aula translocação bacteriana

TRANSLOCAO BACTERIANA

P r o f . J O O B A T I S T A T A J R AC I R U R G I O G E R A L

M E S T R E E M C I R U R G I A

domingo, 16 de janeiro de 2011

HISTRICO


TRAUMA X SEPSE X DMOS


EVIDNCIASSIRS PERSISTENTE E DMOS

SIRS E DMOS

AUSNCIA DE FOCO IDENTIFICVEL.

Paciente Grande Queimado


Manuteno de um estado inflamatrio

persistente e desregulado?

R E S P O N S V E I S ? ? ? ? ?

QUAL O MOTOR DESTE ESTADO ???


TRANSLOCAO A BARREIRA INTESTINAL

LINFONODOS

ESTREIS

VEIA PORTA

Mecanismos de proteo


estudos de translocao

Modelo Crescimentobacteriano

Quebra de

barreiraDefesa

Antibiticos + - -

Queimadura - + +/-

Choque - + ?

Ictercia - - +

IsquemiaHeptica

- + ?


EVIDNCIAS MODELOS EXPERIMENTAIS

Translocation of Certain Indigenous Bacteria from the Gastrointestinal Tract to the Mesenteric Lymph Nodes and Other Organs in a Gnotobiotic Mouse Model RODNEY D. BERG* AND ALVA W. GARLINGTONDepartment of Microbiology and Immunology, Louisiana State University Medical Center, School of Medicine in Shreveport, Shreveport, Louisiana 71130

INFECTION AND IMMUNITY, Feb. 1979, p. 403411

Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis.

Masayuki Fukata,1 Kathrin S. Michelsen,2 Rajaraman Eri,3 Lisa S. Thomas,3 Bing Hu,4 Katie Lukasek,3 Cynthia C. Nast,4 Juan Lechago,4 Ruliang Xu,1,5 Yoshikazu Naiki,2 Antoine Soliman,2 Moshe Arditi,2 and Maria T. Abreu1

Am J Physiol Gastrointest Liver Physiol 288: G1055-G1065, 2005;


TRANSLOCAO BACTERIANACONCEITO

Deslocamento de bactrias ou seus produtos atravs do TGI para stios estreis.

Wolochow et al 1966.

Berg e Garlington 1979.


COMPONENTES DA BARREIRA INTESTINAL

Peristalse

Muco

Epitlio

Renovao epitelial

Abbreviations usedAJ: Adherens junction

BBDP: BioBreeding diabetes-prone ratCD: Crohns disease

EPEC: Enteropathogenic Escherichia coliHA/P: Hemagglutinin proteaseHRP: Horseradish peroxidaseIBD: Inflammatory bowel diseaseiIEL: Intestinal intraepithelial lymphocyte

iIL-9Tg: Intestinal IL-9 transgenic miceJAM: Junctional adhesion molecule

MAPK: Mitogen-activated protein kinaseMLC: Myosin light chain

MLCK: Myosin light chain kinaseMUPP1: Multi-PDZ domain protein-1NSAID: Nonsteroidal anti-inflammatory drug

NO: Nitric oxidePALS1: Protein associated with Lin Seven-1PATJ: PALS1-associated proteinPDZ: Postsynaptic density-95/Drosophila

disc large/Zonula occludens-1 proteinPKC: Protein kinase CSTAT: Signal transducer and activator of transcriptionTER: Transepithelial resistanceTJ: Tight junctionZO: Zonula occludensZot: Zonula occludens toxin

function is to act as a selective filter, allowing the translocation ofessential dietary nutrients, electrolytes, and water from theintestinal lumen into the circulation.1,3-5 The intestinal epitheliummediates selective permeability through 2 major routes: transepi-thelial/transcellular and paracellular pathways (Fig 1).6 Transcel-lular permeability is generally associated with solute transportthrough the epithelial cells and predominantly regulated by selec-tive transporters for amino acids, electrolytes, short-chain fattyacids, and sugars.3-5 Paracellular permeability is associated withtransport in the space between epithelial cells and is regulatedby intercellular complexes localized at the apical-lateral mem-brane junction and along the lateral membrane.7 Contact betweenintestinal epithelial cells includes 3 components that can be iden-tified at the ultrastructural level: desmosomes, adherens junctions(AJs), and tight junctions (TJs; Fig 2).8 The adhesive junctionalcomplexes consist of transmembrane proteins that link adjacentcells to the actin cytoskeleton through cytoplasmic scaffoldingproteins. The AJs and desmosomes are thought to be more impor-tant in the mechanical linkage of adjacent cells.9-11 The TJs, onthe other hand, are the apical-most junctional complex andresponsible for sealing the intercellular space and regulatingselective paracellular ionic solute transport.6,12-14 The AJ andTJ complexes are also important in the regulation of cellularproliferation, polarization, and differentiation.6,11-16

STRUCTURAL COMPONENTS OF JUNCTIONALCOMPLEXESAJs

AJs (also known as zonula adherens) are protein complexes onthe lateral membrane that occur at points of cell-cell contact(Fig 2). They are formed by interactions between transmembraneproteins, intracellular adaptor proteins, and the cytoskeleton. The

major AJs are formed by cadherin-catenin interactions.E-cadherins (calcium-dependent adhesion molecules) are type Isingle-transmembrane-spanning glycoproteins that possess an in-tracellular C-terminus and extracellular N-terminus. The extra-cellular domain forms homotypical interactions with cadherinsof neighboring cells to promote cell-cell adhesion. The intracellu-lar domain contains a catenin-binding domain that interacts withmembers of the armadillo repeat superfamily, b-, g-, andp120-catenin.11,17-21 The catenins link the AJ to the cytoskeletalnetwork through direct binding to the C-terminal domain ofF-actin or indirectly through interactions with other adaptorproteins, such as afadin.22-26 Cadherin-catenin complexes are im-portant not only for linking adjacent cells but also for maintainingcell polarity, regulating epithelial migration and proliferation, andforming other adhesive complexes, such as desmosomes.19,21,27 Insupport of this, downregulation of E-cadherin in the intestinalepithelium weakens cell-cell adhesion and has been linked withperturbed intestinal epithelial proliferation and migration.28,29

Nectin-afadin interactions form another important AJcomplex.21,30,31 Nectins (nectin-1 through nectin-4) are immuno-globulin-like proteins that undergo homophilic and heterophilicinteractions with nectins on adjacent cells.32 Nectins can interactwith the cytoskeleton through afadin, an F-actinbinding protein,or alternatively through interactions with other F- or a-actinbinding proteins, including ponsin/SH3P12, vinculin, and afadinDIL domaininteracting protein.33-37

TJsTJs are the apical-most adhesive junctional complexes in

mammalian epithelial cells and form a continuous belt-like ringaround epithelial cells at the border between the apical and lateralmembrane regions (Fig 2).8 TJs are dynamic, multiprotein com-plexes that function as a selective/semipermeable paracellularbarrier, which facilitates the passage of ions and solutes throughthe intercellular space while preventing the translocation of lumi-nal antigens, microorganisms, and their toxins. The evolution ofTJ biology emerged in the 1960swith the development of electronmicroscopy. Analysis of epithelial cells revealed a series of appar-ent fusions where the space between adjacent epithelial cells waseliminated.6,38,39 These so-called kissing points are morpho-logically different from AJs and desmosomes, where adjacent

FIG 1. Pathways of epithelial permeability. Transcellular permeability isassociated with solute or water movement through intestinal epithelialcells. Paracellular permeability is associated with movement in the inter-cellular space between epithelial cells and is regulated by TJs localized atthe junction of the apical-lateral membranes.

J ALLERGY CLIN IMMUNOL

JULY 2009

4 GROSCHWITZ AND HOGAN

Tipos de permeabilidade e funes de proteo.

MECNICO

Groschwitz e Hogan. J Allergy Clin Immunol 2009


COMPONENTES DA BARREIRA INTESTINAL

Aquisio de flora materna. desenvolvimento imune

Equilbrio como proteo. Antagonismo bacteriano.

Resistncia a colonizao.

Inibio de contato.

MICROBIOLOGIA


FUNO DA MICROBIOTA

Como o TGI consegue distinguir bactrias comensais das patognicas?

Qual a importncia do sistema comensal no desenvolvimento imunolgico em mamferos?


Janine L. Coombes & Fiona PowrieNature Reviews Immunology 8, 435-446 (June 2008)

GALT

SISTEMAIMUNE DE

DEFESA


Potenciais rotas de translocao bacteriana

BLUK113-Langnas November 26, 2007 17:58

CHAPTER 2

Figure 2.1 Cross-section through jejunum(left) and ileum (right) showing crypt-villusstructure. (Images kindly supplied byP. Domizio.)

proximal large intestine, up to the distal third of thetransverse colon, is also from branches of the supe-rior mesenteric artery. However, the distal large intes-tine receives its blood supply via branches of the infe-riormesenteric artery. Corresponding veins drain bloodfrom both the small and large intestine into the portalcirculation. It can be seen that the superior mesentericartery and vein are of paramount importance to bloodsupply to and from the intestine and is the reason whyinfarction and thrombosis are so devastating.

Microscopic structureBoth the small and large intestine are composed of fourdistinct layers: themucosa, the submucosa, themuscu-laris externa, and the serosa. The mucosa consists of alayer of columnar epitheliumbelowwhich lies the lam-ina propria, a loose connective tissue layer containingblood vessels, lymphatics, and some lymphoid tissue.In the small intestine, the mucosa forms fingerlike pro-jections known as villi. These are most pronounced inthe ileum and serve to increase small intestinal surfacearea about tenfold. Each villus contains a dense capil-lary network, which lies just below the epithelium, andblind-ending lymphatic vessels, lacteals, which draininto lymphatics forming a plexus in the lamina pro-pria. In between and in continuity with the villi are theintestinal crypts (Figure 2.1). These crypts contain in-testinal epithelial stem cells which allow repopulationand repair of the small intestinal mucosa. Although themucosa of the colon does not form villi, stem cells re-main located in crypts.

Scattered throughout the lamina propria and sub-mucosa of the small intestine, predominantly theileum, are visible aggregates of lymphoid tissue knownas Peyers patches. The mucosa overlying the Peyerspatches is flattened and contains numerous antigen-sampling M cells. In the colon, the lamina propria con-tains larger numbers of smaller lymphoid nodules. Thelamina propria is separated from the submucosa by athin inner circular layer and an outer longitudinal layerof smooth muscle known as the muscularis mucosae.The submucosa contains a network of blood vessels andlymphatics as well as groups of neurons, which makeup Meissners plexus.The muscularis externa, like the muscularis mu-

cosae, consists of an inner circular layer and an outerlongitudinal layer of smooth muscle. In the large intes-tine, the outer smooth muscle layer forms three bandsknown as teniae coli. Between the inner and outersmooth muscle layers are blood vessels and lymphat-ics, and neurons which make up Auerbachs plexus.The outer serosa is a thin layer of loose connective tis-sue containing vasculature and lymphatics, covered onthe outer surface by mesothelium.

Mucosal cell typesThere are five main epithelial cell lineages, all of whichare derived from stem cells in the intestinal crypts.The most plentiful cells in the mucosal epithelium arecolumnar enterocytes, which make up the absorptivesurface of the intestine. The apical surface of these cells

14

FgadoCorao

Linfonodo

Linfa

Vascular

INVASO

Ducto torcico


EVOLUO DOS ESTUDOS

FOCO NA INFECO

FOCO NO HOSPEDEIRO


COMPONENTES ESTRUTURAIS

Desmosomas -laterais

Junes aderentes (AJ) -Laterais

Tight junction (TJ) - Apicais

Citoesqueleto

cell membranes remain 15 to 20 nm apart.6 Since these initial ob-servations, TJs have been found to consist of 4 unique families oftransmembrane proteins: occludin, claudins, junctional adhesionmolecules (JAMs), and tricellulin.

The extracellular domains of transmembrane TJ proteins inadjacent cells anastomose to form the TJ seal. These interactionsinclude those involving proteins in the same membrane (in cis)and those involving proteins in adjacent cells (in trans). Inaddition, TJ proteins can form either homophilic (with the sameprotein) or heterophilic (between nonidentical TJ proteins) inter-actions. Similar to the AJs, the intracellular domains interact withvarious scaffolding proteins, adaptor proteins, and signalingcomplexes to regulate cytoskeletal attachment, cell polarity, cellsignaling, and vesicle trafficking (Fig 3). The intracellular regionsof AJs possess postsynaptic density-95/Drosophila disc large/Zo-nula occludens-1 protein (PDZ)binding domains, which recruitand interact with PDZ domaincontaining proteins. The PDZ do-main is a common structural domain of 80 to 90 amino acids thatfunctions to anchor transmembrane proteins to the cytoskeleton.The intracellular domains can also interact with nonPDZ-bindingdomaincontaining proteins, such as cingulin, which can interactwith junctional membrane proteins, the actin cytoskeleton, andsignaling proteins.40 The complex network of intracellular proteininteractions is also known as the cytoplasmic plaque.

TJ FORMATION IN THE GASTROINTESTINAL (GI)TRACT

The intestinal epithelium forms the largest and most importantbarrier between our internal and external environments. Thebarrier is maintained by the expression of AJs and TJs, includingcadherins, claudins, occludin, and JAM proteins, which sealtogether adjacent cells and provide cytoskeletal anchorage (Fig3).41 Expression of junctional proteins in the intestine is highlyregulated and dependent on the intestinal compartment (smallor large intestine), villus/crypt localization, and cell membranespecificity (apical, lateral, or basolateral). The complex patternof TJ expression in the intestine is related to the specific functionsof a particular intestinal region and localization. Expression of AJand TJ proteins is also regulated by means of phosphorylation.42-53

Phosphorylation can either promote TJ formation and barrierfunction or alternatively promote TJ protein redistribution andcomplex destabilization.54,55

OccludinThe first TJ-specific integral membrane protein identified was

occludin.56 Occludin is expressed predominately at TJs in epithe-lial and endothelial cells but also by astrocytes, neurons, and den-dritic cells.56-59 Occludin (60-82 kd) is a tetraspanning integralmembrane protein with 2 extracellular loops, a short cytoplasmicN-terminus, and a long cytoplasmic C-terminus.11,13 Functionalanalysis indicates that the extracellular loops and transmembranedomains of occludin regulate selective paracellular permeability.Intracellularly, the C-terminus interacts with the PDZ domaincontaining protein zonula occludens (ZO-1), which is requiredto link occludin to the actin cytoskeleton (Fig 3).60,61

Several occludin isoforms have been identified and arethought to be a result of alternative mRNA splicing.62,63 Nota-bly, several splice variants demonstrate altered subcellulardistribution and interaction with other TJ molecules.62,63

Analysis of the splice variants revealed that the cytoplasmicC-terminal domain is essential for the intracellular traffickingof occludin to the lateral cell membrane and that the fourth trans-membrane domain is critical for targeting occludin to the TJ andfor ZO-1 interactions.63

The function of occludin is not fully delineated; however,in vitro and in vivo data suggest a role for occludin in the regula-tion of paracellular permeability.64,65 Notably, the major allergenof the house dust mite Der p 1 has been found to proteolyticallycleave occludin, leading to the disruption of the TJ complexand increased paracellular permeability.66 Furthermore, hydro-cortisone treatment of bovine retinal endothelial cells increasedoccludin expression 2-fold and enhanced monolayer barrier prop-erties.67 Although occludin is an important constituent of TJs, TJformation and paracellular permeability barrier function are notdependent on occludin. Experimental analyses of occludin2/2

mice demonstrated equivalent numbers and organization of TJsand similar paracellular ion conductance as seen in wild-typemice.42 Furthermore, epithelial transport and barrier functionwere normal in occludin2/2mice.43 In addition to regulating par-acellular permeability, there is evidence suggesting that occludinis involved in cellular adhesion.68 Expression of occludin inoccludin2/2 rat fibroblasts conferred cellcell adhesion thatwas abrogated by synthetic peptides corresponding to the firstextracellular loop of occludin, underscoring the importance ofthis region of occludin in cell adhesion.69

In vitro analysis suggests that occludin localization to theTJ complex is regulated by phosphorylation. Several potentialphosphorylation sites at tyrosine, serine, and threonine residuesof occludin have been identified, and regulation of occludinphosphorylation is proposed to occur by kinases, including thenonreceptor tyrosine kinase c-Yes and protein kinase C (PKC),and phosphatases, including the serine/threonine protein phos-phatase 2A.70,71 PKCh, a novel protein kinase predominantly ex-pressed in the intestinal epithelium, has been shown to directlyphosphorylate occludin at threonine residues (T403 and T404).Blockade of PKCh-mediated occludin phosphorylation disruptedjunctional distribution of occludin and ZO-1 and compromisedepithelial barrier function.72 These data suggest that occludinphosphorylation regulates occludinZO-1 interactions and themaintenance of intact TJ complexes and paracellular barrierfunction.

FIG 2. Overview of intestinal epithelial junctional complexes. The intestinalepithelium consists of a single layer of polarized epithelial cells. Adjacentcells are connected by 3 main junctional complexes: desmosomes, AJs,and TJs. Desmosomes are localized dense plaques that are connected tokeratin filaments. AJs and TJs both consist of transcellular proteinsconnected intracellularly through adaptor proteins to the actin cytoskele-ton. The collection of proteins in the junctional complexes forms cytoplas-mic plaques.


VOLUME 124, NUMBER 1

GROSCHWITZ AND HOGAN 5



COMPONENTES ESTRUTURAIS DA BARREIRA

AJ -Caderinas/ cateninas.

TJ - Ocludinas,claudinas, molculas de adeso funcional (JAMs)

ClaudinsClaudins are 20- to 27-kd integral membrane proteins with 4

hydrophobic transmembrane domains, 2 extracellular loops, andN- and C-terminal cytoplasmic domains.7,73-75 The extracellularloops are critical for homophilic and/or heterophilic, TJ protein-protein interactions and the formation of ion-selective channels.7

The intracellular C-terminal domain is involved in anchoringclaudin to the cytoskeleton through interactions with PDZ-bind-ing domain proteins, including ZO-1, ZO-2, and ZO-3.76-78 Cur-rently, 24 distinct claudin family gene members have beenidentified in human subjects, with a number of orthologues ex-pressed in other species.6,79 They exhibit distinct tissue-, cell-,and developmental stagespecific expression patterns.80-85

Claudin-claudin interactions between adjacent cells can beeither homophilic or heterophilic.86,87 Homophilic interactionshave been demonstrated for claudin-1, claudin-2, claudin-3, clau-din-5, claudin-6, claudin-9, claudin-11, claudin-14, and claudin-19. On the other hand, heterophilic interactions are morerestricted and primarily have been observed with claudin-3,which can interact with claudin-1, claudin-2, and claudin-5.86 No-tably, there is specificity in heterophilic transinteractions. For ex-ample, transfection of fibroblasts with claudin-1, claudin-2, andclaudin-3 led to claudin-3 interactions with both claudin-1 andclaudin-2; however, no interactions between claudin-1 and clau-din-2 were observed.87 These selective interactions are thoughtto explain the diversity in TJ formations and provide a molecularbasis for tissue-specific heterogeneity of barrier function.75

Recent studies with claudin-deficient mice also provide cor-roborative data supporting a role for claudins in the regulation ofbarrier function (Table I). Claudin 12/2 mice die within 1 day ofbirth because of significant transepidermal water loss.44 In addi-tion, transgenic overexpression of claudin-6 in the epidermis dis-rupted TJ formation and increased epithelial permeability.45

Notably, experimental data suggests that claudins can have differ-ential effects on paracellular permeability. For example, introduc-tion of claudin-2 into MDCK I cells that express claudin-1 andclaudin-4 induces a decrease in transepithelial resistance (TER),whereas transfection of claudin-3 had no effect, suggesting thatclaudin-2 markedly decreased claudin-1/claudin-4based TJstrand tightness.88 In support of these data, recent experimentalevidence suggests that claudins can form size- and charge-specificparacellular channels. Transfection of claudin-8 into MDCK IIcells that lack endogenous claudin-8 significantly reduced para-cellular movement of monovalent and divalent cations without af-fecting anion and uncharged solute movement.89 Experimentalanalyses suggest that the first extracellular loop of claudins playan important role in determining charge selectivity. Interchangingof the first or both extracellular domains of claudin-4 on claudin-2profoundly decreased the ion conductance of Na1 relative toCl2.90 Furthermore, substitution of a negatively charged lysineto a positively charged aspartic acid (K65D) within the loop ofclaudin-15 caused an increase in Na1 permeability, whereas mu-tation in the same region of 3 positively charged amino acids tonegatively charged aspartic acid, arginine, and aspartic acid

FIG 3. TJs are localized to the apical-lateral membrane junction. They consist of integral transmembraneproteins (occludin, claudins, and JAMs) that interact in the paracellular space with proteins on adjacentcells. Interactions can be homophilic (eg, claudin-1/claudin-1) or heterophilic (eg, claudin-1/claudin-3). Theintracellular domains of transmembrane proteins interact with PDZ domaincontaining adaptor proteinsthat mechanically link the TJ complex to the actin cytoskeleton. TJ proteins are regulated by means ofphosphorylation by kinases, phosphatases, and other signaling molecules.


JULY 2009


Ocludinas - permeabilidade paracelular.Claudinas - canais seletivos inicos.JAMs -Imunoglobulina de adeso leucocitria.



MUTAES DETECTADAS EM TRANSLOCAO

signal transducer and activator of transcription (STAT)-6 activa-tion, blocked IL-4/IL-13induced barrier dysfunction.110 How-ever, studies in STAT-62/2 mice identified a role for STAT-6signaling in IL-4 and IL-13mediated intestinal epithelial bar-rier dysfunction.47 Moreover, IL-4 and IL-13induced alteredpermeability, glucose absorption, and chloride secretion wereattenuated in STAT-62/2 mice compared with values seen inwild-type mice.47

The anti-inflammatory cytokine IL-10 has also been shown toregulate intestinal barrier function.48,49 Stimulation of ileal seg-ments from Sprague-Dawley rats with IL-10 enhanced intestinalelectroneutral sodium and chloride absorption and inhibited stim-ulated chloride secretion.113 In addition, treatment of T84 epithe-lial cell monolayers with IL-10 blocked IFN-ginduced epithelialpermeability.114 These results suggest that IL-10 plays a protec-tive role in intestinal barrier function. In support of this, micedeficient in IL-10 have increased intestinal permeability.48 Nota-bly, IL-102/2 mice spontaneously develop chronic intestinal in-flammation, which is manifested by symptoms commonlyassociated with Crohns disease (CD), including weight loss, mu-cosal hyperplasia, and chronic enterocolitis.50 These data suggestthat increased permeability might predispose IL-102/2 mice tointestinal inflammation and colitis. Consistent with this hypothe-sis, increased permeability in IL-102/2micewas observed beforedisease onset.48

Mechanistic studies to delineate IL-10mediated intestinalpermeability have implicated the zonulin pathway and TNF-a.Remarkably, inhibition of the zonulin receptor in IL-102/2 miceled to decreased intestinal permeability and reduced colonicTNF-a secretion ex vivo and abrogated the spontaneous develop-ment of colitis.49 These findings further support a role for

increased intestinal permeability in the development of intestinalinflammation and disease and a possible role for zonulin. Thezonulin/zonulin receptor pathway is thought to regulate TJformation through PKC-dependent actin reorganization.115

Whether decreased intestinal barrier function in IL-102/2

mice is primarily due to an inherent defect in the zonulin/zonulinreceptor pathway or, alternatively, a consequence of increasedexpression of cytokines, such as IFN-g and TNF-a, remains tobe delineated.

Immune cellsT cells. Anti-CD3inducedCD41T-cell activation inmice pro-

motes an increase in transcellular and paracellular intestinal perme-ability and the release of proinflammatory cytokines, such as IFN-gand TNF-a (Fig 4).116,117 Furthermore, injection of mice withTNF-a provokes a breakdown in intestinal barrier function, diar-rhea, and PKCa-dependent inhibition of Na1/H1 exchange.118

T cells regulate transcellular permeability through the downregula-tion of Na1/K1ATPase and disruption of Na1 absorption, Na1-glucose cotransport, and inducible Cl2 secretion,116,117 whereasdysregulation of the paracellular permeability pathway is mediatedthrough MLCK-dependent TJ disruption.117

g/d-positive intestinal intraepithelial lymphocytes (iIELgd1),which are closely associated with the basolateral side of intestinalepithelial cells, have also been implicated in intestinal barriermaintenance.119 In response to enteric parasitic infestation,mice deficient in iIELgd1T cells have abnormal claudin-3, occlu-din, and ZO-1 localization; decreased occludin phosphorylation;and abnormal epithelial TJ formation.119 Notably, the alterationsin intestinal barrier function could be attributed to a single subset

TABLE I. Transgenic or knockout mice and effects on intestinal barrier function

Protein Transgenic or knockout Function Phenotype References

Occludin Gene deletion TJ protein No change in TJs or permeability 42, 43Claudin-1 Gene deletion TJ protein Die within 1 d of birth 44Claudin-6 Epidermis transgenic TJ protein Disrupted TJ formation

and increased epithelialpermeability

45

JAM-A Gene deletion TJ protein Increased intestinal permeability 46Increased claudin-10 andclaudin-15 expression

Increased susceptibility to DSScolitis

IL-9 Intestinal transgenic Cytokine Increased intestinal mast cell 52Increased intestinal permeabilityIncreased susceptibility to oralantigen sensitization andanaphylaxis

IL-10 Gene deletion Cytokine Increased permeability;spontaneously have chroniccolitis similar to CD

48-50

STAT-6 Gene deletion Signaling molecule Protected against IL-4 and IL-13induced intestinal epithelialbarrier dysfunction

47

Mcpt1 Gene deletion Mast cell protease Protected against T spiralisinfestationinduced intestinalepithelial barrier dysfunction

51

MLCK Transgenic Signaling molecule Increased intestinal permeability 53Increased onset and severity ofimmune-related colitis

DSS, Dextran sulphate sodium; mMCP-1, murine mast cell protease 1.


JULY 2009




DISFUNO DA BARREIRA INTESTINAL

Fatores exgenos.

Apoptose epitelial e capacidade de regenerao.

Citocinas sistmicas

Atividade imune intestinal

Sistema nervoso entrico


ESTUDO MOLECULARFATORES PREDISPONENTES

energy production associated with increased intestinal permeabil-ity and an increase in serum endotoxin levels.212

The immunosuppressive activity of tacrolimus is through theinhibition of calcineurin, which is critical for IL-2induced T-cell activation.219 Inhibition of IL-2 has been shown to promotethe TH2 immune response.

220 TH2 cells secrete IL-4, IL-5, and-13, which promote IgE-mediated allergic inflammation and setthe stage for food antigen sensitization and the induction of foodallergies. There are most likely several mechanisms involved inthe pathogenesis of food allergies in tacrolimus-immunosup-pressed patients and increased intestinal permeability appearsto be an important mediator to facilitate presentation of food an-tigens to the immune system and oral antigen sensitization.We have recently provided experimental evidence supporting

a role for altered intestinal permeability in oral antigen sensi-tization and the development of food allergies in mice.52 Wegenerated a transgenic mouse that overexpresses the cytokineIL-9 specifically in the enterocytes of the small intestine(iIL-9Tg mice). A consequence of transgenic overexpressionof IL-9 was a pronounced intestinal mastocytosis and alteredintestinal permeability.52 Repeated oral administration of oval-bumin to iIL-9Tg BALB/c mice and not wild-type mice pro-moted the development of antigen-specific IgE, CD41IL-41

T cells, and symptoms of a food-induced allergic response inthe absence of prior systemic sensitization or the use of adju-vant. Pharmacologic mast cell depletion in iIL-9Tg mice wasfound to restore intestinal permeability to levels comparablewith those seen in wild-type mice. Remarkably, reconstitutionof barrier function and decreased intestinal permeability iniIL-9Tg mice prevented orally induced antigen sensitization.52

These findings suggest that increased intestinal permeabilityfacilitates enhanced antigen uptake and the oral induction offood antigen sensitization.Intestinal barrier dysfunction is also thought to contribute to the

severity of food allergeninduced clinical symptoms. Oral chal-lenge of subjects with food allergy with food allergen induced anincrease in the lactulose/mannitol ratio in urine.190,206 The levelof intestinal barrier dysfunction positively correlated with the se-verity of clinical symptoms.206 Notably, treatment of the group

with food allergy with sodium cromoglycate, a mast cell stabi-lizer, before ingestion of food allergen significantly reduced lac-tulose permeability compared with that seen in subjectschallenged with food allergen not receiving sodium cromogly-cate, indicating a role for mast cells in dietary antigeninduced in-testinal epithelial barrier dysfunction.190

Consistent with clinical observations, animal models of GIanaphylaxis and food allergy have also demonstrated increasedintestinal permeability after oral antigen challenge.52,221,222 In-traluminal challenge of egg-sensitized rats with egg albumininduced a 15-fold increase in uptake of 51C-labeled EDTA com-pared with that seen in rats treated with unrelated protein.125

Studies using mast celldeficient animals or pharmacologicagents to deplete mast cells have provided corroborativeevidence demonstrating that mast cells are critical for alteredintestinal barrier function during food-induced allergicreactions.52,126,127,222 Increased permeability after antigen chal-lenge has been shown to initially be the result of increased an-tigen uptake and translocation through the transcellular route, asevidenced by an increase in horseradish peroxidase (HRP)containing endosomes within minutes of HRP challenge inrats that had been sensitized.223 The second phase, which occursafter sensitization and is mast cell dependent, was associatedwith a disruption in the TJs and an increase in paracellularpermeability.223 Collectively, these studies suggest a role foraltered intestinal barrier function in patients with food allergy.Furthermore, these studies suggest a role for mast cells in theregulation of intestinal barrier dysfunction in patients withfood allergy.

IBDThe IBDs CD and ulcerative colitis are chronic, relapsing-

remitting inflammatory diseases. An emerging model of thepathogenesis of IBD suggests there are 3 essential factors: (1) abreakdown in intestinal barrier function; (2) exposure of luminalcontents to immune cells in the lamina propria; and (3) anexaggerated immune response.224 However, it is currently unclearwhich factor is responsible for initiating this self-perpetuating

TABLE II. Diseases associated with altered TJ protein expression and intestinal epithelial barrier function

Disease state TJ proteins Inflammatory cell/cytokine Proposed mechanism References

Food allergy Not defined Mast cells Mast cellmediateddegradation of TJ proteins

52, 190

IBD Y Claudin-3, claudin-4,claudin-5, and claudin-8

[ Claudin-2

CD41 T cells IL-10, IFN-g,and TNF-a and MLCK

IFN-g and TNF-amediatedactivation of MLCKleading to TJ disruptionand dysregulation ofclaudin and occludinexpression

49, 53, 104, 105, 191, 192

Celiac disease Occludin and ZO-1 Zonulin Gliadin-induced zonulinsecretion by intestinalepithelial cells

193, 194

Zonulin-induceddownregulation ofoccludin/ZO-1

Diabetes Not defined Zonulin Increased zonulin secretion 195, 196Stress Occludin and ZO-1 Mast cells and corticotrophin-

releasing hormoneDestabilization andredistribution of occludin/ZO-1

197-201


JULY 2009




REGULAOIMUNE

DAS FUNES DE

BARREIRAINTESTINAL

Alterao de fluxo inico, permeabilidade e redistribuio de molculas de adeso.

of iIELgd1 lymphocytes: T cells expressing Vg71 encoded T cellreceptors. Reconstitution of mice deficient in iIELgd1 T cellswith Vg71 iIELs restored epithelial barrier function.119

Mast cells. Mast cells are present in all compartments of theGI tract.120 On activation, they release a powerful array ofinflammatory mediators, including histamine, 5-hydroxytrypta-mine, neutral proteases (tryptases, chymases, and carboxypepti-dase A), prostaglandins, leukotrienes, platelet-activating factor,and multiple cytokines, including TNF-a, IL-3, IL-4, IL-5, IL-6, and GM-CSF.121-123 Employing models of food allergy or hel-minthic infestation (Nippostrongylus brasiliensis or Trichinellaspiralis), investigators have demonstrated mast cell involvementin intestinal barrier function.124 Intraluminal challenge of egg al-buminsensitized rats induced a 15-fold increase in uptake of51Cr-labeled EDTA compared with that seen in rats treated withunrelated protein.125 The antigen-induced decreased barrier func-tion was associated with mast cell degranulation and an increasein the short-circuit current, a measure of net ion transport.125 Theimportance of mast cells was demonstrated by the absence ofchanges in barrier function in mast celldeficient mice sensitizedand challenged with egg albumin, which was restored by bonemarrow reconstitution.126,127 Furthermore, several mast cell

mediators have been shown to modulate intestinal epithelial iontransport. Pretreatment of egg albuminsensitized rats with hista-mine H1 or 5-hydroxytryptamine 2 receptor antagonists signifi-cantly reduced oral antigeninduced short-circuit currentalterations.126,128

Experimental analyses employing models of parasitic infesta-tion have identified a role for mast cellderived proteases inintestinal barrier function.51 Murine infestation with the entericnematode T. spiralis induced intestinal mastocytosis, occludindegradation, and increased intestinal permeability.51 The altera-tions in barrier function were demonstrated to be mast cell depen-dent as depletion of mast cells with a neutralizing antic-kitantibody ablated intestinal epithelial barrier dysfunction.51 Simi-larly, mice deficient in the murine mast cell protease-1 (Mcpt1)were also resistant to T. spiralis infestationinduced intestinal ep-ithelial barrier dysfunction. Mcpt1 mediated increase in intestinalpermeability during T spiralis infection was linked to occludindegradation.51

Eosinophils. Increased eosinophils and eosinophil granularproteins, including major basic protein, eosinophil peroxidase,and eosinophilic cationic protein, are often associated with IBDand altered barrier function.129-132 In vitro coculture of T84

FIG 4. Immune regulation of intestinal barrier function. T cellderived IFN-g and TNF-a inhibit MLCK-mediated phosphorylation of MLC, leading to TJ junction disruption and intestinal barrier dysfunction. IFN-g can also promote the redistribution of TJ proteins, JAM-A, occludin, claudin-1, and claudin-4 from theapical TJ border by means of a micropinocytosis process. TNF-a and IFN-g can alternatively disrupt TJstability and increase intestinal permeability through dysregulation of occludin expression. IL-4 and IL-13induced increase in intestinal permeability is mediated through induction of epithelial apoptosis andexpression of the pore-forming TJ protein claudin-2. IL-4, but not IL-13, regulates ion conductance throughdownregulation of epithelial cystic fibrosis transmembrane conductance regulator Cl2 channel expression.Intraepithelial iIELgd1 lymphocytes (T-cell receptor Vg71) iIEL cells are important in serine phosphorylationof occludin and TJ stabilization. Mast cell mediators, including the cytokine TNF-a, mast cell protease1 (mcpt-1), and lipid mediators, including histamine, platelet-aggregating factor, and prostaglandins,promote increased Cl2 conductance and increase intestinal permeability. Mcpt1 degrades the TJ proteinoccludin, altering barrier function. Eosinophil-derived major basic protein downregulates TJ protein occlu-din-1 expression in colonic epithelial cells. MBP, Major basic protein.


VOLUME 124, NUMBER 1

GROSCHWITZ AND HOGAN 9


Relao de defesa:Linfcito/EpitlioIntestino delgado: 1 para 10Intestino Grosso: 2-5 por 100


Controle neural do tgi

Mazzone,A.;Fraguai.; Neurogastroenterology. Gastroenterol Clin N Am, 2007

the gut occurs by secretomotor and vasomotor neurons, respectively. The cellbodies of these neurons reside in the submucosal plexus [9].

Interneurons are defined as ascending or descending based on whether theirprocesses run orally or anally. They integrate information from IPANs and, ingeneral, relay the information to enteric motor neurons. At least one type ofascending and three types of descending interneurons have been described inthe small intestine of guinea pig [10]. Ascending interneurons are mainly cho-linergic, whereas descending motor neurons have a varied and complex neuro-chemistry [11]. Ascending interneurons and the three types of the descendingones participate in local motility reflexes. A fourth type of descending interneu-ron conducts the migrating myenteric complexes (MMC). Ascending interneu-rons project to other myenteric neurons, whereas descending interneurons alsoinnervate the submucosal plexus.

A fourth class of enteric neurons, intestinofugal afferent neurons (IFANs),have their cell bodies within the myenteric plexus but send their processesout of the gut wall to form synapses with the inferior and superior mesentericganglia and the celiac ganglion (collectively known as prevertebral ganglia)[12]. IFANs carry efferent signals from the gut and they work as mechanore-ceptors that detect changes in gut volume [12]. Primary IFANs transmit directlyto the prevertebral ganglia without synaptic interruption, whereas another

Fig. 1. Types of neurons in the enteric nervous system. 1. interneuron; 2. excitatory longitudi-nal muscle motor neuron; 3. myenteric intrinsic primary afferent neuron; 4. inhibitory longitu-dinal muscle motor neuron; 5. intestinofugal neuron; 6. myenteric plexus interstitial cell ofCajal; 7. excitatory circular muscle motor neuron; 8. inhibitory circular muscle motor neuron;9. circular muscle interstitial cell of Cajal; 10. cholinergic secretomotor (nonvasodilator)neuron; 11. cholinergic secretomotor neuron; 12. noncholinergic vasomotor neuron; 13.submucosal intrinsic primary afferent neuron; 14. mucosal cell; 15. enterochromaffin cell.PVG, prevertebral ganglia. (Adapted from Furness JB. The enteric nervous system. BlackwellPublishing: Oxford, UK; 2006; p. 30; with permission.)

501CONCEPTS IN THE CELLULAR CONTROL OF GI MOTILITY

Cho

ChoVasomotor Cajal

Cajal

Aferente

Aferente

Interneurnio

EnterocromafinCel mucosa

Intestino -Fugal

Motores


Sistema nervoso entrico

Regulao de absoro e secreo.

Regulao de fluxo sangneo

Regulao de motilidade

Regulao da atividade imune

Regulao de atividade regenerativa


Regenerao e tipos celulares intestinais

Entercitos

Clulas produtoras de muco.

Clulas de Paneth

Enterocromafins

Stem cells

BLUK113-Langnas November 26, 2007 17:58

CHAPTER 2

TA cell

Paneth cellBasementmembrane

Stem cell

Mesenchymalcell

Figure 2.2 Intestinal crypt. Stem cells(red) are found at the crypt bases. Stemcell progeny (yellow) known as transitamplifying (TA) cells migrate up the cryptundergoing further replication anddifferentiation. Basement membrane andmesenchymal cells (green) surround thestem cell niche. (Reprinted from Spradlinget al. [5], with permission from MacmillanPublishers Ltd.)

earlier, division may be symmetrical, resulting in ei-ther two daughter cells or two stem cells. If the formeroccurs, then that stem cell will be lost from the crypt.This ultimately may lead to all cells in a crypt being de-scendants of a single stem cell. Conversely, if divisionresults in two stem cells then the total number of stemcells in the crypt will increase. It has been proposedthat increases in stem cell number may be triggers forcrypt fission, a process vital to intestinal growth andrepair after injury.

Stem cell plasticityStem cell plasticity refers to the ability of organ-specificstem cells from one tissue to produce cells of a differ-ent lineage and tissue. For example, adult bone mar-row cells may be able to engraft into other tissues anddifferentiate into cell types specific to that organ, suchas hepatocytes or skeletal myocytes. There is increasingevidence to support this hypothesis, especially fromob-servations that Y-chromosome containing cells can beseen in the tissues of female recipients of bone marrowfrommale donors, although it has been argued that thismay represent cell fusion rather than stem cell plas-ticity. In the small and large intestine, bone marrow

engraftment and differentiation into mesenchymalcells have been demonstrated in humans and mice [7].The possibility that bone marrow derived cells couldbe used to populate and regenerate intestinal mucosais clearly exciting and of particular relevance to the fieldof tissue engineering.

Tissue engineering of intestinal mucosa

Tissue engineering offers a novel approach to the treat-ment of short bowel syndrome. The basic concept is toseed intestinal tissue onto artificial, biodegradable scaf-folds which will support the growth and developmentof organized intestinal neomucosa. The use of autol-ogous cells to create biocompatible tissue-engineeredintestinal constructs has several benefits, including thestimulation of natural biological mechanisms for repairand remodeling, complete biocompatibility, the poten-tial for further growth, and, of course, the avoidance ofproblems associated with transplantation. Potentially,the engineering of small intestine as a functional ab-sorptive area could render patients with short bowelsyndrome independent of parenteral nutrition.

16


Regulao neural do sistema imune do tgi

VIA ADRENRGICA VIA COLINRGICA

ATIVIDADE PR-INFLAMATRIA

ATIVIDADEANTI-INFLAMATRIA

Estudos em trauma craniano.

jects may, at least in part, result from increasedvagal activity and subsequent suppression of theimmune response (figure).

DISCUSSION The hypothesis postulated above isbased on evidence obtained from studies with dif-ferent research objects. To our knowledge, nostudies are available that test this hypothesis di-rectly. Since the cholinergic anti-inflammatorypathway can be pharmacologically modulated inhumans, such treatment in patients with TBIcould represent a novel, promising approach toprevent infections in this specific group of pa-tients.

Although elevated intracranial pressure ap-pears to be an important link in the parasympa-thetic anti-inflammatory response following TBI,other mechanisms have also been suggested. Re-cently, the role of endogenous mediators in sys-temic inflammation in the absence of infection50

was discussed. Toll-like receptors (TLRs) arewell-known for their role in recognition of exoge-nous pathogens and subsequent triggering of im-mune responses. Excessive or inappropriateactivation of TLRs can lead to systemic inflam-mation and shock, similar to that observed in sep-tic patients. In addition to recognizing outsideinvaders, TLRs, as well as other receptors, alsorecognize endogenous damage signals termedalarmins, such as heat-shock proteins and

HMGB1. Tissue trauma, for instance resultingfrom TBI, elicits release of these damagesignals.51-53 Consequently, trauma can trigger theimmune response and this could explain the sys-temic inflammatory reaction in the absence of in-fection often observed in these patients. Anincrease in vagal activity may counteract thismechanism by attenuating the release of endoge-nous inflammatory mediators. In this context, itis noteworthy that ex vivo treatment of humanmonocytes with nicotine (stimulation of the cho-linergic anti-inflammatory pathway) resulted indownregulation of TLR4 expression in humanmonocytes.54 This observation may also explainthe attenuated production of proinflammatorycytokines in blood of trauma patients that wasstimulated ex vivo with LPS, the primary ligandfor TLR4.15 In this prospect, increased vagal ac-tivity may represent a negative feedback mecha-nism counteracting the massive systemicproinflammatory response. This notion is sup-ported by animal studies in which increased vagalactivity resulted in attenuation of inflammation,prevention of development of shock, and de-creased mortality after experimental inflamma-tion.38,39,44 In this view, the resultant and sustainedimmune paralysis is merely a side-effect of thenegative feedback loop. However, the animalstudies mentioned before have all been performedusing models of sterile inflammation. In animalexperiments using live microbial sepsis, activa-tion of the cholinergic anti-inflammatory path-way resulted in worsened survival, impairedmigration of neutrophils to the inflamed area,and increased outgrowth of bacteria.40,43,55 More-over, human data on heart rate variability in theacute phase after TBI indicates that increasedHF/LF ratios are associated with poor outcomeand higher mortality.31,56,57 These results suggestthat increased vagal activity is beneficial in sterileinflammation induced by LPS or chemical com-pounds that evoke inflammation, but may impairthe bodys ability to combat an infection with livebacteria.

In contrast to aforementioned studies, also adecreased HF power and decreased HF/LF ratioshave been reported in patients with TBI.19,21,58

However, these studies evaluated patients in thepost-acute (chronic) phase of TBI (!30 days).Along these lines, patients in the chronic phase ofsubarachnoid hemorrhage demonstrated similarHF/LF ratios compared to control subjects, whileHF/LF ratios were increased in the acute phase, asdiscussed before.33 In the chronic phase of TBI orsubarachnoid hemorrhage, patients usually do

Figure Traumatic brain injury and vagal activity

Traumatic brain injury increases vagal tone directly or as aresult of elevated intracranial pressure. Subsequently, vagalactivity attenuates inflammation via the cholinergic anti-inflammatory pathway. As a result, susceptibility toward in-fections increases, resulting in worse outcome.

Neurology 70 February 5, 2008 483


CONCLUSES

As funes de Barreira so influenciadas por:

1. Equilbrio microbiolgico.

2.Atividades das clulas dendrticas.

3.Atividade neural

4.Estado de manuteno mecnico do epitlio.

5.Capacidade Regenerativa


O INTESTINO BOMBA

A SEMENTE DA DESTRUIO NA SEPSE E TRAUMA


OBRIGADO

[email protected]


aula translocação bacteriana

Documents