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GASTROENTEROLOGY 2009;136:425–440

EVIEWS IN BASIC AND CLINICALASTROENTEROLOGY

John P. Lynch and David C. Metz, Section Editors

oles of Infection, Inflammation, and the Immune System in Cholesterolallstone Formation

IRK J. MAURER,*,‡ MARTIN C. CAREY,* and JAMES G. FOX‡,§

‡

Division of Gastroenterology, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston; and Division of Comparative Medicinend §Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

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holesterol gallstone formation is a complex processediated by genetic and environmental factors. Until

ecently, the role of the immune system in the patho-enesis of cholesterol gallstones was not considered aalid topic of research interest. This review collates andnterprets an extensive body of basic literature, some ofhich is not customarily considered to be related to

holelithogenesis, describing the multiple facets of themmune system that appear to be involved in choles-erol cholelithogenesis. A thorough understanding ofhe immune interactions with biliary lipids and chole-ystocytes should modify current views of the pathogen-sis of cholesterol gallstones, promote further researchn the pathways involved, and lead to novel diagnosticools, treatments, and preventive measures.

ile is a complex aqueous colloidal system that is essen-tial for a wide range of physiologic functions, including

he excretion of lipids from the organism and intestinal fatbsorption.1,2 Bile is formed primarily in hepatic canaliculi,mall (1–2 �m) spaces formed between the tight junctionsf hepatocytes. It is composed of water, electrolytes, and aariety of lipid solutes dispersed in mixed micelles andesicles including bile salts, phospholipids (�96% beingixed phosphatidylcholines), and cholesterol, as well as

roteins and bilirubin conjugates.3 Phospholipids and bilealts are essential for the removal from the organism oftherwise insoluble cholesterol molecules in an aqueousnvironment by solubilizing the sterol in mixed micelles,omposed of the catabolic product bile salts, and unilamel-ar vesicles.2–4 Bile is transported from the canaliculi alongubules of increasingly greater diameter until it egresses intohe gut at the midduodenum. In health, about half theecreted bile is stored, concentrated, and slightly acidified inhe gallbladder during the interdigestive interval. The gall-ladder is connected to the biliary tree via the cystic duct,hich functions simultaneously as a gallbladder filling and

mptying conduit.3

Alterations in the relative or absolute proportions ofholesterol, phospholipids, and bile salts can lead to phaseeparation of cholesterol from solution in bile. Most fre-uently these changes result from excess secretion of cho-

esterol from the liver.1–3 As the absolute cholesterol con-entration increases, the excess cholesterol phase separates,orming unilamellar vesicles with biliary phospholipidsFigure 1). Under suitable physicochemical conditions,hese can aggregate to form multilamellar vesicles (lamellariquid crystals), and eventually cholesterol monohydraterystals can separate from these and aggregate in the gall-ladder.2,4 These crystals can progress to form cholesterolallstones by agglomeration within a gallbladder-secreteducin gel. Formation of cholesterol gallstones invariably

ccurs adjacent to the gallbladder wall, where cholesterolonohydrate crystals nucleate in the gelled mucin glyco-

rotein scaffolding.2,4–6 The majority of cholesterol gall-tones form in the gallbladder; however, in rare situationsspecially and perhaps uniquely from genetic deficiency ofiliary phospholipid secretion,7 the stones can form any-here along the biliary tree, even intrahepatically.1–3 Al-

hough a relative excess of cholesterol is necessary for for-ation of cholesterol gallstones, it is not sufficient. Biliary

ludge, composed of agglomerated cholesterol crystals of anltrasonically detectable size (�50 �m) suspended in mucinel, is frequently observed and can progress to form choles-erol gallstones.8–10 Lee et al found that in 18% of patientstudied by ultrasonography for a mean of more than 3ears, biliary sludge disappeared completely; in 60% of theatients, the biliary sludge disappeared and reappeared.10

Abbreviations used in this paper: EGF, epidermal growth factor;GFR, epidermal growth factor receptor; FXR, farnesoid X receptor; IL,

nterleukin; LPS, lipopolysaccharide; MyD88, myeloid differentiationrotein 88; TGF, transforming growth factor; TLR, toll-like receptor;NF, tumor necrosis factor; PCR, polymerase chain reaction.

© 2009 by the AGA Institute0016-5085/09/$36.00

doi:10.1053/j.gastro.2008.12.031

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426 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

The study of cholesterol gallstones relies on multipleethodologies, including physical-chemical studies ofodel bile and ex vivo bile systems, biliary lipid secretory

tudies, animal models with particular focus on choles-erol gallstone (Lith) genes, and epidemiologic studiesnalyzing susceptible and resistant human popula-ions.2,11–14 These studies provide the basis for our un-erstanding of the epidemiology and pathogenesis ofholesterol gallstones. Formation of cholesterol gall-tones is principally a polygenic trait with significantontributions from the environment.3,15 Although thesetudies provide a strong basis for our understanding ofhe etiology and pathogenesis of cholesterol gallstones,

any issues remain unresolved. For example, why do soew individuals with phase-separated and aggregated cho-esterol monohydrate crystals (such as the biliary sludgehat is frequently observed in pregnant women) proceedo gallstones?8 –10 Additionally, why do genetically iden-ical mouse models challenged with the same lithogenic

igure 1. The physical-chemical processes involved in formation of cholhospholipid (green), and bile salt (purple) molecules are shown, along withalts combine by hydrophobic interactions to form mixed micelles (micelnilamellar vesicles would be �5–10 times larger than micelles (�40 Å in raholesterol concentration in gallbladder bile increases principally from hepaupersaturated bile usually implies that phase separation of excess chohospholipids (mostly �95% phosphatidylcholine). In the most common

iquid crystals, which are visible by low-power polarizing microscopy. Fromucleate heterogeneously, usually in a mucin gel. The dotted arrow indicateicelles. The solid resulting cholesterol monohydrate crystals are a polymoassage of time. Once the nucleation sequence has occurred and solid choontinuously supersaturated. Gallbladder dysmotility and mucin gel formatrystals and contribute to their agglomeration and growth into macroscop

iet but housed at different research institutions display c

uch marked differences in their proclivity to form cho-esterol gallstones?16

We proposed that the differences in the prevalence ofallstones in genetically identical mice might arise fromifferences in their colonization status with gastrointes-inal microbes. Initially, we analyzed the ability of entero-epatic Helicobacter spp to contribute to lithogenicity and

dentified several enterohepatic Helicobacter spp that con-ributed to cholesterol gallstone nucleation and otherelicobacter strains that did not.16,17 The host immune

ystem might be the primary mechanism whereby theserganisms (and perhaps others) promote cholesterol gall-tone formation as well as mucin gel and gallbladdernflammation. Subsequent studies showed that the re-ponse of the adaptive immune system was important,nd probably essential, because Rag-deficient mice, whichack mature T and B cells, rarely (�10%) develop choles-erol gallstones.18 A number of previous studies showednvariably that gallbladder inflammation occurred con-

l gallstones. The classic physical-chemical symbols for cholesterol (pink),acromolecular structures they form. Cholesterol, phospholipids, and biled cholesterol and phospholipids form unilamellar vesicles. Normally thebut for illustration purposes they are depicted here nonproportionately. Asersecretion of cholesterol, the true supersaturated state forms transiently.

rol from micelles has occurred, forming unilamellar vesicles with biliarytion sequence, unilamellar vesicles fuse to form multilamellar vesicles, or

se, plate-like cholesterol monohydrate crystals (solid cholesterol crystals)cholesterol can occasionally phase separate directly from supersaturatedthe classic cholesterol monohydrate plates into which they transform withol crystals have formed, the phase sequence is not repeated if bile remainsso contribute to the aggregation of the plate-like cholesterol monohydratelesterol gallstones.

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February 2009 CHOLESTEROL GALLSTONE FORMATION 427

tone formation; none, however, conclusively showedhat this process was a primary contributing factor ratherhan a secondary effect of the lithogenic process.19 –23

This review focuses on the immune aspects of the patho-enesis of cholesterol gallstones and examines the role ofnfection, inflammation, and the response of the immuneystem during the formation of cholesterol gallstones.here is an extensive body of immunity literature related

o cholesterol gallstones as well as a number of studiesnvolving mouse models of infection, inflammation, andmmunity. This review describes the critical contribu-ions of the gallbladder epithelium and the immuneystem to modulation of cholesterol gallstone formationnd progression, rather than the nonimmunologic fac-ors (hypersecretion of cholesterol, alterations in cho-esterol to bile salt and phospholipid ratios and concen-rations, and so on) that are pivotal factors in theathogenesis of cholesterol gallstones but have been re-iewed elsewhere.3,24 Most of the immunologic factorsescribed in this review occur in conjunction with theundamental prolithogenic alterations in liver and bile;ithout these alterations, inflammation would not be

ufficient to induce cholesterol supersaturation of bilend gallstone formation. The causes of biliary tree in-ammation, as they relate to cholesterol gallstone dis-ase, are far from being fully elucidated; nonetheless, theiterature indicates that some noninfective inflammationccurs secondary to physical-chemical alterations of bile

cholesterol supersaturation and early stages in the nucle-tion and phase-separation sequences; Figure 1), whereasthers occur independently of biliary lipid composition.

Biliary Epithelium and ImmunityAnatomically, the biliary tree is often divided into

ntrahepatic and extrahepatic portions that include theallbladder epithelium. However, with respect to biliaryissue and disease, this distinction appears artificial whenescribing the interactions of the biliary epithelium withhe immune system; it appears that biliary epithelial cellsrom either location have similar responses to immuno-enic stimuli.25,26

Biliary epithelial cells participate in both innate anddaptive immunity.27–29 Briefly, the innate immune sys-em induces no immunologic memory to an antigen butesponds instead to a variety of evolutionarily conservedatterns present in foreign antigens. In addition to spe-ific cellular subsets that include neutrophils, macro-hages, eosinophils, basophils, mast cells, and naturaliller cells, a variety of proteins also participate in innate

mmunity, including the complement cascade, cytokines,nd proteins of the acute phase response.30 –32 In con-rast, the adaptive immune response induces “memory”o foreign antigens and is mediated by both B and T cells.ollowing activation, the adaptive immune response canlso stimulate the production of a variety of cytokines

nd the recruitment of inflammatory cells.33–36 There is t

verlap in these 2 pathways; perturbation of either canlter the ability of the other to respond appropriately tostimulus.36

Biliary epithelial cells express all known toll-like recep-ors (TLR), which mediate antigen-pattern recognition—aey response of the innate immune system.28,37–39 Addi-ionally, myeloid differentiation protein 88 (MyD88),hich is an important downstream effector of the TLRathway, is also present in these cells.28 Biliary epithelialells that express dominant-negative forms of MyD88ave increased susceptibility to infection with the intra-ellular biliary pathogen Cryptosporidium parvum, indicat-ng that the TLR/MyD88 system functions in biliarypithelial cells.39 Additionally, C parvum infection up-egulates �-defensins in infected wild-type cells, but �-de-ensin production is ablated in cells with reduced levelsf TLR2, TLR4, or nuclear factor �B, indicating an es-ential role for defensins in protection of cholangiocytesnd, by implication, bile itself from infection.38

The relationship between biliary epithelial cells and thedaptive immune response requires a variety of receptor-igand interactions that occur between epithelial cells, Tells, and “professional” antigen-presenting cells such asendritic cells. Human biliary epithelium cell lines con-titutively express HLA class I (major histocompatibilityomplex class I) antigens but only low levels of class IIntigens (Figure 2).25,26 However, after stimulation withroinflammatory cytokines, the cells express a variety ofLA class II antigens, including HLA-DR, HLA-DP, andLA-DQ (Figure 2).25 Essentially, this expression profile

hows that biliary epithelial cells are likely to interactith both CD8� T cells (cytotoxic T cells) and CD4� T

ells (helper T cells). In addition to these primary im-une receptors, biliary epithelial cells also possess a

ariety of costimulatory molecules necessary for func-ional interaction with these immune cells, includingD40, intercellular adhesion molecule 1, lymphocyte

unction-associated antigen 3, and FAS (Figure 2).29,40 – 45

hese protein expression profiles indicate that the biliarypithelium actively participates in adaptive immunityhrough interaction with T cells and local antigen-pre-enting cells.

The second aspect of adaptive immunity involves the-cell response, mediated through immunoglobulins

Igs). All Ig classes, including IgA, IgG, and IgM, areresent in low concentrations in bile.29 However, biliarypithelial cells have only minimal participation in Igecretion. In humans, IgA is synthesized by plasma cellshat line the bile ducts and then is bound to the poly-

eric Ig receptor at the basolateral surface of cholangio-ytes.46 – 48 IgA is then transcytosed to the apical surfacef the cells and secreted into bile. This intracellular trans-ort system has an important role in protecting theiliary tree from intracellular pathogens by binding thesentigens in transit.49 Similar to IgA secretion, IgM is

ransported via the polymeric Ig receptor, but in contrast,

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428 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

gG is secreted using the FcRn.49 –51 Igs that reach intralu-inal bile aid in protecting bile from colonization with

astrointestinal pathogens.52,53

Aside from expressing the necessary receptors andownstream activators for participation in the immuneesponse, biliary epithelial cells also secrete a variety ofytokines and undergo phenotypic changes in responseo these inflammatory mediators. Cultured murine cho-ecystocytes (gallbladder epithelial cells) express Tnf-�,cl5 (RANTES), and Mip-2 (CXCL2) (Figure 2).54 Follow-

ng treatment with lipopolysaccharide (LPS), these cellsncrease expression of Mip-2 and Tnf-�. Additionally, ex-ression of Il-1�, Il-6, and Mcp1 is induced followingxposure to LPS (Figure 2).54 Interestingly IL-10, the Th2nd T-regulatory cytokine, is not expressed in resting orPS-stimulated gallbladder epithelium.54 It has been es-ablished that the biliary epithelium possess receptors forome of these cytokines and chemokines (including tu-

or necrosis factor [TNF]-�), indicating that these se-reted cytokines can act in both an autocrine and aaracrine manner (Figure 2).29,55 The addition of exoge-ous cytokines to cultured biliary epithelial cells induceshenotypic changes in these cells, supporting an auto-rine role for some of these cytokines (Figure 2). Forxample, the addition of TNF-�, LPS, interleukin (IL)-1�,nd prostaglandin E2 to cultured human cholecystocyteslters the transport of sodium and chloride and dimin-shes the absorptive function of the gallbladder epithe-ium.56 Studies in guinea pigs (Cavia porcellus) with ligatedile ducts support these in vitro findings.57 Specifically,he instillation of IL-1 or LPS into the gallbladder in-uces gallbladder wall inflammation and stimulates the

igure 2. Receptors and ligands expressed by biliary epithelial cells. The rxtracellularly) expressed by biliary epithelial cells are shown in the unstimulahat interact with these biliary expressed proteins are also shown. CCL, cheXC ligand; CXCR, chemokine CXC receptor; LFA, lymphocyte function-a

elease of myeloperoxidase, prostaglandin E2, and water b

nto the gallbladder lumen.57 Systemic administration ofytokines during chemotherapeutic treatment is also as-ociated with changes in the biliary epithelium. For ex-mple, IL-2 administration to humans causes acalculousholecystitis.58 – 60 The administration of transformingrowth factor (TGF)-� to cultured rabbit (Oryctolagusuniculi) gallbladder epithelium induces a mesenchymal,brotic phenotype.61 This in vitro phenomenon appearso have an in vivo correlate because TGF-� expression isssociated with fibrosis and inflammation during forma-ion of cholesterol gallstones in humans.62 Finally, hista-

ines released during mast cell degranulation induceallbladder smooth muscle contraction.63

There are several disease states associated with im-une-mediated destruction of the biliary epithelium: pri-ary biliary cirrhosis, primary sclerosing cholangitis, and

raft-versus-host disease.40,42,64 – 67 The biliary epitheliumlearly participates in cellular and humoral immunityhrough antigen presentation, cytokine and chemokineroduction, and Ig transport. Moreover, biliary epithelialells possess all of the necessary cellular componentsequired to participate in the innate immune system;erturbations of innate immunity appear to predisposeo biliary infection.28,37–39

Role of Infection, Inflammation, and theImmune System in Formation ofCholesterol GallstonesIgsIn vitro studies with model bile created with var-

ous concentrations of purified lipids have shown that

ors (displayed on the basolateral surface of the cell) and ligands (displayedght panel) and stimulated (left panel) state. The receptors, ligands, and cellse CC motif ligand; CCR, chemokine CC motif receptor; CXCL, chemokineiated antigen; MHC, major histocompatibility complex.

eceptted (ri

iliary Igs, particularly IgM and IgG, promote the nucle-

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tion of solid cholesterol crystals.68,69 Interestingly, theource of the Igs appears to influence the ability of theseroteins to nucleate solid cholesterol crystals from bile.gM and IgA from commercial sources are incapable ofucleating supersaturated bile, whereas commerciallyvailable IgG has pronucleating activity.68,70 The ana-omic source of the Igs also appears to influence thebility of these molecules to cause cholesterol crystalhase separation. Specifically, biliary Igs seem to promoteolid cholesterol nucleation more than Igs isolated fromlood.70 Of the biliary Igs, IgM most potently nucleatesupersaturated bile, whereas IgG has slightly less facilitynd IgA has very little activity.68 –70

Prospective studies utilizing animal models generallyo not support a role for Igs in the pathogenesis ofholesterol gallstones. AKR or C57L mice fed a lithogeniciet undergo an initial decrease, followed by a temporary

ncrease, in biliary IgM and IgA levels.22 In both strains ofice, the total amount of IgG remained relatively un-

hanged. Van Erpecum et al concluded that Igs are notikely to participate in formation of cholesterol gallstonesecause both gallstone-resistant (AKR) and gallstone-usceptible (C57L) mice displayed alterations in their Igevels when placed on lithogenic diets.22 Actually, gall-tone-resistant AKR mice had higher concentrations ofiliary Igs than the gallstone-susceptible C57L mice.22

lthough these changes appeared to negate any impor-ant role that Igs had in formation of cholesterol gall-tones, data interpretation was complicated by pro-ounced differences in cholesterol saturation index (auantifiable measurement of the relative amount of cho-

esterol in bile) values between C57L mice (�2.0) andKR mice (�1.5).71 Therefore, the variable contributionsf differing cholesterol saturation indexes of biliary lipidsost likely confounded these results. Moreover, results

f human studies indicated that Igs did not contribute toormation of cholesterol gallstones; in ex vivo bile sam-les from individuals with or without gallstones, thereas no correlation between biliary Igs and cholesterol

rystal detection times.72

One limitation of these studies is that the investigatorsocused primarily on the effect of Ig concentrations onolid cholesterol monohydrate crystal nucleation. Al-hough Ig concentrations could be important, it is likelyhat other factors are relevant to this physical-chemicalnteraction. For example, the antigen specificity of the Igs

ay be crucial. Indeed, Fab fragments isolated from pa-ients with cholesterol gallstones accelerated cholesterolrystal observation times in vitro, whereas commerciallyvailable Fab fragments did not.73 These data indicatehat antigen-specific Fab fragments might contribute toholesterol nucleation in vitro.73 Therefore, merely quan-ifying Ig concentrations in bile appears to provide lim-ted functional information.

Recent studies have shown that mice that have only T

ells (that lack B cells and Ig) develop gallstones at a b

reater frequency than wild-type mice. In contrast, miceossessing only B cells do not recapitulate the prevalencef gallstones observed in wild-type mice.18

Mucin GlycoproteinsBiliary mucin glycoproteins are the most thor-

ughly analyzed biliary proteins involved in the patho-enesis of cholesterol gallstones. Mucin genes that arexpressed in human biliary epithelia include MUC1,

UC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6.74

ucin gel accumulation appears to be important forormation of cholesterol gallstones in humans and innimal models.5,75–79 Moreover, these proteins promoteolid cholesterol-phase nucleation in vitro.76,80 – 83 In vitrond in vivo data suggest that mucin gel accumulationrecedes and promotes formation of cholesterol gall-tones by nucleating cholesterol crystals from (liquidrystal) phase-separated bile.

In cultured cholangiocytes, many mucin genes are reg-lated by inflammatory mediators. Studies in 2 differentell culture systems have shown that mucin gel accumu-ation and mucin gene expression increased followingxogenous addition of LPS and TNF-�. In culturedholangiocytes, expression of Muc2 and Muc5ac increased

igure 3. Immune system–stimulated production of MUC2. Variousathogen-associated molecular patterns (PAMPs), through their inter-ction with TLR molecules, increase the production of mucin 2 (MUC2).portion of this increase seems to be due to increased production of

audal type homeobox transcription factor 2 (CDX2), which feeds backo stimulate MUC2 production. Further, LPS and TNF-� stimulate theroduction of MUC2. In the case of LPS, this stimulation seems to beediated through the TNF-� pathway because inhibition of TNF-�

locks the effects of LPS treatment on MUC2 production.

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430 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

ollowing exposure to LPS or TNF-� (Figures 3 and 4).84

tilizing the same cell culture system, the investigatorshowed that TNF-� stimulated protein kinase C, whichppeared to signal increased production of Muc2 anduc5ac (Figures 3 and 4).84 In the case of Muc3, its

roduction was increased by LPS, but not TNF-�, possi-ly due to an interaction of these molecules with CD14Figure 5).84 In another study, LPS and other pathogen-ssociated molecular patterns were found to interact withLR2 or TLR4 on the surface of biliary epithelium.85 This

nteraction induced expression of Cdx2, a gene whoseroduct is associated with intestinal metaplasia of epi-helial cells; CDX2 in turn induced expression of Muc2Figure 3).85 Furthermore, MUC2 protein expression isncreased in the gallbladders of humans with cholesterol

igure 4. The points at which the immune system stimulates produc-ion of MUC5AC. As described for mucin 2, LPS stimulation leads toroduction of mucin 5AC (MUC5AC) in a TNF- � dependent manner

not shown). TNF-� acts via protein kinase C (PKC)-dependent andGF-mediated pathways to up-regulate MUC5AC production. In thease of the EGF pathway, this up-regulation results from the productionf apical EGF receptors and subsequent binding to apical EGF.

allstones.86 p

Choi et al87 obtained similar results utilizing culturedanine (Canis familiaris) gallbladder epithelial cells. Theddition of LPS from any of 3 bacterial species stimu-ated mucin production in cultured dog cholecystocytes.here appeared to be differences in the ability of LPS

rom different bacterial species to induce mucin secre-ion. For example, LPS from Escherichia coli produced thereatest stimulation, whereas LPS from Pseudomonaseruginosa stimulated the least mucin production. Fur-hermore, the addition of TNF-� to the culture mediaromoted mucin production, albeit to a lesser degreehan LPS. The diminished response to TNF-� could haveeen due to the use of human rather than canine TNF-�.he investigators noted that the changes in mucin accu-ulation were not due to cytotoxicity of either LPS orNF-�, because cell viability assays demonstrated sur-ival of the cultured cells.87

Recent studies have shown that TNF-� regulatesUC5AC expression via an epidermal growth factor

EGF)-mediated pathway (Figure 4).88 Stimulation of

igure 5. Stimulation of mucin 3 (MUC3) production by LPS via CD14nd likely TLR molecules. Stimulation of MUC3 by LPS is independentf TNF-�, in contrast to MUC2 and MUC5AC. It is currently unclearhat downstream pathways are important in LPS stimulation of MUC3

roduction.

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allbladder epithelium with TNF-� induced productionf the EGF receptor (EGFR). EGF then binds to theGFR and this interaction induces expression of Muc5ac,resumably through a second messenger protein88; inhi-ition of the EGF-EGFR pathway prevented productionf MUC5AC.88

Findings from these in vitro and in vivo studies indicatehat production and secretion of the mucin glycoproteinsre influenced by inflammatory mediators. TNF-�, patho-en-associated molecular patterns, and their second mes-engers are likely to be only “the tip of the iceberg” withegard to inflammatory mediators that stimulate mucinroduction by the gallbladder. Perhaps a more importantuestion to ask is not whether inflammatory mediatorslter mucin production, but rather under what patho-hysiological situations do inflammatory mediators exertheir effects on the biliary epithelium?

Other Putative Pronucleating BiliaryProteinsMany other proteins nucleate cholesterol mono-

ydrate crystals in vitro.72,89,90 Some of these putativeronucleating agents include haptoglobin, phospho-

ipase C, �1-acid glycoprotein, albumin, and aminopepti-ase N.72,89,90 In general, in vivo studies that analyzed theole of putative pronucleating proteins have been ham-ered by a number of factors: (1) large-scale proteomicnalysis has only recently been undertaken to systemati-ally analyze the protein complexity of gallbladder bile,91

2) prior methods used to identify quantitative differ-nces in these biliary proteins were relatively insensitivend might be inadequate to demonstrate significant dif-erences in concentration, and (3) concentrations of in-ividual proteins might not be important, but rather aumulative or cooperative effect of multiple proteinsould be required to promote phase separation of crystalsr liquid crystals in bile and lead to formation of choles-erol gallstones. Moreover, there is little understanding ofhe physical-chemical interactions between these pro-eins. Large-scale, sensitive proteomic screens are becom-ng easier to perform and more widely used in patho-hysiology and translational research; utilization of theseechniques will likely increase our understanding of whatole, if any, the myriad of secreted biliary proteins maylay in formation of cholesterol gallstones.

Gallbladder InflammationIn both animal models and humans, formation of

holesterol gallstones is preceded by histopathologic al-erations in the gallbladder wall that indicate inflamma-ion. These changes include edema, increased gallbladderall thickness, the presence of inflammatory cells, and

emodeling of the epithelium with concomitant increasesn TGF-� production.19 –21,62,92,93 These alterations areccompanied often by changes in gallbladder contractil-

ty and modifications in the ability of the gallbladder c

pithelium to transport a variety of substances.2,21,23,94,95

urthermore, lithogenic bile can alter mechanisms asso-iated with cytoprotection in the gallbladder muscle,emonstrated by the ability of the gallbladder muscle toespond to reactive oxygen species.96

A systematic histopathologic study23 showed that57L mice, which are susceptible to gallstones, increaseallbladder wall thickness progressively when fed a litho-enic diet. In contrast, AKR mice, which are resistant toallstones, display only a mild increase in thickness. Bothtrains accumulate subepithelial inflammatory cells anddema; however, C57L mice do so to a significantlyreater degree. These changes coincide with decreases inhe gallbladder expression of the aquaporin genes Aqp1nd Aqp8.23 These findings suggest that during forma-ion of cholesterol gallstones, the gallbladder undergoesrogressive changes that ultimately result in decreasedotility, increased edema, and altered transport func-

ions. The farnesoid X receptor (FXR) is a critical tran-cription factor that alters biliary lipid composition.92 Inxr�/� mice, the gallbladder becomes thickened, edema-ous, and infiltrated with granulocytes following 1 weekf lithogenic diet feeding; these changes were not observed

n wild-type mice.92 Additionally, levels of potentially cyto-oxic hydrophobic bile salts, such as deoxycholate conju-ates, were significantly increased in Fxr�/� mice.92

It is likely that phase-separated cholesterol crystals andydrophobic bile salts are responsible, in part, for theistopathologic changes noted in the murine gallblad-er during formation of cholesterol gallstones. Ex vivonalysis of gallbladders from patients with cholesterolallstones revealed smooth muscle cells that containedxcess cholesterol in their plasma membranes.96 Further-ore, the isolated muscle tissues displayed less contrac-

ility and decreased levels of scavengers of reactive oxygenpecies.96 These changes appeared to be associated withysfunction of the receptor for prostaglandin E2; prosta-landin E2 binding to its receptor usually has a cytopro-ective effect. Others have reported correlations betweenuman gallbladder muscle dysfunction, increases in theholesterol saturation index of bile, and the histopatho-ogic inflammation score of the gallbladder.97 Addition-lly, oxysterols are capable of inducing apoptosis in cul-ured gallbladder epithelial cells.98,99 The mechanismhat mediates apoptosis appears to involve incorporationf oxysterols into the mitochondria of epithelial cells andubsequent cytochrome c activation.99

Based on these studies, it is not possible to determinehether the functional and histopathologic changes thatccur in the gallbladder are due to cytotoxicity or inflam-ation; however, studies in the prairie dog model of

holelithogenesis indicated that inflammation is an es-ential element of cholesterol gallstone formation.100

pecifically, prairie dogs fed high doses of acetylsalicyliccid (aspirin) failed to form gallstones, whereas 100% of

ontrols developed gallstones.100 Because both groups

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upersaturated their gallbladder biles, this study showshat cholesterol supersaturation is necessary but not suf-cient for formation of cholesterol gallstones. Therefore,he proinflammatory effects of solid cholesterol itself,nd likely other proinflammatory mediators, have anntegral role in cholelithogenesis. However, subsequenttudies failed to fully recapitulate these findings; it isifficult to make direct comparisons between these typesf studies because they all used different doses of acetyl-alicylic acid, dosing regimens, parameters of measureduccess, and lithogenic diets.101–105

Further evidence for the role of the immune system inhe pathogenesis of cholesterol gallstones arose fromtudies with Rag-deficient mice, which do not have ma-ure T or B cells (Rag mice). Both wild-type and Rag-eficient mice exhibited supersaturated gallbladder biles,videnced by the phase separation of liquid crystals; how-ver, only the wild-type mice had a high prevalence ofholesterol gallstones.18 Gallstone formation in this caseppeared to be due in part to a potent Th1 immuneesponse, which accompanied the formation of solid cho-esterol monohydrate crystals in the gallbladder. Theseata indicate that most likely solid cholesterol crystalsromote inflammation via the adaptive immune system,pecifically via T-cell function.18

Hydrophobic bile salts promote inflammation and ap-ptosis in vitro and are likely to do so in vivo.106 –108 Inontrast, the hydrophilic bile acid ursodeoxycholic acidecreases mucosal inflammation and biliary protein con-entrations.109 In C57L mice infected with Helicobacterpp, the relative concentration of hydrophobic bile saltssuch as deoxycholate conjugates) increased in hepaticile whereas that of hydrophilic bile salts (such as �- and-muricholate conjugates) decreased compared with un-

nfected mice.110 It appears that one downstream effect ofydrophobic bile salts is the induction of proinflamma-ory cytokines.108 An increase in hydrophobic bile salts,rincipally deoxycholate conjugates, is a common finding

n patients with cholesterol gallstone disease and in an-mal models; it is likely that bile salt–mediated inflam-

ation is also important in cholelithogenesis.111–114

Bacterial Colonization of the Biliary Tractand Formation of Cholesterol GallstonesBacteria have historically been implicated in the

athogenesis of cholesterol gallstones; however, a defin-tive cause-and-effect relationship has never been estab-ished.115–117 A major factor contributing to this confu-ion is the chronic nature of cholecystolithiasis. Theormation of cholesterol gallstones requires a long time,ften decades. When gallstones do form, they do notisappear spontaneously except by migration into theuodenum, which is invariably followed by reforma-ion.118 –120 Furthermore, after cholesterol gallstonesorm in a patient, they do not always lead to symptoms;

hen they do, clinical expression can also take years.2,4,121 t

n prospective mouse models, organisms need not colo-ize the gallbladder to promote formation of cholesterolallstones.16 Therefore, the identification of bacteria inile or gallbladder tissue of patients with cholesterolallstones does not mean that these organisms causedhe gallstones. More likely, the bile, gallbladder mucosa,nd biliary motility might have been altered to allow forubsequent bacterial colonization. Furthermore, bacteriahat might have been present at the time of initial stoneormation could have been cleared and therefore notetected by conventional culture techniques. Another

ssue is that some bacteria alter the composition of bileirectly via �-glucuronidase, cholyl-glycyl hydrolase, phos-holipase A1, or urease activities, thereby promoting cal-ium bilirubinate (brown pigment stone) formation.122

Despite these deficiencies, numerous studies have iden-ified bacteria in bile, cholesterol stones, and gallbladderissue from patients with cholesterol gallstones.123–127

ome propose that bacteria, through their �-glucuroni-ase activity, are responsible for nucleation and calciumalt deposition.128,129 Although this hypothesis is biolog-cally plausible, no studies have shown conclusively thatt occurs. In fact, in vitro studies utilizing E coli oralcium salts have failed to induce cholesterol crystalucleation from supersaturated bile.130

Recently, there has been a great deal of interest in aossible connection between the gastric pathogen Helico-acter pylori and cholesterol gallstones.17,131–134 Interest-ngly, growth of this organism is inhibited by bile saltsoth in vivo and in vitro, and chemotactic assays showhat bile salts actually repel the organism,17,135,136 find-ngs that contradict the ability of H pylori to colonize aealthy gallbladder. Nonetheless, a number of groupslaimed to have identified H pylori DNA in biliary tissuend gallstones.131,132,134,137 However, there are severalroblems with these studies. First, some samples wereollected at endoscopic retrograde cholangiopancreatog-aphy134; because H pylori colonizes the gastric mucosa of

ore than half the world’s population, the statisticalhances for sample contamination by gastric H pylori areigh. Additionally, 16S ribosomal RNA genus-specificrimers are commonly used to identify these organisms

n polymerase chain reaction (PCR) analysis, despite theact that these primer sets can amplify other non–H pylorielicobacters.16,17 Furthermore, sequence evaluation ofhese primer products is generally inadequate to speciate

pylori properly, because of high homology with non–pylori Helicobacter spp.138 For example, Avenaud et al139

dentified H pylori–like organisms based on sequencing ofhe 16S ribosomal RNA gene from human liver samples;owever, rigorous follow-up tests proved that these or-anisms were an unclassified Helicobacter spp.139 The de-elopment of species-specific primers to other regions ofhe genome may alleviate some of these concerns. None-heless, the efficacy of species-specific primers depends on

heir precise level of specificity. Finally, these organisms

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ould invade the biliary tree following alterations in theiliary microenvironment and especially biliary dysmotil-

ty. Supporting this hypothesis is a recent study showinghat H pylori DNA was present in gallbladder bile ofatients with chronic cholecystitis, consistent with obstruc-ion, but not in asymptomatic patients with cholesterolallstones.131

In addition to the gastric pathogen H pylori, a variety ofnterohepatic helicobacters (eg, Helicobacter bilis and Hel-cobacter hepaticus) exist.140,141 These reside in the smallnd large intestines and canalicular spaces of the liver ofxperimentally and naturally infected animals, includingumans.140,141 DNA from these organisms was detected

n nonwhite patients with chronic cholecystitis, gall-tones, and malignant biliary tract diseases.142,143 Studiesith experimental animal models indicate that these en-

erohepatic helicobacters promote formation of choles-erol gallstones in vivo.16 In contrast, in the same exper-mental model, H pylori did not promote formation ofholesterol gallstones.17 Interestingly, a follow-up studyhowed that urease-positive Helicobacter spp can precipi-ate calcium salts in vitro.144 The investigators of thistudy proposed that this activity might contribute, ateast in part, to the ability of these organisms to promoteholesterol gallstones by inducing the formation of aalcium salt nidus, because monoinfection with urease-ositive helicobacters or coinfection with at least onerease-positive helicobacter is necessary to induce choles-erol gallstones.16,144

Because PCR was used to identify Helicobacter spp andther bacteria in many of these studies, data interpreta-ion can be challenging. PCR can detect relatively smalluantities of bacterial DNA, a marker for colonizationith organisms that might not be identified with stan-ard culture methods. Unfortunately, the presence ofacterial DNA at the time of examination does not meanhat it contributed to formation of gallstones. Theoretically,nalyzing the core of stones might provide more reliableata on the inciting cause; however, due to the sensitiveature of PCR, the mere act of trying to isolate the corean contaminate it with nucleic acid from the externaltone surface or from other sources. Additionally, notudies have analyzed the ability of cholesterol gallstoneso absorb DNA.145 If stones are capable of imbibingNA, then regardless of the analysis (be it core or exter-al), the DNA detected might represent only the mostecent DNA encountered. Furthermore, when multipleNA fragments are present and nonspecific primers aresed (designed to amplify 16S ribosomal RNA genes

rom multiple bacteria), there is the possibility that theNA present at the highest concentration was preferen-

ially amplified.146 Because PCR amplification is nonlin-ar, even a small bias in initial amplification can lead toogarithmic differences in the final DNA concentration.nfortunately, the most prevalent bacteria are not nec-

ssarily the most important bacteria or might not be t

mportant at all. The use of more discriminatory PCRechniques such as those utilized to analyze complexnvironmental samples could circumvent this problem inhe future.147,148

One of the classic bacterial pathogens of the biliaryree in humans is Salmonella enterica ser Typhi, whichauses typhoid fever.149,150 This organism crosses the intes-inal epithelial barrier, invades macrophages, and spreadsystemically.151 After colonizing the liver, the organisman be shed into the gallbladder and chronically colonizehe gallbladder wall (3%–5% of those infected).149,150 Al-hough chronic colonization with S typhi is associatedith gallstones and gallbladder cancer,149,150 it is not

lear whether this organism contributes to stone forma-ion or alternatively if the presence of gallstones pro-

otes chronic colonization. Recent evidence indicateshat S typhi forms bacterial biofilms on the surface ofholesterol gallstones.151 This biofilm formation wouldllow for chronic colonization and protect the organismuring antibiotic treatment. Biofilm formation is depen-ent on the presence of bile because organisms culturedithout bile do not readily form biofilms.151 This indi-

ates that this organism is exquisitely adapted to survivaln a gallbladder that contains cholesterol gallstones. Fur-hermore, biofilm formation is reduced when pebbles orlass beads are utilized instead of cholesterol gallstones,ndicating that both bile and cholesterol gallstones aressential for biofilm formation. Moreover, colonizationn the presence of cholesterol gallstones requires expres-ion of several bacterial genetic components.151,152 Theseata argue that cholesterol gallstones promote chronicolonization with S typhi rather than S typhi–promotingallstones. Regardless of whether gallstones promote col-nization or vice versa, the concomitant presence of gall-tones and S typhi markedly promotes the risk of gall-ladder cancer, making the interaction between S typhind cholesterol gallstones an important topic of clinicalnterest.149,151

It is not clear whether there is a causative role foracteria in formation of cholesterol gallstones in hu-ans. It is likely that some bacteria identified in humansith cholesterol gallstones are at least partially responsi-le for those gallstones due to their ability to promote

nflammation and precipitate calcium salts. Unfortu-ately, most of these studies are primarily descriptive and

ail to thoroughly analyze the organisms identified. Forxample, it is important to determine whether any of therganisms possess virulence factors that would allow forheir invasion or colonization of a nondiseased biliaryree. Additional information could be garnered by cultur-ng these organisms and utilizing them to prospectivelynfect mouse models. For example, when the C57L/J

ouse model is acquired directly from The Jackson Lab-ratory (Bar Harbor, ME) and the mice are housed inicroisolator cages under specific pathogen-free condi-

ions and free of enzootic Helicobacter spp, they rarely

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434 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

�10%) develop cholesterol gallstones. However, pur-oseful infection with some enterohepatic strains of Hel-

cobacter spp significantly increases the prevalence of gall-tones to a high of 80%.16,17 It will prove valuable to testther putative lithogenic organisms utilizing this modelystem approach, bearing in mind the caveat that for thessay to be effective the organisms must be capable ofolonizing the mouse strain being utilized.

Chronic Inflammatory Conditions andGallstonesChronic hepatitis C virus (HCV) infection is asso-

iated with cholesterol and pigment gallstone forma-ion.153–156 Chronic hepatic damage might be responsibleor this effect, leading to deranged liver function andirrhosis.155,156 However, recent studies have shown thatCV infection, independent of liver cirrhosis, is associatedith increased prevalence of cholesterol gallstones.153,154

CV can replicate in the gallbladder epithelium,157,158

nd damage of the biliary tree was noted in 22%–91% ofiopsy specimens from patients with HCV.154 It is there-ore possible that the association between noncirrhoticatients with HCV and gallstones depends on HCV rep-

ication in biliary epithelia, leading to local inflamma-ion, alterations in gallbladder contraction, and inflam-

ation-mediated alterations in mucin gene production.nterestingly, one such study reported a correlationetween HCV and peaks in the prevalence of gallstones attime points. The first peak occurred in patients between

he ages of 31 and 40 years (7% gallstone prevalence) andhe second peak occurred much later, at 61–70 years ofge (16% gallstone prevalence).154 Perhaps the first peakepresents the direct effect of HCV infection itself,hereas the second peak represents the effect of chronic

iver dysfunction and hepatic cirrhosis. Future studiessing biochemical and molecular techniques that focusn changes in the gallbladder itself could provide greater

nsight into the mechanistic relationships between HCVnfection and formation of cholesterol gallstones.

Patients with Crohn’s disease (CD) are at increased riskor the development of gallstones.159 –162 Results varyegarding which factors correlate with pigment or cho-esterol gallstone formation. Factors mentioned includege, female sex, site of disease, surgical resection, anduration of disease (independent of age).160,161,163 Some

nvestigators noted that patients with CD with ileal dis-ase or colonic resection supersaturated their bile withholesterol, probably because of alterations in cholesterolnd bile acid absorption from the small intestine.164,165

owever, several studies have described a decrease in theholesterol saturation in bile of patients with CD, espe-ially when the ileum is involved.166,167 Furthermore, bilecid composition is altered in patients with CD, andhese alterations are often characterized by a decrease ineoxycholate and an increase in ursodeoxycholate conju-

ates.164,166,167 These changes seem paradoxical, because t

rsodeoxycholate enrichment would seem to favor pre-ention of cholesterol gallstones.118,168,169 Unlike patientsith CD, patients with ulcerative colitis are not at in-

reased risk for cholesterol gallstones.159,161 Gallstoneormation might vary between patients with ulcerativeolitis and patients with CD because of differences innflammatory changes.170 –172 Although not true in allases, CD is most commonly associated with a Th1 proin-ammatory response whereas ulcerative colitis is moreften associated with a Th2 response.170 –172 No studiesave described the gallbladder inflammatory profile ofatients with either ulcerative colitis or CD; this wouldrovide insight into the relationship of CD, inflamma-ion, and gallstones.

Inflammation, Cholesterol Metabolism, andGallstonesInflammation, and more specifically the acute phase

esponse, produces marked alterations in the metabolism ofvariety of proteins and lipids; these changes are extensive

nd beyond the scope of this review (see Heller et al172 forleading account). However, metabolic changes of the

iver that alter cholesterol and bile acid metabolism canirectly alter the composition of bile. Most studies ofhese phenomena were conducted using exogenous, acutedministration of cytokines or LPS,173–177 which causesapid (minutes to hours), relatively short-lived (24 hoursr less) alterations in hepatic gene transcription andranslation. Although these changes are significant, it isot clear if they are maintained for prolonged periods

days to weeks).176 In the case of cholesterol gallstoneormation this could prove to be very important, becausehort-lived (hours to days) alterations in bile composi-ion are less likely to promote disease than prolongednd sustained alterations.

With this proviso in mind, it is clear from these studieshat acute inflammation does alter the hepatic metabo-ism of both cholesterol and bile salts. In rodents, admin-stration of LPS or proinflammatory cytokines (IL-1,NF) raises serum cholesterol levels and increases theroduction of HMG-CoA reductase at both the transcrip-ion and translational levels.173,175,178 This change aug-

ents de novo cholesterol synthesis. However, the in-rease in cholesterol production is rather modest becausether enzymes involved in cholesterol biosynthesis, in-luding squalene synthase and enzymes downstream ofMG-CoA reductase in the mevalonate pathway, areown-regulated.173,177–179 Some investigators proposehat these changes maintain adequate cholesterol synthe-is while redirecting mevalonate metabolites into non-terol pathways.176 This hypothesis is supported by thebserved increases in dolichol phosphate and glycosylatedlasma proteins during the acute phase response.180,181 Inontrast, primates decrease their serum cholesterol levelsn response to a variety of proinflammatory media-

ors.182–184 Unfortunately, the mechanism underlying

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his change is less well understood but might arise fromlterations in cholesterol secretion or ApoB synthe-is.185,186

Profound alterations in bile salt production also occurnder these conditions. Specifically, LPS down-regulateshe classic and alternative pathways of bile salt synthesisy decreasing production of CYP7A1 (classic) and CYP7B1r CYP27A1 (alternative) proteins.187,188 Additionally, ex-genous administration of LPS decreases hepatocellularptake and secretion of bile salts by down-regulatingasolateral and apical bile salt transporters.189 –193 Noturprisingly, because bile salts stimulate the biliary secre-ion of both phospholipids and cholesterol, administra-ion of LPS also decreases expression of phospholipidMDR2) and cholesterol transporters (ABCG5/ABCG8)n the canalicular (apical) membranes of the hepato-ytes.190,193,194

These observations indicate that acute infections wouldecrease cholesterol secretion into bile due to intrahe-atic cholestasis. Because formation of cholesterol gall-tones requires only a relative excess of cholesterol, thesehanges may or may not be lithogenic. Additionally, thetudies do not necessarily reflect the events followinghronic immune and inflammatory stimulation. In fact,uring chronic (10 weeks) infection with enterohepaticelicobacter spp, hepatic bile salt secretion rate and bileow increase.110 Interestingly, infection also alters theomposition of the bile salt pool, rendering it moreydrophobic consistent with a proinflammatory re-ponse. These findings highlight the differences betweencute studies involving exogenous administration of cy-okines and LPS and those studies involving chronicnfection. Furthermore, these data highlight the need fortudies with pathogens that cause either or both chronicnd acute infections, which could provide entirely differ-nt results than studies utilizing exogenous cytokine andPS administration.

Lith (LITH) GenesLarge genetic screens have identified a variety of

ouse Lith loci; products of genes at this loci promoteholesterol cholelithogenesis.3,93 Most of these allelespan large chromosomal regions, and the genes respon-ible for gallstone proclivity have not been identified (seeyons et al93 for a detailed review of Lith alleles and theirotential to encode inflammatory mediators). Unfortu-ately, the Lith loci identified to date occur on virtuallyvery mouse chromosome and span relatively large re-ions encompassing numerous genes14; the proinflam-atory genes are likely to be located in many, if not most,

f these loci. Therefore, a more plausible and productivepproach to study the role of inflammation in gallstoneathogenesis would be to utilize currently availableransgenic and knockout mouse models, with alter-tions in genes important in inflammation and immune

esponses, and compare these with their wild-type coun-

erparts. With the creation of these new models, theenetic loci involved in gallstone pathogenesis will be-ome more refined and can be used to study the influencef potential roles of inflammation and immunity onormation of cholesterol gallstones.

ConclusionsThe gallbladder, despite its simple structure and

unction, is a complex organ. Like most organs, when itecomes inflamed and damaged, it loses its ability toerform concentrative, pH modification, absorptive, andontractive functions. Unlike most other organs in theody, the gallbladder can be exposed to large concentra-ions of free (unesterified) cholesterol and potentiallyytotoxic detergent-like bile salts. These molecules, asell as other proinflammatory molecules, can induceotent inflammatory responses under certain circum-tances.23,106 –108 Such inflammatory damage, through aariety of undetermined mediators, then induces gall-ladder hypomotility and promotes the production ofronucleating agents.23,84,87 These effects appear to beue, at least in part, to T cells and a proinflammatoryh1 immune response.18 Furthermore, inflammation, at

east in short-term studies, modifies hepatic lipid metab-lism and secretion, which could promote cholelithogen-sis by altering biliary cholesterol secretion and bile con-entration.173,175,178 These findings raise the possibilityhat inflammatory mediators or their subsets are sero-ogic biomarkers of cholesterol gallstone formation. Ifdentified, these biomarkers could prove valuable for di-gnosis and interventional therapy. Specifically, abate-ent of prolonged immune stimulation might prove

seful in the prevention or treatment of cholesterol gall-tones, either alone or in conjunction with other lipid-ltering therapies.

It is important to note that many of the studies de-cribed in this review relied principally on observationsing genetically homozygous mice (�99% identifymong genomes). In contrast, even the most consanguin-ous of human populations are relatively heterogeneous.he role of inflammation in human cholesterol gallstoneisease is therefore likely to be subtle and involve a wideariety of overlapping factors. Both environmental (expo-ure to pathogens and proinflammatory conditions) andenetic immune factors (polymorphisms in genes thatromote the response to immune stimuli) are likely toontribute to pathogenesis. Clearly, much remains to beearned in this regard, and further in vitro, animal model,nd human studies are likely to clarify many of thesexciting possibilities.

References

1. Carey MC, Lamont JT. Cholesterol gallstone formation. 1. Phys-ical-chemistry of bile and biliary lipid secretion. Prog Liver Dis1992;10:139–163.

2. Carey MC. Pathogenesis of gallstones. Am J Surg 1993;165:

410–419.

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436 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

3. Paigen B, Carey MC. Gallstones. In: King RA, Rotter JI, MotulskyAG, eds. Genetic basis of common diseases. 2nd ed. New York,NY: Oxford University Press, 2002:298–335.

4. Carey MC. Pathogenesis of gallstones. Recenti Prog Med 1992;83:379–391.

5. Lee SP, LaMont JT, Carey MC. Role of gallbladder mucus hyper-secretion in the evolution of cholesterol gallstones. J Clin Invest1981;67:1712–1723.

6. Wang DQ, Paigen B, Carey MC. Phenotypic characterization ofLith genes that determine susceptibility to cholesterol choleli-thiasis in inbred mice: physical-chemistry of gallbladder bile. JLipid Res 1997;38:1395–1411.

7. Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect inadults with symptomatic intrahepatic and gallbladder choles-terol cholelithiasis. Gastroenterology 2001;120:1459–1467.

8. Carey MC, Cahalane MJ. Whither biliary sludge? Gastroenterol-ogy 1988;95:508–523.

9. Lee SP, Hayashi A, Kim YS. Biliary sludge: curiosity or culprit?Hepatology 1994;20:523–525.

10. Lee SP, Maher K, Nicholls JF. Origin and fate of biliary sludge.Gastroenterology 1988;94:170–176.

11. Lammert F, Matern S. The genetic background of cholesterolgallstone formation: an inventory of human lithogenic genes.Curr Drug Targets Immune Endocr Metabol Disord 2005;5:163–170.

12. Portincasa P, Moschetta A, Palasciano G. Cholesterol gallstonedisease. Lancet 2006;368:230–239.

13. van Erpecum KJ. Biliary lipids, water and cholesterol gallstones.Biol Cell 2005;97:815–822.

14. Wang DQ, Afdhal NH. Genetic analysis of cholesterol gallstoneformation: searching for Lith (gallstone) genes. Curr Gastroen-terol Rep 2004;6:140–150.

15. Katsika D, Grjibovski A, Einarsson C, et al. Genetic and environ-mental influences on symptomatic gallstone disease: a Swed-ish study of 43,141 twin pairs. Hepatology 2005;41:1138–1143.

16. Maurer KJ, Ihrig MM, Rogers AB, et al. Identification of chole-lithogenic enterohepatic helicobacter species and their role inmurine cholesterol gallstone formation. Gastroenterology 2005;128:1023–1033.

17. Maurer KJ, Rogers AB, Ge Z, et al. Helicobacter pylori andcholesterol gallstone formation in C57L/J mice: a prospectivestudy. Am J Physiol Gastrointest Liver Physiol 2006;290:G175–G182.

18. Maurer KJ, Rao VP, Ge Z, et al. T-cell function is critical formurine cholesterol gallstone formation. Gastroenterology 2007;133:1304–1315.

19. Rege RV, Prystowsky JB. Inflammation and a thickened mucuslayer in mice with cholesterol gallstones. J Surg Res 1998;74:81–85.

20. Baig SJ, Biswas S, Das S, et al. Histopathological changes ingallbladder mucosa in cholelithiasis: correlation with chemicalcomposition of gallstones. Trop Gastroenterol 2002;23:25–27.

21. Hofmann AF. Pathogenesis of cholesterol gallstones. J ClinGastroenterol 1988;10(Suppl 2):S1–S11.

22. van Erpecum KJ, Wang DQ, Lammert F, et al. Phenotypiccharacterization of Lith genes that determine susceptibility tocholesterol cholelithiasis in inbred mice: soluble pronucleat-ing proteins in gallbladder and hepatic biles. J Hepatol 2001;35:444–451.

23. van Erpecum KJ, Wang DQ, Moschetta A, et al. Gallbladderhistopathology during murine gallstone formation: relation tomotility and concentrating function. J Lipid Res 2006;47:32–41.

24. Strasberg SM, Harvey PR. Biliary cholesterol transport and pre-cipitation: introduction and overview of conference. Hepatology

1990;12:1S–5S.

25. Auth MK, Keitzer RA, Scholz M, et al. Establishment and immu-nological characterization of cultured human gallbladder epithe-lial cells. Hepatology 1993;18:546–555.

26. Rumin S, Loreal O, Drenou B, et al. Patterns of intermediatefilaments, VLA integrins and HLA antigens in a new human biliaryepithelial cell line sensitive to interferon-gamma. J Hepatol 1997;26:1287–1299.

27. Harada K, Ohba K, Ozaki S, et al. Peptide antibiotic humanbeta-defensin-1 and -2 contribute to antimicrobial defense ofthe intrahepatic biliary tree. Hepatology 2004;40:925–932.

28. Harada K, Ohira S, Isse K, et al. Lipopolysaccharide activatesnuclear factor-kappaB through toll-like receptors and relatedmolecules in cultured biliary epithelial cells. Lab Invest 2003;83:1657–1667.

29. Reynoso-Paz S, Coppel RL, Mackay IR, et al. The immunobiologyof bile and biliary epithelium. Hepatology 1999;30:351–357.

30. Akira S, Uematsu S, Takeuchi O. Pathogen recognition andinnate immunity. Cell 2006;124:783–801.

31. Fritz JH, Girardin SE. How Toll-like receptors and Nod-like recep-tors contribute to innate immunity in mammals. J Endotoxin Res2005;11:390–394.

32. Karin M, Lawrence T, Nizet V. Innate immunity gone awry: linkingmicrobial infections to chronic inflammation and cancer. Cell2006;124:823–835.

33. Banyer JL, Hamilton NH, Ramshaw IA, et al. Cytokines in innateand adaptive immunity. Rev Immunogenet 2000;2:359–373.

34. Fagarasan S. Intestinal IgA synthesis: a primitive form of adap-tive immunity that regulates microbial communities in the gut.Curr Top Microbiol Immunol 2006;308:137–153.

35. Firestein GS. The T cell cometh: interplay between adaptiveimmunity and cytokine networks in rheumatoid arthritis. J ClinInvest 2004;114:471–474.

36. Flajnik MF, Du Pasquier L. Evolution of innate and adaptiveimmunity: can we draw a line? Trends Immunol 2004;25:640–644.

37. Harada K, Isse K, Sato Y, et al. Endotoxin tolerance in humanintrahepatic biliary epithelial cells is induced by upregulation ofIRAK-M. Liver Int 2006;26:935–942.

38. Chen XM, O’Hara SP, Nelson JB, et al. Multiple TLRs are ex-pressed in human cholangiocytes and mediate host epithelialdefense responses to Cryptosporidium parvum via activation ofNF-kappaB. J Immunol 2005;175:7447–7456.

39. Rogers KA, Rogers AB, Leav BA, et al. MyD88-dependent path-ways mediate resistance to Cryptosporidium parvum infection inmice. Infect Immun 2006;74:549–556.

40. Aoki CA, Bowlus CL, Gershwin ME. The immunobiology of pri-mary sclerosing cholangitis. Autoimmun Rev 2005;4:137–143.

41. Adams DH, Afford SC. The role of cholangiocytes in the devel-opment of chronic inflammatory liver disease. Front Biosci2002;7:e276–e285.

42. Adams DH, Afford SC. Effector mechanisms of nonsuppurativedestructive cholangitis in graft-versus-host disease and allograftrejection. Semin Liver Dis 2005;25:281–297.

43. Bloom S, Fleming K, Chapman R. Adhesion molecule expressionin primary sclerosing cholangitis and primary biliary cirrhosis.Gut 1995;36:604–609.

44. Howell CD, Li J, Chen W. Role of intercellular adhesion mole-cule-1 and lymphocyte function-associated antigen-1 during non-suppurative destructive cholangitis in a mouse graft-versus-hostdisease model. Hepatology 1999;29:766–776.

45. Korlipara LV, Leon MP, Rix DA, et al. Development of a flowcytometric assay to quantify lymphocyte adhesion to cytokine-stimulated human endothelial and biliary epithelial cells. J Im-munol Methods 1996;191:121–130.

46. Kaetzel CS. The polymeric immunoglobulin receptor: bridginginnate and adaptive immune responses at mucosal surfaces.

Immunol Rev 2005;206:83–99.

REV

IEW

SIN

BA

SIC

AN

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February 2009 CHOLESTEROL GALLSTONE FORMATION 437

47. Mostov KE. Transepithelial transport of immunoglobulins. AnnuRev Immunol 1994;12:63–84.

48. Norderhaug IN, Johansen FE, Schjerven H, et al. Regulation ofthe formation and external transport of secretory immunoglobu-lins. Crit Rev Immunol 1999;19:481–508.

49. Rojas R, Apodaca G. Immunoglobulin transport across polarizedepithelial cells. Nat Rev Mol Cell Biol 2002;3:944–955.

50. Dickinson BL, Claypool SM, D’Angelo JA, et al. Ca2�-dependentcalmodulin binding to FcRn affects immunoglobulin G transportin the transcytotic pathway. Mol Biol Cell 2008;19:414–423.

51. Blumberg RS, Koss T, Story CM, et al. A major histocompatibilitycomplex class I-related Fc receptor for IgG on rat hepatocytes.J Clin Invest 1995;95:2397–2402.

52. Brown WR, Kloppel TM. The liver and IgA: immunological, cellbiological and clinical implications. Hepatology 1989;9:763–784.

53. McQueen CE, Boedeker EC, Le M, et al. Mucosal immuneresponse to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody. Infect Immun 1992;60:206–212.

54. Savard CE, Blinman TA, Choi HS, et al. Expression of cytokineand chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipo-polysaccharides. BMC Gastroenterol 2002;2:23.

55. Shi JS, Zhou LS, Han Y, et al. Expression of tumor necrosisfactor and its receptor in gallstone and gallbladder carcinomatissue. Hepatobiliary Pancreat Dis Int 2004;3:448–452.

56. Rege RV. Inflammatory cytokines alter human gallbladder epi-thelial cell absorption/secretion. J Gastrointest Surg 2000;4:185–192.

57. Prystowsky JB, Rege RV. Interleukin-1 mediates guinea pig gall-bladder inflammation in vivo. J Surg Res 1997;71:123–126.

58. Chung-Park M, Kim B, Marmolya G, et al. Acalculus lymphoeosi-nophilic cholecystitis associated with interleukin-2 and lympho-kine-activated killer cell therapy. Arch Pathol Lab Med 1990;114:1073–1075.

59. Dickey KW, Barth RA, Stewart JA. Recurrent transient gallblad-der wall thickening associated with interleukin-2 chemotherapy.J Clin Ultrasound 1993;21:58–61.

60. Powell FC, Spooner KM, Shawker TH, et al. Symptomatic inter-leukin-2-induced cholecystopathy in patients with HIV infection.AJR Am J Roentgenol 1994;163:117–121.

61. Mori M, Miyazaki K. Factors affecting morphogenesis of rabbitgallbladder epithelial cells cultured in collagen gels. Cell TissueRes 2000;300:331–344.

62. Koninger J, di Mola FF, Di Sebastiano P, et al. Transforminggrowth factor-beta pathway is activated in cholecystolithiasis.Langenbecks Arch Surg 2005;390:21–28.

63. Hemming JM, Guarraci FA, Firth TA, et al. Actions of histamineon muscle and ganglia of the guinea pig gallbladder. Am JPhysiol Gastrointest Liver Physiol 2000;279:G622–G630.

64. He XS, Ansari AA, Ridgway WM, et al. New insights to theimmunopathology and autoimmune responses in primary biliarycirrhosis. Cell Immunol 2006;239:1–13.

65. Alba LM, Angulo P, Lindor KD. Primary sclerosing cholangitis.Minerva Gastroenterol Dietol 2002;48:99–113.

66. Cullen S, Chapman R. Primary sclerosing cholangitis. Autoim-mun Rev 2003;2:305–312.

67. Koukoulis GK, Shen J, Karademir S, et al. Cholangiocytic apo-ptosis in chronic ductopenic rejection. Hum Pathol 2001;32:823–827.

68. Harvey PR, Upadhya GA, Strasberg SM. Immunoglobulins asnucleating proteins in the gallbladder bile of patients with cho-lesterol gallstones. J Biol Chem 1991;266:13996–14003.

69. Harvey PR, Upadhya GA. A rapid, simple high capacity choles-

terol crystal growth assay. J Lipid Res 1995;36:2054–2058.

70. Upadhya GA, Harvey PR, Strasberg SM. Effect of human biliaryimmunoglobulins on the nucleation of cholesterol. J Biol Chem1993;268:5193–5200.

71. Paigen B, Schork NJ, Svenson KL, et al. Quantitative trait locimapping for cholesterol gallstones in AKR/J and C57L/J strainsof mice. Physiol Genomics 2000;4:59–65.

72. Miquel JF, Nunez L, Amigo L, et al. Cholesterol saturation, notproteins or cholecystitis, is critical for crystal formation in hu-man gallbladder bile. Gastroenterology 1998;114:1016–1023.

73. Lipsett PA, Hildreth J, Kaufman HS, et al. Human gallstonescontain pronucleating nonmucin glycoproteins that are immuno-globulins. Ann Surg 1994;219:25–33.

74. Andrianifahanana M, Moniaux N, Batra SK. Regulation of mucinexpression: mechanistic aspects and implications for cancerand inflammatory diseases. Biochim Biophys Acta 2006;1765:189–222.

75. Lammert F, Wang DQ, Wittenburg H, et al. Lith genes controlmucin accumulation, cholesterol crystallization, and gallstoneformation in A/J and AKR/J inbred mice. Hepatology 2002;36:1145–1154.

76. LaMont JT, Smith BF, Moore JR. Role of gallbladder mucin inpathophysiology of gallstones. Hepatology 1984;4:51S–56S.

77. Lee KT, Liu TS. Mucin gene expression in gallbladder epithe-lium. J Formos Med Assoc 2002;101:762–768.

78. Wang HH, Afdhal NH, Gendler SJ, et al. Targeted disruption ofthe murine mucin gene 1 decreases susceptibility to cholesterolgallstone formation. J Lipid Res 2004;45:438–447.

79. Wang HH, Afdhal NH, Gendler SJ, et al. Evidence that gallblad-der epithelial mucin enhances cholesterol cholelithogenesis inMUC1 transgenic mice. Gastroenterology 2006;131:210–222.

80. Afdhal NH, Niu N, Gantz D, et al. Bovine gallbladder mucinaccelerates cholesterol monohydrate crystal growth in modelbile. Gastroenterology 1993;104:1515–1523.

81. Levy PF, Smith BF, LaMont JT. Human gallbladder mucin accel-erates nucleation of cholesterol in artificial bile. Gastroenterol-ogy 1984;87:270–275.

82. Smith BF. Gallbladder mucin as a pronucleating agent for cho-lesterol monohydrate crystals in bile. Hepatology 1990;12:183S–186S; discussion 186S–188S.

83. Yamasaki T, Chijiiwa K, Endo M. Isolation of mucin from humanhepatic bile and its induced effects on precipitation of choles-terol and calcium carbonate in vitro. Dig Dis Sci 1993;38:909–915.

84. Zen Y, Harada K, Sasaki M, et al. Lipopolysaccharide inducesoverexpression of MUC2 and MUC5AC in cultured biliary epithe-lial cells: possible key phenomenon of hepatolithiasis. Am JPathol 2002;161:1475–1484.

85. Ikeda H, Sasaki M, Ishikawa A, et al. Interaction of Toll-likereceptors with bacterial components induces expression ofCDX2 and MUC2 in rat biliary epithelium in vivo and in culture.Lab Invest 2007;87:559–571.

86. Sakamoto H, Mutoh H, Ido K, et al. A close relationship betweenintestinal metaplasia and Cdx2 expression in human gallblad-ders with cholelithiasis. Hum Pathol 2007;38:66–71.

87. Choi J, Klinkspoor JH, Yoshida T, et al. Lipopolysaccharide fromEscherichia coli stimulates mucin secretion by cultured doggallbladder epithelial cells. Hepatology 1999;29:1352–1357.

88. Finzi L, Barbu V, Burgel PR, et al. MUC5AC, a gel-forming mucinaccumulating in gallstone disease, is overproduced via an epi-dermal growth factor receptor pathway in the human gallblad-der. Am J Pathol 2006;169:2031–2041.

89. Little TE, Madani H, Lee SP, et al. Lipid vesicle fusion inducedby phospholipase C activity in model bile. J Lipid Res 1993;34:211–217.

90. Offner GD, Gong D, Afdhal NH. Identification of a 130-kilodaltonhuman biliary concanavalin A binding protein as aminopepti-

dase N. Gastroenterology 1994;106:755–762.

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OEN

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438 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

91. Zhou H, Chen B, Li RX, et al. Large-scale identification of humanbiliary proteins from a cholesterol stone patient using a pro-teomic approach. Rapid Commun Mass Spectrom 2005;19:3569–3578.

92. Moschetta A, Bookout AL, Mangelsdorf DJ. Prevention of cho-lesterol gallstone disease by FXR agonists in a mouse model.Nat Med 2004;10:1352–1358.

93. Lyons MA, Wittenburg H. Susceptibility to cholesterol gallstoneformation: Evidence that LITH genes also encode immune-re-lated factors. Biochim Biophys Acta 2006;1761:1133–1147.

94. Portincasa P, Di Ciaula A, Vendemiale G, et al. Gallbladdermotility and cholesterol crystallization in bile from patients withpigment and cholesterol gallstones. Eur J Clin Invest 2000;30:317–324.

95. Lee SP. Lessons from experimental cholelithiasis: gallbladderand mucosa, nonsteroidal antiinflammatory drugs, and gall-stones. Gastroenterology 1991;101:857–860.

96. Xiao ZL, Amaral J, Biancani P, et al. Impaired cytoprotectivefunction of muscle in human gallbladders with cholesterolstones. Am J Physiol Gastrointest Liver Physiol 2005;288:G525–G532.

97. van de Heijning BJ, van de Meeberg PC, Portincasa P, et al.Effects of ursodeoxycholic acid therapy on in vitro gallbladdercontractility in patients with cholesterol gallstones. Dig Dis Sci1999;44:190–196.

98. Haigh WG, Lee SP. Identification of oxysterols in human bile andpigment gallstones. Gastroenterology 2001;121:118–123.

99. Seo DW, Choi HS, Lee SP, et al. Oxysterols from human bileinduce apoptosis of canine gallbladder epithelial cells in mono-layer culture. Am J Physiol Gastrointest Liver Physiol 2004;287:G1247–G1256.

00. Lee SP, Carey MC, LaMont JT. Aspirin prevention of cholesterolgallstone formation in prairie dogs. Science 1981;211:1429–1431.

01. Adamek HE, Buttmann A, Weber J, et al. Can aspirin preventgallstone recurrence after successful extracorporeal shockwavelithotripsy? Scand J Gastroenterol 1994;29:355–359.

02. Broomfield PH, Chopra R, Sheinbaum RC, et al. Effects ofursodeoxycholic acid and aspirin on the formation of lithogenicbile and gallstones during loss of weight. N Engl J Med 1988;319:1567–1572.

03. Cohen BI, Mosbach EH, Ayyad N, et al. Aspirin does not inhibitcholesterol cholelithiasis in two established animal models.Gastroenterology 1991;101:1109–1116.

04. Kurata JH, Marks J, Abbey D. One gram of aspirin per day doesnot reduce risk of hospitalization for gallstone disease. Dig DisSci 1991;36:1110–1115.

05. Li YF, Russell DH, Myers SI, et al. Gallbladder contractility inaspirin- and cholesterol-fed prairie dogs. Gastroenterology1994;106:1662–1667.

06. Araki Y, Andoh A, Bamba H, et al. The cytotoxicity of hydrophobicbile acids is ameliorated by more hydrophilic bile acids in intes-tinal cell lines IEC-6 and Caco-2. Oncol Rep 2003;10:1931–1936.

07. Yui S, Saeki T, Kanamoto R, et al. Characteristics of apoptosisin HCT116 colon cancer cells induced by deoxycholic acid.J Biochem (Tokyo) 2005;138:151–157.

08. Lamireau T, Zoltowska M, Levy E, et al. Effects of bile acids onbiliary epithelial cells: proliferation, cytotoxicity, and cytokinesecretion. Life Sci 2003;72:1401–1411.

09. Kano M, Shoda J, Irimura T, et al. Effects of long-term ursode-oxycholate administration on expression levels of secretorylow-molecular-weight phospholipases A2 and mucin genes ingallbladders and biliary composition in patients with multiplecholesterol stones. Hepatology 1998;28:302–313.

10. Maurer KJ. A systematic evaluation of the role of infection,

immunity and inflammation in cholesterol gallstone pathogene-

sis [doctoral thesis]. Cambridge, MA: Massachusetts Instituteof Technology, 2007:159.

11. Paumgartner G, Pauletzki J, Sackmann M. Ursodeoxycholic acidtreatment of cholesterol gallstone disease. Scand J Gastroen-terol Suppl 1994;204:27–31.

12. Paumgartner G, Sauerbruch T. Gallstones: pathogenesis. Lan-cet 1991;338:1117–1121.

13. Henkel A, Wei Z, Cohen DE, et al. Mice overexpressing hepaticAbcb11 rapidly develop cholesterol gallstones. Mamm Genome2005;16:903–908.

14. Portincasa P, van de Meeberg P, van Erpecum KJ, et al. Anupdate on the pathogenesis and treatment of cholesterol gall-stones. Scand J Gastroenterol Suppl 1997;223:60–69.

15. Merselis JG, Jr., Kaye D, Connolly CS, et al. The typhoid carrierstate: quantitative bacteriology and preliminary observations ontherapy. East Afr Med J 1964;41:219–227.

16. Rains AJ, Barson GJ, Crawford N, et al. A chemical and bacte-riological study of gallstones. The presence of an actinomycete.Lancet 1960;2:614–618.

17. Scott AJ, Khan GA. Origin of bacteria in bileduct bile. Lancet1967;2:790–792.

18. Salen G. Gallstone dissolution therapy with ursodiol. Efficacyand safety. Dig Dis Sci 1989;34:39S–43S.

19. Hellstern A, Leuschner U, Benjaminov A, et al. Dissolution ofgallbladder stones with methyl tert-butyl ether and stone recur-rence: a European survey. Dig Dis Sci 1998;43:911–920.

20. Pauletzki J, Holl J, Sackmann M, et al. Gallstone recurrenceafter direct contact dissolution with methyl tert-butyl ether. DigDis Sci 1995;40:1775–1781.

21. Lamont JT, Carey MC. Cholesterol gallstone formation. 2.Pathobiology and pathomechanics. Prog Liver Dis 1992;10:165–191.

22. Cahalane MJ, Neubrand MW, Carey MC. Physical-chemicalpathogenesis of pigment gallstones. Semin Liver Dis 1988;8:317–328.

23. Lee DK, Tarr PI, Haigh WG, et al. Bacterial DNA in mixedcholesterol gallstones. Am J Gastroenterol 1999;94:3502–3506.

24. Swidsinski A, Lee SP. The role of bacteria in gallstone patho-genesis. Front Biosci 2001;6:E93–E103.

25. Swidsinski A, Ludwig W, Pahlig H, et al. Molecular geneticevidence of bacterial colonization of cholesterol gallstones.Gastroenterology 1995;108:860–864.

26. Wu XT, Xiao LJ, Li XQ, et al. Detection of bacterial DNA fromcholesterol gallstones by nested primers polymerase chain re-action. World J Gastroenterol 1998;4:234–237.

27. Kawai M, Iwahashi M, Uchiyama K, et al. Gram-positive cocciare associated with the formation of completely pure choles-terol stones. Am J Gastroenterol 2002;97:83–88.

28. Vitetta L, Sali A, Moritz V, et al. Bacteria and gallstone nucle-ation. Aust N Z J Surg 1989;59:571–577.

29. Vitetta L, Best SP, Sali A. Single and multiple cholesterol gall-stones and the influence of bacteria. Med Hypotheses 2000;55:502–506.

30. Whiting MJ, Watts JM. Cholesterol gallstone pathogenesis: astudy of potential nucleating agents for cholesterol crystal for-mation in bile. Clin Sci (Lond) 1985;68:589–596.

31. Farshad S, Alborzi A, Malek Hosseini SA, et al. Identification ofHelicobacter pylori DNA in Iranian patients with gallstones.Epidemiol Infect 2004;132:1185–1189.

32. Monstein HJ, Jonsson Y, Zdolsek J, et al. Identification ofHelicobacter pylori DNA in human cholesterol gallstones. ScandJ Gastroenterol 2002;37:112–119.

33. Myung SJ, Kim MH, Shim KN, et al. Detection of Helicobacterpylori DNA in human biliary tree and its association with hepa-

tolithiasis. Dig Dis Sci 2000;45:1405–1412.

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February 2009 CHOLESTEROL GALLSTONE FORMATION 439

34. Silva CP, Pereira-Lima JC, Oliveira AG, et al. Association of thepresence of Helicobacter in gallbladder tissue with cholelithia-sis and cholecystitis. J Clin Microbiol 2003;41:5615–5618.

35. Mathai E, Arora A, Cafferkey M, et al. The effect of bile acids onthe growth and adherence of Helicobacter pylori. Aliment Phar-macol Ther 1991;5:653–658.

36. Worku ML, Karim QN, Spencer J, et al. Chemotactic response ofHelicobacter pylori to human plasma and bile. J Med Microbiol2004;53:807–811.

37. Kawaguchi M, Saito T, Ohno H, et al. Bacteria closely resem-bling Helicobacter pylori detected immunohistologically and ge-netically in resected gallbladder mucosa. J Gastroenterol 1996;31:294–298.

38. O’Rourke JL, Solnick JV, Neilan BA, et al. Description of ‘Can-didatus Helicobacter heilmannii’ based on DNA sequence anal-ysis of 16S rRNA and urease genes. Int J Syst Evol Microbiol2004;54:2203–2211.

39. Avenaud P, Marais A, Monteiro L, et al. Detection of Helicobac-ter species in the liver of patients with and without primary livercarcinoma. Cancer 2000;89:1431–1439.

40. Fox J. Enterohepatic Helicobacters: natural and experimentalmodels. Ital J Gastroenterol Hepatol 1998;30(Suppl 3):S264–S269.

41. Solnick JV, Schauer DB. Emergence of diverse Helicobacterspecies in the pathogenesis of gastric and enterohepatic dis-eases. Clin Microbiol Rev 2001;14:59–97.

42. Fox JG, Dewhirst FE, Shen Z, et al. Hepatic Helicobacter speciesidentified in bile and gallbladder tissue from Chileans withchronic cholecystitis. Gastroenterology 1998;114:755–763.

43. Matsukura N, Yokomuro S, Yamada S, et al. Association be-tween Helicobacter bilis in bile and biliary tract malignancies: H.bilis in bile from Japanese and Thai patients with benign andmalignant diseases in the biliary tract. Jpn J Cancer Res 2002;93:842–847.

44. Belzer C, Kusters JG, Kuipers EJ, et al. Urease induced calciumprecipitation by Helicobacter species may initiate gallstone for-mation. Gut 2006;55:1678–1679.

45. Sanabria JR, Upadhya GA, Harvey RP, et al. Diffusion of sub-stances into human cholesterol gallstones. Gastroenterology1994;106:749–754.

46. Bruce IJ. Nucleic acid amplification mediated microbial identifi-cation. Sci Prog 1993;77:183–206.

47. Davis RE, Jomantiene R, Kalvelyte A, et al. Differential amplifi-cation of sequence heterogeneous ribosomal RNA genes andclassification of the ‘Fragaria multicipita’ phytoplasma. Micro-biol Res 2003;158:229–236.

48. King S, McCord BR, Riefler RG. Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soilbacteria. J Microbiol Methods 2005;60:83–92.

49. Dutta U, Garg PK, Kumar R, et al. Typhoid carriers amongpatients with gallstones are at increased risk for carcinoma ofthe gallbladder. Am J Gastroenterol 2000;95:784–787.

50. Lai CW, Chan RC, Cheng AF, et al. Common bile duct stones: acause of chronic salmonellosis. Am J Gastroenterol 1992;87:1198–1199.

51. Prouty AM, Schwesinger WH, Gunn JS. Biofilm formation andinteraction with the surfaces of gallstones by Salmonella spp.Infect Immun 2002;70:2640–2649.

52. Prouty AM, Gunn JS. Comparative analysis of Salmonella en-terica serovar Typhimurium biofilm formation on gallstones andon glass. Infect Immun 2003;71:7154–7158.

53. Bini EJ, McGready J. Prevalence of gallbladder disease amongpersons with hepatitis C virus infection in the United States.Hepatology 2005;41:1029–1036.

54. Chang TS, Lo SK, Shyr HY, et al. Hepatitis C virus infectionfacilitates gallstone formation. J Gastroenterol Hepatol 2005;

20:1416–1421.

55. Elzouki AN, Nilsson S, Nilsson P, et al. The prevalence ofgallstones in chronic liver disease is related to degree of liverdysfunction. Hepatogastroenterology 1999;46:2946–2950.

56. Sheen IS, Liaw YF. The prevalence and incidence of cholecys-tolithiasis in patients with chronic liver diseases: a prospectivestudy. Hepatology 1989;9:538–540.

57. Loriot MA, Bronowicki JP, Lagorce D, et al. Permissiveness ofhuman biliary epithelial cells to infection by hepatitis C virus.Hepatology 1999;29:1587–1595.

58. Nouri Aria KT, Sallie R, Sangar D, et al. Detection of genomicand intermediate replicative strands of hepatitis C virus in livertissue by in situ hybridization. J Clin Invest 1993;91:2226–2234.

59. Bargiggia S, Maconi G, Elli M, et al. Sonographic prevalence ofliver steatosis and biliary tract stones in patients with inflam-matory bowel disease: study of 511 subjects at a single center.J Clin Gastroenterol 2003;36:417–420.

60. Fraquelli M, Losco A, Visentin S, et al. Gallstone disease andrelated risk factors in patients with Crohn disease: analysis of330 consecutive cases. Arch Intern Med 2001;161:2201–2204.

61. Kratzer W, Haenle MM, Mason RA, et al. Prevalence of choleli-thiasis in patients with chronic inflammatory bowel disease.World J Gastroenterol 2005;11:6170–6175.

62. Whorwell PJ, Hawkins R, Dewbury K, et al. Ultrasound survey ofgallstones and other hepatobiliary disorders in patients withCrohn’s disease. Dig Dis Sci 1984;29:930–933.

63. Hutchinson R, Tyrrell PN, Kumar D, et al. Pathogenesis of gallstones in Crohn’s disease: an alternative explanation. Gut1994;35:94–97.

64. Pereira SP, Bain IM, Kumar D, et al. Bile composition in inflam-matory bowel disease: ileal disease and colectomy, but notcolitis, induce lithogenic bile. Aliment Pharmacol Ther 2003;17:923–933.

65. Freudenberg F, Carey MC. Hepatobiliary manifestations in in-flammatory bowel disease. In: Gut-Liver Interactions: Basic andClinical Concepts. Blumberg RS, Gangl A, Manns MP, et al, eds.Dordrecht, The Netherlands: Springer, 2005:165–176.

66. Lapidus A, Einarsson C. Bile composition in patients with ilealresection due to Crohn’s disease. Inflamm Bowel Dis 1998;4:89–94.

67. Lapidus A, Einarsson K. Effects of ileal resection on biliary lipidsand bile acid composition in patients with Crohn’s disease. Gut1991;32:1488–1491.

68. Hofmann AF. Medical dissolution of gallstones by oral bile acidtherapy. Am J Surg 1989;158:198–204.

69. Jungst D, Brenner G, Pratschke E, et al. Low-dose ursodeoxy-cholic acid prolongs cholesterol nucleation time in gallbladderbile of patients with cholesterol gallstones. J Hepatol 1989;8:1–6.

70. Bamias G, Sugawara K, Pagnini C, et al. The Th1 immunepathway as a therapeutic target in Crohn’s disease. Curr OpinInvestig Drugs 2003;4:1279–1286.

71. Heller F, Florian P, Bojarski C, et al. Interleukin-13 is the keyeffector Th2 cytokine in ulcerative colitis that affects epithelialtight junctions, apoptosis, and cell restitution. Gastroenterology2005;129:550–564.

72. Heller F, Fuss IJ, Nieuwenhuis EE, et al. Oxazolone colitis, a Th2colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 2002;17:629–638.

73. Feingold KR, Pollock AS, Moser AH, et al. Discordant regulationof proteins of cholesterol metabolism during the acute phaseresponse. J Lipid Res 1995;36:1474–1482.

74. Hardardottir I, Grunfeld C, Feingold KR. Effects of endotoxin onlipid metabolism. Biochem Soc Trans 1995;23:1013–1018.

75. Hardardottir I, Moser AH, Memon R, et al. Effects of TNF, IL-1,

and the combination of both cytokines on cholesterol metabo-

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1

1

1

1

1

1

1

1

1

1

1

1

1

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IEWS

INBA

SICA

ND

CLIN

ICA

LG

ASTR

OEN

TERO

LOG

Y

440 MAURER ET AL GASTROENTEROLOGY Vol. 136, No. 2

lism in Syrian hamsters. Lymphokine Cytokine Res 1994;13:161–166.

76. Khovidhunkit W, Kim MS, Memon RA, et al. Effects of infectionand inflammation on lipid and lipoprotein metabolism: mecha-nisms and consequences to the host. J Lipid Res 2004;45:1169–1196.

77. Memon RA, Shechter I, Moser AH, et al. Endotoxin, tumornecrosis factor, and interleukin-1 decrease hepatic squalenesynthase activity, protein, and mRNA levels in Syrian hamsters.J Lipid Res 1997;38:1620–1629.

78. Feingold KR, Hardardottir I, Memon R, et al. Effect of endotoxinon cholesterol biosynthesis and distribution in serum lipopro-teins in Syrian hamsters. J Lipid Res 1993;34:2147–2158.

79. Yoo JY, Desiderio S. Innate and acquired immunity intersect in aglobal view of the acute-phase response. Proc Natl Acad SciU S A 2003;100:1157–1162.

80. Mookerjea S, Coolbear T, Sarkar ML. Key role of dolichol phos-phate in glycoprotein biosynthesis. Can J Biochem Cell Biol1983;61:1032–1040.

81. Sarkar M, Mookerjea S. Differential effect of inflammation anddexamethasone on dolichol and dolichol phosphate synthesis.Biochem Cell Biol 1988;66:1265–1269.

82. Auerbach BJ, Parks JS. Lipoprotein abnormalities associatedwith lipopolysaccharide-induced lecithin: cholesterol acyltrans-ferase and lipase deficiency. J Biol Chem 1989;264:10264–10270.

83. Ettinger WH, Miller LD, Albers JJ, et al. Lipopolysaccharide andtumor necrosis factor cause a fall in plasma concentration oflecithin: cholesterol acyltransferase in cynomolgus monkeys. JLipid Res 1990;31:1099–1107.

84. Sammalkorpi K, Valtonen V, Kerttula Y, et al. Changes in serumlipoprotein pattern induced by acute infections. Metabolism1988;37:859–865.

85. Ettinger WH, Varma VK, Sorci-Thomas M, et al. Cytokines de-crease apolipoprotein accumulation in medium from Hep G2cells. Arterioscler Thromb 1994;14:8–13.

86. Schectman G, Kaul S, Mueller RA, et al. The effect of inter-feron on the metabolism of LDLs. Arterioscler Thromb 1992;

12:1053–1062.

87. Feingold KR, Spady DK, Pollock AS, et al. Endotoxin, TNF, andIL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels andactivity. J Lipid Res 1996;37:223–228.

88. Memon RA, Moser AH, Shigenaga JK, et al. In vivo and in vitroregulation of sterol 27-hydroxylase in the liver during the acutephase response. potential role of hepatocyte nuclear factor-1.J Biol Chem 2001;276:30118–126.

89. Green RM, Beier D, Gollan JL. Regulation of hepatocyte bile salttransporters by endotoxin and inflammatory cytokines in ro-dents. Gastroenterology 1996;111:193–198.

90. Hartmann G, Cheung AK, Piquette-Miller M. Inflammatory cyto-kines, but not bile acids, regulate expression of murine hepaticanion transporters in endotoxemia. J Pharmacol Exp Ther 2002;303:273–281.

91. Moseley RH, Wang W, Takeda H, et al. Effect of endotoxin onbile acid transport in rat liver: a potential model for sepsis-associated cholestasis. Am J Physiol 1996;271:G137–G146.

92. Trauner M, Arrese M, Lee H, et al. Endotoxin downregulates rathepatic ntcp gene expression via decreased activity of criticaltranscription factors. J Clin Invest 1998;101:2092–2100.

93. Vos TA, Hooiveld GJ, Koning H, et al. Up-regulation of themultidrug resistance genes, Mrp1 and Mdr1b, and down-regu-lation of the organic anion transporter, Mrp2, and the bile salttransporter, Spgp, in endotoxemic rat liver. Hepatology 1998;28:1637–1644.

94. Tygstrup N, Bangert K, Ott P, et al. Messenger RNA profiles inliver injury and stress: a comparison of lethal and nonlethal ratmodels. Biochem Biophys Res Commun 2002;290:518–525.

Received July 16, 2008. Accepted December 8, 2008.Address requests for reprints to: Kirk J. Maurer, DVM, PhD, Cornell

enter for Animal Resources and Education, College of Veterinaryedicine, Cornell University, Ithaca, New York. e-mail: km429@

ornell.edu; fax: (607) 253-3527.M.C.C. and J.G.F. contributed equally to this work.K.J.M. was supported by grant K08-DK07728, J.G.F. was supported

y grants P30-ES02109 and R01-CA067529, and M.C.C. was sup-orted by grants R01-DK073687 and R37-DK036588.

The authors disclose no conflicts.