Pathogenesis Of Cholesterol Gallstones Research Articles

Inhibiting miRNA-146a suppresses mouse gallstone formation by regulating LXR/megalin/cubilin-media cholesterol absorption

BackgroundmiRNA has been implicated in regulating cholesterol homeostasis, a critical factor in gallstone formation. Here, we focused on elucidating the role of miR-146a in this pathological process. MethodsC57BL/6 mice were fed with lithogenic diet (LD) and injected with miR-146 antagomir (anta-146) via the tail vein for various weeks. The gallbladders and liver tissues were collected for cholesterol crystal imaging, gallstone mass quantification, and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and metabolic parameters in serum and bile were assessed by ELISA. A 3′ UTR reporter gene assay was used to verify the direct target genes for miR-146. The relative expression of metabolism genes was analyzed by quantitative real-time PCR and immunoblotting. ResultsmiR-146a-5p expression was reduced in mice and clinical samples with gallstones. Anta-146 treatment effectively prevented LD-induced gallstone formation in mice without hepatic and cholecystic damage. The mice treated with anta-146 exhibited beneficial alterations in bile cholesterol and bile acids and lipid levels in the blood. A key biliary cholesterol transporter-Megalin was identified as a direct target of miR-146. Anta-146 administration upregulated megalin expression, thereby ameliorating impaired gallbladder cholesterol absorption associated with the LXR-megalin/cubilin pathway. ConclusionThe data demonstrates that miR-146 modulates gallbladder cholesterol absorption by targeting megalin, and prevents the pathogenesis of cholesterol gallstones.

Gallstones: The thing in itself.

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Research progress on the immune microenvironment of the gallbladder in patients with cholesterol gallstones.

Cholesterol gallstones are very common in hepatobiliary surgery and have been studied to a certain extent by doctors worldwide for decades. However, the mechanism of cholesterol gallstone formation is not fully understood, so there is currently no completely effective drug for the treatment and prevention of cholesterol gallstones. The formation and development of cholesterol gallstones are caused by a variety of genetic and environmental factors, among which genetic susceptibility, intestinal microflora disorders, impaired gallbladder motility, and immune disorders are important in the pathogenesis of cholesterol gallstones. This review focuses on recent advances in these mechanisms. We also discuss some new targets that may be effective in the treatment and prevention of cholesterol gallstones, which may be hot areas in the future.

Intestinal flora imbalance affects bile acid metabolism and is associated with gallstone formation

BackgroundThe gut microbiota participates in the metabolism of substances and energy, promotes the development and maturation of the immune system, forms the mucosal barrier, and protects the host from pathogen attacks. Although the pathogenesis of cholesterol gallstones is still not clear, studies have suggested that gut microbiota dysbiosis plays an important role in their formation.MethodsMicrobial DNA from faeces of normal control patients and those of patients with calculi was subjected to 16S rRNA gene sequencing to detect gene expression changes in intestinal microbes. ELISA kits were used to measure free bile acids, secondary bile acids and coprostanol according to the manufacturer’s instructions. The relationship between flora and their metabolites was then analysed.ResultsIn the gallstone group, the diversity of intestinal bacteria and the abundances of certain phylogroups were significantly decreased (p < 0.05), especially Firmicutes (p < 0.05), the largest phylum represented by the gut microbiota. This study found an increase in free bile acids (p < 0.001) and secondary bile acids (p < 0.01) in the enterohepatic circulation. Bile salt hydrolase activity was not related to the abundances of BSH-active bacteria. 7a-dehydroxylating gut bacteria were significantly increased (p < 0.01), whereas cholesterol-lowering bacteria were significantly reduced (p < 0.05). The Ruminococcus gnavus group could be used as a biomarker to distinguish the gallstone group from the control group.ConclusionWe conclude that intestinal flora imbalance affects bile acid and cholesterol metabolism and is associated with gallstone formation.

Recent Advances in the Critical Role of the Sterol Efflux Transporters ABCG5/G8 in Health and Disease.

Cardiovascular disease is characterized by lipid accumulation, inflammatory response, cell death, and fibrosis in the arterial wall and is the leading cause of morbidity and mortality worldwide. Cholesterol gallstone disease is caused by complex genetic and environmental factors and is one of the most prevalent and costly digestive diseases in the USA and Europe. Although sitosterolemia is a rare inherited lipid storage disease, its genetic studies led to identification of the sterol efflux transporters ABCG5/G8 that are located on chromosome 2p21 in humans and chromosome 17 in mice. Human and animal studies have clearly demonstrated that ABCG5/G8 play a critical role in regulating hepatic secretion and intestinal absorption of cholesterol and plant sterols. Sitosterolemia is caused by a mutation in either the ABCG5 or the ABCG8 gene alone, but not in both simultaneously. Polymorphisms in the ABCG5/G8 genes are associated with abnormal plasma cholesterol metabolism and may play a key role in the genetic determination of plasma cholesterol concentrations. Moreover, ABCG5/G8 is a new gallstone gene, LITH9. Gallstone-associated variants in ABCG5/G8 are involved in the pathogenesis of cholesterol gallstones in European, Asian, and South American populations. In this chapter, we summarize the latest advances in the critical role of the sterol efflux transporters ABCG5/G8 in regulating hepatic secretion of biliary cholesterol, intestinal absorption of cholesterol and plant sterols, the classical reverse cholesterol transport, and the newly established transintestinal cholesterol excretion, as well as in the pathogenesis and pathophysiology of ABCG5/G8-related metabolic diseases such as sitosterolemia, cardiovascular disease, and cholesterol gallstone disease.

The Role of Diet in the Pathogenesis of Cholesterol Gallstones.

Cholesterol gallstone disease is a major health problem in Westernized countries and depends on a complex interplay between genetic factors, lifestyle and diet, acting on specific pathogenic mechanisms. Overweigh, obesity, dyslipidemia, insulin resistance and altered cholesterol homeostasis have been linked to increased gallstone occurrence, and several studies point to a number of specific nutrients as risk- or protective factors with respect to gallstone formation in humans. There is a rising interest in the identification of common and modifiable dietetic factors that put the patients at risk of gallstones or that are able to prevent gallstone formation and growth. In particular, dietary models characterized by increased energy intake with highly refined sugars and sweet foods, high fructose intake, low fiber contents, high fat, consumption of fast food and low vitamin C intake increase the risk of gallstone formation. On the other hand, high intake of monounsaturated fats and fiber, olive oil and fish (ω-3 fatty acids) consumption, vegetable protein intake, fruit, coffee, moderate alcohol consumption and vitamin C supplementation exert a protective role. The effect of some confounding factors (e.g., physical activity) cannot be ruled out, but general recommendations about the multiple beneficial effects of diet on cholesterol gallstones must be kept in mind, in particular in groups at high risk of gallstone formation.

Mouse models of gallstone disease.

The establishment of mouse models of gallstones, and the contribution of mouse models to genetic studies of gallstone disease, as well as the latest advances in the pathophysiology of gallstones from mouse experiments are summarized. The combined uses of genomic strategies and phenotypic studies in mice have successfully led to the identification of many Lith genes, which pave the way for the discovery of human LITH genes. The physical-chemical, genetic, and molecular biological studies of gallstone disease in mice with knockout or transgene of specific target genes have provided many novel insights into the complex pathophysiological mechanisms of this very common hepatobiliary disease worldwide, showing that interactions of five primary defects play a critical role in the pathogenesis of cholesterol gallstones. Based on mouse studies, a new concept has been proposed that hepatic hypersecretion of biliary cholesterol is induced by multiple Lith genes, with insulin resistance as part of the metabolic syndrome interacting with cholelithogenic environmental factors to cause the phenotype. The mouse model of gallstones is crucial for elucidating the physical-chemical and genetic mechanisms of cholesterol crystallization and gallstone formation, which greatly increase our understanding of the pathogenesis of this disease in humans.

Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function.

Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation.

Interstitial Cajal-Like Cell: A New Player in Cholelithiasis?

To the Editor: Th e incidence of gallstones is relatively high in the developed countries, with ~ 10 % of the whole population being aff ected. Even though there is a lack of clear hypothesis on gallstone formation, a few factors are proved to be responsible for this process: cholesterol supersaturation, hydrophobic bile salts, pronucleating proteins, mucus hypersecretion with gel formation in the gallbladder and disrupted gallbladder motility. Th e gallbladder dysmotility seems to be a “ trigger ” event in the pathogenesis of cholesterol gallstones, providing the time necessary for the precipitation of cholesterol microcrystals from bile supersaturated with cholesterol. Considering increasing knowledge of the role of interstitial cells of Cajal in the regulation of gastrointestinal tract (GI) motility as well as in the pathogenesis of multiple GI disorders, and the fact that these cells were recently described in human gallbladder and biliary tract, we wondered whether interstitial cells of Cajal (or interstitial Cajal-like cells — ICLCs — as they are called when localized in extraintestinal organs) are present in gallbladder wall in patients with cholelithiasis and whether their density decreases or perhaps remains intact. We used indirect double immunofl uorescence method to visualize ICLCs. A characteristic feature of ICLCs is the expression of transmembrane tyrosine kinase receptor proteins, including the c-Kit receptor (CD117), which enables the identifi cation of ICLCs. Unfortunately, the CD117 is present in the mast cells as well, and for that reason we stained slides with anti-mast cell tryptase. ICLCs were defi ned as c-Kitpositive nucleated cells that lacked mast cell tryptase expression ( Figure 1 ). ICLCs were observed throughout the gallbladder, including the fundus, body (corpus), and neck, although they were predominantly located in the corpus, almost exclusively within the muscularis propria. ICLCs had a centrally located nucleus and were mostly fusiform in shape; however, sparse, round tryptase / c-Kit-positive cells were also present ( 1 ). In our study (2), we reported for the fi rst time that interstitial Cajal-like cells are lost or damaged in patients suff ering from cholelithiasis. We observed the cells positive for the c-Kit receptor (CD117) in the gallbladder wall in all cases examined. ICLCs were most frequently located in the muscularis propria. Th e density of ICLCs in the muscularis propria was signifi cantly lower in the patients with gallstones than the density observed in the controls (26.24 ± 10.89 vs. 56.29 ± 13.35 cell mm − 2 in the muscularis propria, P < 0.001). Th e published data on ICLCs in the human gallbladder are still very limited. ICLCs were described in the wall of the human gallbladder by Hinescu et al. ( 3 ) and in the bile ducts by Ahmadi et al. ( 4 ). Both teams reported that ICLCs in the gallbladder formed a cellular network that is likely involved in biliary tract motility. Consequently, animal studies suggested the potential role of ICLCs in functional disturbances of the gallbladder. So far, we have not found any works published on ICLCs in the context of pathogenesis of gallstone in humans. Our studies revealed that ICLCs play a role in the pathogenesis of gallstone disease. Furthermore, from the currently fi nished research ( 5 ) we conclude that bile content may infl uence these cells. It implies some possible future medical interventions .

Influence of Ca2+ on cholesterol crystallization from supersaturated model biles

The quaternary system of bile salt (BS)–lecithin (L)–cholesterol (Ch)–water has been mainly employed to simulate the in vitro formation of cholesterol gallstones. In this work, a quinary system of bile salt (BS)–lecithin (L)–cholesterol (Ch)–inorganic salt (Ca2+)–water was developed to study the nucleation of cholesterol crystals from a thermodynamic standpoint. The accurate concentration of each component in the freshly prepared biles and after incubation was analyzed by high performance liquid chromatography (HPLC). The micelles, vesicles and crystals formed in the biles were characterized by quasielastic light scattering (QLS) and polarizing microscope, respectively. The results show that, under different concentrations of above five components at 37°C the model biles can finally disperse as one phase (micelles), two phases (micelles and vesicles, micelles and crystals) or three phases (micelles, vesicles and crystals). The mean diameter of BS–L micelles in the one phase region is about 5.7nm, whereas that of Ch–L vesicles in the phase region of micelles and vesicles is about 362.4nm. In addition, the introduction of Ca2+ will decrease the solubility of cholesterol in model biles and thus will shorten the nucleation time of cholesterol crystals, which indicates that Ca2+ plays an important role in the pathogenesis of cholesterol gallstones.

Nutritional factors (nutritional aspects) in biliary disorders: Bile acid and lipid metabolism in gallstone diseases and pancreaticobiliary maljunction

Nutritional factors play a key role in the pathogenesis of biliary diseases such as gallstones and pancreaticobiliary maljunction. Gallstones are primarily classified into cholesterol stone and pigment stone according to the major composition. Cholesterol gallstone formation is very likely based upon supersaturated bile formation, and pigment stones are formed in bile rich in bilirubin. Thus, defects of hepatic metabolism of lipids and organic anions lead to biliary stones. Here, the recent understanding of cholesterol gallstone pathogenesis is elaborated. On the other hand, there is another important link of biliary lipid degradation to serious biliary disease, namely pancreaticobiliary maljunction. Lysophosphatidylcholine (lysoPC), a derivative of phosphatidylcholine hydrolysis by phospholipase A2, is a highly abundant bioactive lipid mediator present in circulation as well as in bile. Increases in bile of lysoPC and phospholipase A2 have been reported in pancreaticobiliary maljunction and considered to be the major risk factor for biliary tract cancers. Further, oxidized fatty acids have been established as a potent ligand for G2A, a member of G protein-coupled receptor family that mediates a diverse array of biological processes including cell growth and apoptosis. Thus, both of lysoPC and free fatty acids are supposed to play an important role through G2A in biliary inflammation and carcinogenesis of pancreaticobiliary maljunction. Taken together, nutritional factors, especially lipid compounds, are seemingly crucial in the pathogenesis of biliary diseases, and such a causal relationship is reviewed by mainly authors' previous publications.

An analysis of the role of the indigenous microbiota in cholesterol gallstone pathogenesis.

Background and AimsCholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates.MethodsGenetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1day of age or by rederiving into a germ-free state.ResultsAlterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice.ConclusionsThese data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.

Current Views on Genetics and Epigenetics of Cholesterol Gallstone Disease

Cholesterol gallstone disease, one of the commonest digestive diseases in western countries, is induced by an imbalance in cholesterol metabolism, which involves intestinal absorption, hepatic biosynthesis, and biliary output of cholesterol, and its conversion to bile acids. Several components of the metabolic syndrome (e.g., obesity, type 2 diabetes, dyslipidemia, and hyperinsulinemia) are also well-known risk factors for gallstones, suggesting the existence of interplay between common pathophysiological pathways influenced by insulin resistance, genetic, epigenetic, and environmental factors. Cholesterol gallstones may be enhanced, at least in part, by the abnormal expression of a set of the genes that affect cholesterol homeostasis and lead to insulin resistance. Additionally, epigenetic mechanisms (mainly DNA methylation, histone acetylation/deacetylation, and noncoding microRNAs) may modify gene expression in the absence of an altered DNA sequence, in response to different lithogenic environmental stimuli, such as diet, lifestyle, pollutants, also occurring in utero before birth. In this review, we will comment on various steps of the pathogenesis of cholesterol gallstones and interaction between environmental and genetic factors. The epigenomic approach may offer new options for therapy of gallstones and better possibilities for primary prevention in subjects at risk.

Pathogenesis of cholesterol and pigment gallstones: An update

Phase separation of cholesterol crystals from supersaturated bile is still considered the key event in cholesterol gallstone formation. In this review, we will first provide a basal framework of the interactions between the sterol, bile salts and phospholipids in aqueous solutions and then summarize new developments. The hepatocytic apical membrane harbours specific transport proteins for these lipids. Polymorphisms in the gene encoding the cholesterol transporter ABCG5-G8 have been found to increase overall gallstone risk, whereas functional mutations in the gene encoding the phospholipid floppase ABCB4 lead to the rare clinical syndrome of low phospholipid associated cholelithiasis. Expression of bile salt and phospholipid transport proteins is regulated bij the bile salt nuclear receptor Farnesoid X receptor (FXR), while the Liver X Receptor (LXR) α regulates ABCG5-G8. Although data from murine experiments suggest a critical role of FXR in gallstone formation, its role in human lithogenesis remains controversial. Variants of the gene encoding UGT1A1 (uridine 5'-diphosphate (UDP)-glucuronosyltransferase 1A1) responsible for bilirubin conjugation were recently associated with risk of gallstones as well as stone bilirubin content, suggesting common factors in cholesterol and pigment gallstone pathogenesis.

Relation of Gallbladder Motility to Viscosity and Composition of Gallbladder Bile in Patients with Cholesterol Gallstones

Background/Aims: Increased viscosity and supersaturation of cholesterol in gallbladder bile, as well as an impaired motility of the gallbladder, are considered to be important factors in the pathogenesis of cholesterol gallstones. However, the relation of these parameters has not yet been determined. Material and Methods: Bile viscosity (mPa·s) was measured by rotation viscosimetry and the composition of gallbladder bile was determined using standard methodology. Gallbladder motility was calculated as ejection fraction in percent of total volume 45 min after a test meal using ultrasonography in patients with gallstones prior to elective cholecystectomy. Results: The study included 35 patients with cholesterol gallstones. Viscosity of gallbladder bile ranged between 0.9 and 12.5 mPa·s (median 2.2 mPa·s) and an ejection fraction of the gallbladder of 55.4 ± 18.3% (mean ± SD) was determined. No significant correlation (r = 0.19, p < 0.2) between the 2 parameters could be calculated. Analysis of the composition of gallbladder bile revealed a positive correlation of all components to biliary viscosity but not to the motility of the gallbladder, with the exceptions of a negative correlation (r = 0.39, p < 0.02) between mucin concentration and the ejection fraction at 45 min after the test meal. Conclusions: The motility of the gallbladder appears to be unrelated to the viscosity of gallbladder bile or gallbladder bile composition. The negative correlation between the ejection fraction of the gallbladder and mucin concentration of gallbladder bile suggests that chronic inflammation of the gallbladder wall is associated with both an impaired motility of the gallbladder and increased mucin release into gallbladder bile.

Biliary lipids and cholesterol gallstone disease

Biliary lipids are a family of four dissimilar molecular species consisting of a mixture of bile salts (substituted cholanoic acids), phospholipids, mostly (>96%) diacylphosphatidylcholines, unesterified cholesterol, and bilirubin conjugates known trivially as lipopigments. The primary pathophysiological defect in cholesterol gallstone disease is hypersecretion of hepatic cholesterol into bile with less frequent hyposecretion of bile salts and/or phospholipids. Several other gallbladder abnormalities contribute and include hypomotility, immune-mediated inflammation, hypersecretion of gelling mucins, and accelerated phase transitions; there is also reduced intestinal motility that augments "secondary" bile salt synthesis by the anaerobic microflora. Cholesterol nucleation is initiated when unilamellar vesicles of cholesterol plus biliary phospholipids fuse to form multilamellar vesicles. From these "plate-like" cholesterol monohydrate crystals, the building blocks of macroscopic stones are nucleated heterogeneously by mucin gel. Multiple Lith gene loci have been identified in inbred mice, paving the way for discovery of an ever-increasing number of LITH genes in humans. Because of the frequency of the metabolic syndrome today, insulin resistance and LITH genes all interact with a number of environmental cholelithogenic factors to cause the gallstone phenotype. This review summarizes current concepts of the physical-chemical state of biliary lipids in health and in lithogenic bile and outlines the molecular, genetic, hepatic, and cholecystic factors that underlie the pathogenesis of cholesterol gallstones.

Recent understanding of cholesterol gallstone pathogenesis: implication to non-surgical therapeutic strategy

This article reviews the recent understanding of cholesterol gallstone pathogenesis in light of etiology and molecular mechanisms of the cholesterol gallstone formation process, to provide the future direction of a non-surgical therapeutic strategy. In principle, cholesterol gallstone formation, which is associated with an altered bile salt metabolism, is based primarily upon the impairment of cholesterol metabolism and homeostasis. Cholesterol is eliminated physiologically into bile; thus, the excess cholesterol induces bile metastability due to a relative insufficiency of bile salt, to initiate cholesterol crystal nucleation in the gallbladder with an impaired function. Those crystals grow to become macroscopic stones in the gallbladder mucin gel, accumulated under an arachidonate-prostanoid pathway induced-hypersecretion by the gallbladder wall. These events can be modified by bile salt supplementation, which provides a detergent action. Therefore, oral bile salt administration is a cost-effective, non-surgical therapy under certain circumstances. Understanding the pathogenesis of cholesterol gallstones attributes to the therapeutic guideline based upon scientific and clinical evidence.

Leptin Regulates Gallbladder Genes Related to Gallstone Pathogenesis in Leptin-Deficient Mice

Little is known about the genetic factors that cause alterations in gallbladder motility, cholesterol crystal nucleation, biliary lipids, and, ultimately, cholesterol gallstones. Obese, leptin-deficient (Lep(ob)) mice have large gallbladder volumes with decreased contraction in vitro and are predisposed to cholesterol crystal formation. Leptin administration to these mice causes weight loss and restores gallbladder function. We hypothesize that administration of leptin to Lep(ob) mice would cause weight loss, decrease gallbladder volume, and change gallbladder genes related to gallbladder motility, nucleating factors, and lipid metabolism. Twenty-four 8-week-old Lep(ob) mice were fed a nonlithogenic diet for 4 weeks. Twelve mice received daily IP saline injections, and 12 received 5 mug/g recombinant leptin. Gallbladder mRNA was pooled and analyzed on murine genome microarray chips. Selected genes were confirmed by real-time polymerase chain reaction (PCR) in a second group of mice treated by the same protocol. Leptin-deficient mice given leptin had significant weight loss and reductions in gallbladder volume. These mice had upregulation of the leptin receptor (p = 0.007; PCR = 1.1-fold increase) but downregulation of leptin (p = 0.003; PCR = 13.5-fold decrease). Leptin upregulated the cholecystokinin A receptor (p < 0.001; PCR = 3.1-fold increase), acetylcholine beta2 receptor (p = 0.005), and the Ca+-calmodulin-dependent protein kinase (p = 0.002) genes. Leptin also altered immunoglobulin heavy chain 4 (p = 0.005; PCR = 17.7-fold increase), mucin 3 (p = 0.006), and carboxylesterase (p = 0.016; PCR = 2.5-fold decrease) genes. Leptin downregulated 3-hydroxy 3-methylglutaryl coenzyme A reductase (p = 0.006; PCR = 2.5-fold decrease) and LDL receptor (p = 0.003). Leptin modulates obesity and regulates gallbladder genes related to cholesterol gallstone pathogenesis.

T-Cell Function Is Critical for Murine Cholesterol Gallstone Formation

The formation of cholesterol gallstones is a complex process involving contributions from genes and environmental factors. Although gallbladder inflammation is believed to be common during cholelithogenesis, the role of immunologic factors is unknown. The role of adaptive immunity in cholesterol cholelithogenesis was analyzed utilizing immunocompetent Helicobacter spp.-infected and -uninfected BALB/c and congenic immunodeficient Rag2(-/-) (Rag) mice. Lymphocyte transfer studies were performed to determine which cellular subset was responsible for cholesterol gallstone formation. Also, gallbladder inflammation was quantified to determine the nature of the inflammatory response associated with cholelilithogenesis. When fed a lithogenic diet for 8 weeks, wild-type mice developed significantly more cholesterol gallstones (27%-80% prevalence) than Rag mice ( approximately 5%, P < .05). Helicobacter spp.-infected BALB/cJ mice displayed statistically significant increases in cholesterol gallstone prevalence compared with uninfected mice (81% vs. 39%; P < .05). Transfer of splenocytes or T lymphocytes to Rag2(-/-) mice increased stone prevalence markedly (26% and 40% respectively; P < .05), whereas transfer of B cells was not appreciably cholelithogenic (13%). The adaptive immune response increased the expression of gallbladder Muc genes and accumulation of mucin gel. In addition, T cells and cholesterol monohydrate crystals induced proinflammatory gene expression in the gallbladder, which likely contributes to gallbladder dysfunction. These studies indicate that T cells are critical in murine cholesterol cholelithogenesis. Furthermore, cholesterol monohydrate crystals induce expression of proinflammatory cytokines in a T-cell-dependent fashion. Acquired immunity and inflammation are likely to be crucial factors in cholesterol gallstone pathogenesis, rather then merely the result of cholelithogenesis.

Genetic evidence that apolipoprotein E4 is not a relevant susceptibility factor for cholelithiasis in two high-risk populations

Apolipoprotein E (apoE) isoforms are genetic determinants of interindividual variations in lipid metabolism. To assess whether apoE is a genetic risk factor for cholesterol gallstone disease (GD), we analyzed apoE variants in populations from Chile and Germany, two countries with very high prevalence rates of this disease. ApoE genotypes were determined in Chilean gallstone patients (n = 117) and control subjects (n = 122) as well as in German gallstone patients (n = 184) and matched controls (n = 184). In addition, we studied apoE variants in subgroups of Chilean patients with strong differences in their susceptibility to acquire gallstones: 50 elderly subjects without gallstones in spite of well-known risk factors for this disease (gallstone-resistant) and 32 young individuals with gallstones but without risk factors (gallstone-susceptible). Furthermore, correlation analysis of apoE genotypes with cholesterol crystal formation times, biliary cholesterol saturation index (CSI), and gallstone cholesterol contents was performed in 81 cholecystectomized patients. In this study analyzing the largest sample set available, apoE4 genotype was not associated with an increased frequency of GD in either population. Moreover, in the Chilean population after adjusting for risk factors such as gender, age, body mass index, serum lipids, and glucose, the odds ratio for the association of the apoE4 allele and GD was significantly (P < 0.05) <1. Also, genotypes were not correlated with cholesterol crystal formation time, CSI, or gallstone cholesterol content. In contrast to previous smaller studies, apoE polymorphisms were not associated with susceptibility to cholesterol GD in high-risk populations.