing mucin was most likely due to the suppression of TLR-mediated NF-i?«B activation by MUC1. In a really recent survey conducted by Guang et Al besides showed that MUC1 can rarefy epithelial redness in response to H. pylori infection. Such fading is performed by stamp downing the NF-i?«B tract via suppression of Ii?«Bi?? phosphorylation ( Guang et al. , 2010 ) . Therefore, mucins may be crucially of import to forestall the development of chronic redness.
In this proposed survey, XT between mucin and pattern acknowledgment molecules will be investigated to specify the mechanism of epithelial immune response transition by mucin against pathogens to forestall farther redness, and will concentrate on the MUC1 and MUC13 cell surface mucins.
It is hypothesized that cell surface mucins ( MUC1 and MUC13 ) modulate inflammatory signalling by GI epithelial cells in response to infective bacteriums.
The undertaking aims to characterize the response of GI epithelial cells to:
unrecorded pathogenic bacteriums,
virulency factors of infective bacteriums
TLR- and NOD-ligands,
in the presence and absence of MUC1 or MUC13.
To this terminal, the MUC1 and MUC13 cistrons will be knocked down in stomachic and enteric cell lines utilizing siRNA transfection every bit good as primary gastric and enteric cells will be isolated from Muc1-/-and Muc13-/-knockout mice, severally.
The rule of the experiment is co-culture GI epithelial cells with the pathogens or pathogen-derived substances that can bring on pro-inflammatory cytokine production. With regard to the result of this survey viz. comparing of the inflammatory response in the presence and absence of MUC1 and MUC13, the gastrointestinal cells which express MUC1 or MUC13 and those cells which do non show MUC1 or MUC13 will be used. Then, NF-i?«B activation written text factor adhering checks on atomic infusions and IL-8 ELISA assays on tissue civilization supernatants will be performed to characterize inflammatory signalling by co-cultured epithelial cells.
The proposed survey of MUC1 is well different from the really late published survey by Guang et Al ( Guang et al. , 2010 ) . The gastric cell lines will be used in this survey is MKN7 cells which has an ability to adhere in a uninterrupted polarized bed. This feature of the cell lines is really of import to guarantee bacteriums to attach to the apical surface of the epithelial cells ( Linden et al. , 2007 ) .
To accomplish each purpose, the survey will be conducted as follows:
To characterize the immune response transition of GI cells to pathogenic bacteriums by MUC1 and MUC13
Gastric theoretical account: primary stomachic epithelial cell from Muc1-/- smasher mice and Muc1+/+ wild type mice, MKN7 human stomachic epithelial cells with high MUC1 look and MKN7 Muc1 si RNA transfectant ; Helicobacter. pylori strain J99 ;
Intestinal theoretical account: primary enteric epithelial cell from Muc13-/- smasher mice and Muc13+/+ wild type mice. LS513 human enteric epithelial cells with high MUC13 look and LS513 MUC13 si RNA transfectant ; Campylobacter jejuni strain 81116
Experiment: co-cultures of bacteriums and mammalian epithelial cells with or without mucin look will be established over clip classs 4, 8, 12 and 24 hr with the same figure of bacteriums. Micro-aerobic co-culture conditions are used in this survey.
Readout: NF-i?«B activation ( commercial written text factor check and inflammatory cytokine production ( IL-8 ) utilizing ELISA
To characterize the immune response transition of GI cells to bacterial toxin by MUC1 and MUC13
If the co-cultures of bacteriums and mammalian cells give the important different consequence ( read-out ) , the experiment will be continued farther to look into which portion of the bacteriums that can bring on inflammatory response
Gastric theoretical account: cells and civilization status as for purpose 1. The bacterial virulency factors ( toxin ) will be obtained from Helicobacter pylori J99 lysates and purified CagA and VacA toxin
Intestinal theoretical account: cells and civilization status as for purpose 1. The bacterial virulency factors ( toxin ) will be obtained from Campylobacter jejuni 81116 lysates and purified CDT.
Experiment: co-cultures of bacterial lysates and mammalian epithelial cells with or without mucin look will be established over clip classs 4, 8, 12 and 24 hr as for Aim 1.
Readout: as for Aim 1.
To characterize the immune response transition of GI cells to TLR- and NOD1- and NOD2-ligands
The look of TLRs and NODs on mammalian epithelial cells needs to be investigated first before executing co-culture of mammalian cells with relevant TLR- and NOD-ligand.
Gastric theoretical account: both murine primary gastric cell and cell lines as for Aim 1.
Intestinal theoretical account: both murine primary enteric cell and cell lines as for Aim 1
Experiment: co-cultures of mammalian cells with ligands for relevant TLR, such as Pam3Cys lipopeptide ( for TLR2 ) , LPS ( TLR4 ) , flagellin ( TLR5 ) , CpG DNA ( TLR9 ) , peptidoglycan ( ligand for NOD1 ) and muramyl dipeptide ( NOD2 ) , with the civilization status as for Aim 1
Readout: as for Aim 1.
Statistical analysis: to analyze the important difference in immune response in the presence and absence of MUC1 and MUC13, the consequences will be analysed utilizing Mann Whitney trial.
Techniques that are traveling to be used in this experiment:
Co-culture of epithelial cells and bacteriums
Real clip PCR, to analyze the degree of look of MUC1 and MUC13 in stomachic and enteric epithelial cells, severally.
Enzyme-linked-immunosorbent serologic assay