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The prevalence of cholesterol gallstone disease is increasing, primarily due to the global epidemic of obesity associated with insulin resistance, and this trend leads to a considerable healthcare, financial, and social burden worldwide. Although phospholipids play an essential role in maintaining cholesterol solubility in bile through both mixed micelles and vesicles, little attention has been paid to the impact of biliary phospholipids on the pathogenesis of cholesterol gallstone formation. A reduction or deficiency of biliary phospholipids results in a distinctly abnormal metastable physical-chemical state of bile predisposing to supersaturation with cholesterol. Changes in biliary phospholipid concentrations influence cholesterol crystallization by yielding both liquid crystalline and "anhydrous" crystalline metastable intermediates, evolving into classical parallelogram-shaped cholesterol monohydrate crystals in supersaturated bile. As a result, five distinct crystallization pathways, A-E, have been defined, mainly based on the prime habits of liquid and solid crystals in the physiological or pathophysiological cholesterol saturation of gallbladder and hepatic bile. This review concisely summarizes the chemical structures and physical-chemical properties of biliary phospholipids and their physiological functions in bile formation and cholesterol solubility in bile, as well as comprehensively discusses the latest advances in the role of biliary phospholipids in cholesterol crystallization and growth in gallstone formation, largely based on the findings from clinical and animal studies and in vitro experiments. The insights gleaned from uncovering the cholelithogenic mechanisms are expected to form a fundamental framework for investigating the hitherto elusive events in the earliest stage of cholesterol nucleation and crystallization. This may help to identify better measures for early diagnosis and prevention in susceptible subjects and effective treatment of patients with gallstones.

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Activating transcription factor (ATF)3 is known to have an anti-inflammatory function, yet the role of hepatic ATF3 in lipoprotein metabolism or atherosclerosis remains unknown. Here we show that overexpression of human ATF3 in hepatocytes reduces the development of atherosclerosis in Western-diet-fed Ldlr-/- or Apoe-/- mice, whereas hepatocyte-specific ablation of Atf3 has the opposite effect. We further show that hepatic ATF3 expression is inhibited by hydrocortisone. Mechanistically, hepatocyte ATF3 enhances high-density lipoprotein (HDL) uptake, inhibits intestinal fat and cholesterol absorption and promotes macrophage reverse cholesterol transport by inducing scavenger receptor group B type 1 (SR-BI) and repressing cholesterol 12α-hydroxylase (CYP8B1) in the liver through its interaction with p53 and hepatocyte nuclear factor 4α, respectively. Our data demonstrate that hepatocyte ATF3 is a key regulator of HDL and bile acid metabolism and atherosclerosis.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα ((−/−)) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

Summary Gateway cloning employs the use of the ccdb toxin and has low colony numbers, making it difficult to apply at scale to clone libraries of cDNA vectors. In this protocol, we describe MegaGate, a toxin-less Gateway technology capable of robust cDNA library cloning that is efficient, cheap, and scalable. MegaGate eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning and is 99.8% efficient with high colony numbers. For complete details on the use and execution of this protocol, please refer to Kramme et al. (2021)., Graphical abstract, Highlights • MegaGate is a toxin-less Gateway molecular cloning tool • MegaGate shows 99.8% cloning efficiency and high colony numbers compared with Gateway • MegaGate can clone 300+ genes into barcoded destination vectors in a single pool • MegaGate is universally compatible with existing pENTR libraries and ORF cloning, Gateway cloning employs the use of the ccdb toxin and has low colony numbers, making it difficult to apply at scale to clone libraries of cDNA vectors. In this protocol, we describe MegaGate, a toxin-less Gateway technology capable of robust cDNA library cloning that is efficient, cheap, and scalable. MegaGate eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning and is 99.8% efficient with high colony numbers.

Cholecystokinin (CCK) is an important neuro-intestinal peptide hormone produced by the enteroendocrine I-cells in the upper part of small intestine. Protein- and fat-enriched food plays an important role in triggering CCK secretion from the intestine. Carbohydrates stimulate only small amounts of CCK release. The CCK-1 receptor (CCK-1R) is largely localized in the gallbladder, sphincter of Oddi, pancreas, small intestine, gastric mucosa, and pyloric sphincter, where it is responsible for CCK to regulate multiple digestive processes including gallbladder contraction, pancreatic secretion, small intestinal transit, and gastric emptying. Accumulated evidence clearly demonstrates that CCK regulates gallbladder and small intestinal motility through CCK-1R signaling cascade and the effect of CCK-1R on small intestinal transit is a physiological response for regulating intestinal cholesterol absorption. Disruption of the Cck or the Cck-1r gene in mice significantly increases the formation of cholesterol gallstones by disrupting gallbladder emptying and biliary cholesterol metabolism, as well as promoting intestinal absorption of cholesterol. Abnormalities in gallbladder motility function in response to exogenously administered CCK are found primarily in patients with cholesterol gallstones. Patients with pigment gallstones display an intermediate degree of gallbladder motility defect without gallbladder inflammation and enlarged fasting gallbladder. Dysfunctional gallbladder contractility has been found under several conditions such as pregnancy, obesity, diabetes, celiac disease, and total parenteral nutrition although gallstones are not observed. The gallbladder-specific CCK-1R-selective agonist may lead to an efficacious novel way for preventing gallstone formation by promoting gallbladder emptying, particularly for pregnant women and subjects with dysfunctional gallbladder motility function such as celiac patients, as well as patients with total parenteral nutrition.

Background: Molecular testing (MT) refines risk stratification for thyroid nodules that are indeterminate for cancer by fine needle aspiration (FNA) cytology. Criteria for selecting nodules for MT vary and remain largely untested, raising questions about the best strategy for maximizing the usefulness of MT while minimizing the harms of overtesting. We used a unique data set to examine the effects of repeat FNA cytology-based criteria for MT on management decisions and nodule outcomes. Methods: This was a study of adults (age 25–90 years; 281 women and 72 men) with cytologically indeterminate (Bethesda III/IV) thyroid nodules who underwent repeat FNA biopsy and Afirma Gene Expression Classifier (GEC) testing (N = 363 nodules from 353 patients) between June 2013 and October 2017 at a single institution, with follow-up data collected until December 2019. Subgroup analysis was performed based on classification of repeat FNA cytology. Outcomes of GEC testing, clinical/sonographic surveillance of unresected nodules, and histopathologic diagnoses of thyroidectomies were compared between three testing approaches: (i) Reflex (MT sent on the basis of the initial Bethesda III/IV FNA), (ii) SemiRestrictive (MT sent if repeat FNA is Bethesda I–IV), and (iii) Restrictive (MT sent only if repeat FNA is Bethesda III/IV) testing approaches. Results: Restricting MT to nodules that remain Bethesda III/IV on repeat FNA would have missed 4 low-risk cancers and 3 noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) (collectively 2% of the test population) but would have avoided diagnostic surgery for 42 benign nodules (12% of the test population). The Restrictive testing strategy was more specific (delta 0.126 confidence interval [CI 0.093 to 0.159] and 0.129 [CI 0.097 to 0.161], respectively) but less sensitive (delta −0.339 [CI −0.424 to −0.253] and −0.340 [CI −0.425 to −0.255], respectively) than the Reflex and SemiRestrictive approaches for detecting NIFTP or cancer. Conclusions: Repeat FNA cytology can guide the selection of cytologically indeterminate thyroid nodules that warrant MT. The Restrictive model of performing Afirma GEC only on nodules with two separate biopsies showing Bethesda III/IV cytology would reduce the rate of diagnostic surgery for histologically benign nodules while missing only rare low-risk tumors. Given the low but nontrivial risks of thyroidectomy, the higher specificity of the Restrictive testing approach disproportionately outweighs the potential harms.

Objective Hepatic miR-34a expression is elevated in diet-induced or genetically obese mice and patients with non-alcoholic steatohepatitis (NASH), yet hepatocyte miR-34a's role in the progression of non-alcoholic fatty liver disease (NAFLD) from non-alcoholic fatty liver (NAFL) to NASH remains to be elucidated. Methods Mice overexpressing or deficient in hepatocyte miR-34a and control mice were fed a diet enriched in fats, cholesterol, and fructose (HFCF) to induce NASH. C57BL/6 mice with NASH were treated with an miR-34a inhibitor or a scramble control oligo. The effect of miR-34a on the development, progression, and reversal of NAFLD was determined. Results The hepatocyte-specific expression of miR-34a aggravated HFCF diet-induced NAFLD. In contrast, germline or adult-onset deletion of hepatocyte miR-34a attenuated the development and progression of NAFLD. In addition, pharmacological inhibition of miR-34a reversed HFCF diet-induced steatohepatitis. Mechanistically, hepatocyte miR-34a regulated the development and progression of NAFLD by inducing lipid absorption, lipogenesis, inflammation, and apoptosis but inhibiting fatty acid oxidation. Conclusions Hepatocyte miR-34a is an important regulator in the development and progression of NAFLD. MiR-34a may be a useful target for treating NAFLD., Highlights • Hepatocyte-specific overexpression of miR-34a aggravates high-fat/cholesterol/fructose (HFCF) diet-induced steatohepatitis. • Germline or adult-onset ablation of hepatocyte miR-34a protects against HFCF diet-induced steatohepatitis. • Mir-34a inhibitors reverse non-alcoholic steatohepatitis (NASH). • Mir-34a promotes non-alcoholic fatty liver (NAFL) by inducing lipid absorption and synthesis and inhibiting fatty acid oxidation. • MiR-34a promotes the progression of NAFL to NASH by inducing inflammation, lipotoxicity, and apoptosis.

With the recent advancements in genome editing, next generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop novel algorithms for target identification and modular tools for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for Sequencing-based Target Ascertainment and Modular Perturbation Screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.

The cholecystokinin A receptor (CCKAR) is expressed predominantly in the gallbladder and small intestine in the digestive system, where it is responsible for CCK’s regulation of gallbladder and small intestinal motility. The effect of CCKAR on small intestinal transit is a physiological response for regulating intestinal cholesterol absorption. The Cckar gene has been identified to be an important gallstone gene, Lith13, in inbred mice by a powerful quantitative trait locus analysis. Knockout of the Cckar gene in mice enhances cholesterol cholelithogenesis by impairing gallbladder contraction and emptying, promoting cholesterol crystallization and crystal growth, and increasing intestinal cholesterol absorption. Clinical and epidemiological studies have demonstrated that several variants in the CCKAR gene are associated with increased prevalence of cholesterol cholelithiasis in humans. Dysfunctional gallbladder emptying in response to exogenously administered CCK-8 is often found in patients with cholesterol gallstones, and patients with pigment gallstones display an intermediate degree of gallbladder motility defect. Gallbladder hypomotility is also revealed in some subjects without gallstones under several conditions: pregnancy, total parenteral nutrition, celiac disease, oral contraceptives and conjugated estrogens, obesity, diabetes, the metabolic syndrome, and administration of CCKAR antagonists. The physical–chemical, genetic, and molecular studies of Lith13 show that dysfunctional CCKAR enhances susceptibility to cholesterol gallstones through two primary mechanisms: impaired gallbladder emptying is a key risk factor for the development of gallbladder hypomotility, biliary sludge (the precursor of gallstones), and microlithiasis, as well as delayed small intestinal transit augments cholesterol absorption as a major source for the hepatic hypersecretion of biliary cholesterol and for the accumulation of excess cholesterol in the gallbladder wall that further worsens impaired gallbladder motor function. If these two defects in the gallbladder and small intestine could be prevented by the potent CCKAR agonists, the risk of developing cholesterol gallstones could be dramatically reduced.

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.

Endoscopic mucosal biopsies of the ampulla of Vater (AmpBx) are obtained to histologically assess for dysplasia or carcinoma. However, biopsy material is often scant and a host of factors can induce histologic changes that pose diagnostic challenges. We sought to investigate observer variability in interpretation of AmpBx and the impact clinical data may have on diagnostic interpretation. Thirty-one cases from institutional archives were selected, including 12 cases of reactive atypia (RA), 8 indefinite for dysplasia (ID), and 11 showing low-grade dysplasia (LGD). Slides were independently reviewed at 3 time points with and without clinical information by 6 pathologists who categorized the biopsies RA, ID, or LGD. Following the reviews, intraobserver and interobserver agreement was assessed. Review of AmpBx without clinical data showed fair (κ, 0.27), poor (κ, 0.07), and good (κ, 0.42) interobserver agreement for diagnoses of RA, ID, and LGD, respectively. Interobserver agreement improved for LGD (κ, 0.66 and 0.73) when clinical information was provided; however, agreement remained fair for RA (κ, 0.4 and 0.42) and poor-to-fair for ID (κ, 0.17 and 0.25). When follow-up data were reviewed, all cases that reached unanimous agreement had that diagnosis substantiated by subsequent endoscopic or histologic findings. The same was true of 13 of 19 cases that reached majority consensus. Given the potential clinical consequences of these diagnoses combined with the significant intraobserver and interobserver variability found in this study, we conclude that better-defined diagnostic criteria and consensus reads on difficult cases would assist in the histologic assessment of these challenging cases.

Summary With the recent advancements in genome editing, next-generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop unique algorithms for target identification and a toxin-less Gateway cloning tool, termed MegaGate, for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for sequencing-based target ascertainment and modular perturbation screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.

Background Celiac disease is a chronic, small intestinal, immune-mediated enteropathy caused by a permanent intolerance to dietary gluten in genetically predisposed individuals. Clinical studies have found that intestinal cholecystokinin secretion and gallbladder emptying in response to a fatty meal are impaired before celiac patients start the gluten-free diet (GFD). Design However, it was never really appreciated whether celiac disease is associated with gallstones because there were very few studies investigating the mechanism underlying the impact of celiac disease on the pathogenesis of gallstones. Results We summarize recent progress on the relationship between celiac disease and gallstones and propose that celiac disease is an important risk factor for gallstone formation because defective intestinal cholecystokinin secretion markedly increases susceptibility to cholesterol gallstones via a mechanism involving dysmotility of both the gallbladder and the small intestine. Because GFD can significantly improve the celiac enteropathy, early diagnosis and therapy in celiac patients is crucial for preventing the long-term impact of cholecystokinin deficiency on the biliary and intestinal consequences. When gluten is reintroduced, clinical and histologic relapse often occurs in celiac patients. Moreover, some of the celiac patients do not respond well to GFD. Conclusions It is imperative to routinely examine by ultrasonography whether gallbladder motility function is preserved in celiac patients and monitor whether biliary sludge (a precursor of gallstones) appears in the gallbladder, regardless of whether they are under the GFD program. To prevent gallstones in celiac patients, it is urgently needed to investigate the prevalence and pathogenesis of gallstones in these patients. This article is protected by copyright. All rights reserved.

BACKGROUND AND AIMS: Estrogen is an important risk factor for cholesterol gallstone disease because women are twice as likely as men to form gallstones. The classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in the formation of estrogen-induced gallstones in female mice. The molecular mechanisms underlying the lithogenic effect of estrogen on gallstone formation have become more complicated with the identification of G protein–coupled receptor 30 (GPR30), an estrogen receptor. APPROACH AND RESULTS: We investigated the biliary and gallstone phenotypes in ovariectomized female GPR30(−/−), ERα(−/−), and wild-type mice injected intramuscularly with the potent GPR30-selective agonist G-1 at 0 or 1 μg/day and fed a lithogenic diet for 8 weeks. The activation of GPR30 by G-1 enhanced cholelithogenesis by suppressing expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme for the classical pathway of bile salt synthesis. These metabolic abnormalities led to an increase in biliary cholesterol concentrations in company with hepatic hyposecretion of biliary bile salts, thereby inducing cholesterol-supersaturated gallbladder bile and accelerating cholesterol crystallization. G-1 also impairs gallbladder emptying, leading to sluggish gallbladder motility and promoting the development of biliary sludge in the early stage of gallstone formation. The prevalence rates of gallstones were 80% in wild-type and ERα(−/−) mice treated with G-1 compared to 10% in wild-type mice receiving no G-1. However, no gallstones were formed in GPR30(−/−) mice treated with G-1. CONCLUSIONS: GPR30 produces additional lithogenic actions, working independently of ERα, to increase susceptible to gallstone formation in female mice; both GPR30 and ERα are potential therapeutic targets for cholesterol gallstone disease, particularly in women and patients exposed to high levels of estrogen.

Background Microscopic ileitis and its association with pancolitis in adults with ulcerative colitis (UC) have been described. The incidence of ileitis and associations with colonic disease in pediatric UC have, however, not been thoroughly investigated. This study was undertaken to examine the prevalence of microscopic ileal inflammation at the time of initial diagnosis in a cohort of children with UC. Methods We reviewed colonoscopy and biopsy data at time of diagnosis from 105 children and young adults with treatment naive UC; ileal and colonic mucosal biopsies were available on all patients. Ileal mucosal biopsies were examined for the presence and severity of ileal inflammation, and other histologic features. Concurrently obtained colonic mucosal biopsies were assessed to define the severity, distribution, and extent of disease; endoscopic and clinical follow-up data were reviewed. Results A total of 107 ileal mucosal biopsies and 693 corresponding colonic mucosal biopsies were examined. Seventeen of 105 patients (16%) were found to have ileal inflammation (mean age = 10.4 years, 59% girls), 14 (82%) of whom had histologic pancolitis. The presence of ileal inflammation was significantly associated with endoscopic pancolitis (P = 0.02). The association between histologic pancolitis, severity of active inflammation in the cecum, and ascending colon suggested a possible association with ileal inflammation (P = 0.06, 0.07, and 0.08 respectively), but did not reach statistical significance. Conclusion Patients with new onset UC may have microscopic ileal inflammation at time of diagnosis, even if the terminal ileum appears macroscopically normal. The presence of endoscopic pancolitis is associated with the presence of histologic ileitis. In contrast to existing studies in adults, an association between the presence of ileitis and the histologic severity or the histologic extent of colitis was not observed. Children with microscopic ileitis in the context of UC do not need to be reclassified as "indeterminate colitis" or Crohn disease.

BACKGROUND Ulcerative colitis (UC) is a disease that is normally limited to involvement of the colon. Terminal ileitis in patients with UC with only inactive or mildly active disease has never been investigated. The aim of this prospective study was to determine the prevalence and significance of ileitis among patients with UC enrolled in an endoscopic surveillance program. METHODS The study consisted of 72 patients with UC and 90 healthy controls who underwent surveillance and screening colonoscopy, respectively. The endoscopic and histologic features of the terminal ileum (both groups) and colon (UC group only) were evaluated in a standardized fashion. Extensive clinical and endoscopic information was obtained from the subjects, and these data were compared between patients with UC either with or without ileitis. RESULTS Sixteen of 72 patients with UC (22%) had ileitis compared to only 4 of 90 (4%) of the non-UC controls (P < 0.001). None of the patients had features of backwash. Among patients with UC, the presence of ileitis showed a trend towards correlation with extent of disease, but a significant association with involvement of the colonic side of the ileocecal valve (P = 0.02) was noted. Alcohol use in the week before the colonoscopy was also significant (P = 0.02). There were no other features that were significantly related to ileitis in the patients with UC. Only one UC case with ileitis developed Crohn's disease on follow-up. CONCLUSIONS Ileitis in patients with UC may represent a primary extracolonic manifestation of UC in patients with inactive or mild disease and is not due to backwash.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide and parallels comorbidities such as obesity, metabolic syndrome, dyslipidemia, and diabetes. Recent studies describe the presence of NAFLD in non-obese individuals, with mechanisms partially independent from excessive caloric intake. Increasing evidences, in particular, point towards a close interaction between dietary and environmental factors (including food contaminants), gut, blood flow, and liver metabolism, with pathways involving intestinal permeability, the composition of gut microbiota, bacterial products, immunity, local, and systemic inflammation. These factors play a critical role in the maintenance of intestinal, liver, and metabolic homeostasis. An anomalous or imbalanced gut microbial composition may favor an increased intestinal permeability, predisposing to portal translocation of microorganisms, microbial products, and cell wall components. These components form microbial-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs), with potentials to interact in the intestine lamina propria enriched in immune cells, and in the liver at the level of the immune cells, i.e., Kupffer cells and stellate cells. The resulting inflammatory environment ultimately leads to liver fibrosis with potentials to progression towards necrotic and fibrotic changes, cirrhosis. and hepatocellular carcinoma. By contrast, measures able to modulate the composition of gut microbiota and to preserve gut vascular barrier might prevent or reverse NAFLD.

The metabolic syndrome, by definition, is not a disease but is a clustering of individual metabolic risk factors including abdominal obesity, hyperglycemia, hypertriglyceridemia, hypertension, and low high-density lipoprotein cholesterol levels. These risk factors could dramatically increase the prevalence of type 2 diabetes and cardiovascular disease. The reported prevalence of the metabolic syndrome varies, greatly depending on the definition used, gender, age, socioeconomic status, and the ethnic background of study cohorts. Clinical and epidemiological studies have clearly demonstrated that the metabolic syndrome starts with central obesity. Because the prevalence of obesity has doubly increased worldwide over the past 30 years, the prevalence of the metabolic syndrome has markedly boosted in parallel. Therefore, obesity has been recognized as the leading cause for the metabolic syndrome since it is strongly associated with all metabolic risk factors. High prevalence of the metabolic syndrome is not unique to the USA and Europe and it is also increasing in most Asian countries. Insulin resistance has elucidated most, if not all, of the pathophysiology of the metabolic syndrome because it contributes to hyperglycemia. Furthermore, a major contributor to the development of insulin resistance is an overabundance of circulating fatty acids. Plasma fatty acids are derived mainly from the triglycerides stored in adipose tissues, which are released through the action of the cyclic AMP-dependent enzyme, hormone sensitive lipase. This review summarizes the latest concepts in the definition, pathogenesis, pathophysiology, and diagnosis of the metabolic syndrome, as well as its preventive measures and therapeutic strategies in children and adolescents.

Atherosclerosis is characterized by lipid accumulation, inflammatory response, cell death and fibrosis in the arterial wall, and is a major pathological basis for ischemic coronary heart disease (CHD), which is the leading cause of morbidity and mortality in the USA and Europe. Intervention studies with statins have shown to reduce plasma LDL cholesterol concentrations and subsequently the risk of developing CHD. However, not all the aggressive statin therapy could decrease the risk of developing CHD. Many clinical and epidemiological studies have clearly demonstrated that the HDL cholesterol is inversely associated with risk of CHD and is a critical and independent component of predicting this risk. Elucidations of HDL metabolism give rise to therapeutic targets with potential to raising plasma HDL cholesterol levels, thereby reducing the risk of developing CHD. The concept of reverse cholesterol transport is based on the hypothesis that HDL displays an cardioprotective function, which is a process involved in the removal of excess cholesterol that is accumulated in the peripheral tissues (e.g., macrophages in the aortae) by HDL, transporting it to the liver for excretion into the feces via the bile. In this review, we summarize the latest advances in the role of the lymphatic route in reverse cholesterol transport, as well as the biliary and the non-biliary pathways for removal of cholesterol from the body. These studies will greatly increase the likelihood of discovering new lipid-lowering drugs, which are more effective in the prevention and therapeutic intervention of CHD that is the major cause of human death and disability worldwide.

Compelling evidence has demonstrated that estrogen is a critical risk factor for gallstone formation and enhances cholesterol cholelithogenesis through the hepatic estrogen receptor α (ERα), but not ERβ. To study the lithogenic mechanisms of estrogen through ERα, we investigated whether the deletion of Erα protects against gallstone formation in ovariectomized (OVX) female mice fed a lithogenic diet and treated with 17β-estradiol (E 2 ) at 0 or 6 μg/day for 56 days. Our results showed that the prevalence of gallstones was reduced from 100% in OVX ERα (+/+) mice to 30% in OVX ERα (−/−) mice in response to high doses of E 2 and the lithogenic diet for 56 days. Hepatic cholesterol secretion was significantly diminished in OVX ERα (−/−) mice compared to OVX ERα (+/+) mice even fed the lithogenic diet and treated with E 2 for 56 days. These alterations decreased bile lithogenicity by reducing cholesterol saturation index of gallbladder bile. Immunohistochemical studies revealed that ERα was expressed mainly in the gallbladder smooth muscle cells. High levels of E 2 impaired gallbladder emptying function mostly through the ERα and cholecystokinin-1 receptor pathway, leading to gallbladder stasis in OVX ERα (+/+) mice. By contrast, gallbladder emptying function was greatly improved in OVX ERα (−/−) mice. This markedly retarded cholesterol crystallization and the growth and agglomeration of solid cholesterol crystals into microlithiasis and stones. In conclusion, the deletion of Erα reduces susceptibility to the formation of E 2 -induced gallstones by diminishing hepatic cholesterol secretion, desaturating gallbladder bile, and improving gallbladder contraction function in female mice.

Introduction Cytologic rapid on-site evaluation (ROSE) during minimally invasive biopsy procedures is an increasingly important service provided by cytopathology to increase diagnostic yield and appropriately triage cellular material. Although ROSE can be performed by cytopathologists, cytotechnologists, or cytopathology fellows, few studies have directly compared both procedural and diagnostic outcome measures among different ROSE personnel. Materials and methods We evaluated all transthoracic computed tomography (CT)-guided lung biopsies in which ROSE was performed during a 1-year period at 2 academic institutions with similar patient populations and procedural methods: Dartmouth-Hitchcock Medical Center (DHMC) (where ROSE is performed by cytopathologists) and the Beth Israel Deaconess Medical Center (BIDMC) (where ROSE is rendered by either cytotechnologists or cytopathology fellows). Results A total of 273 CT-guided transthoracic lung biopsies (190 DHMC, 83 BIDMC) were analyzed. There was no major difference in procedure time with respect to ROSE personnel. The repeat procedure rate for nondiagnostic biopsies was similar at DHMC (cytopathologists) and BIDMC (cytotechnologists or cytology fellows) (2.1% versus 2.3%, P = 1.0). Adequacy rates for cytopathologists, cytotechnologists, and cytopathology fellows were comparable ( P = 0.23). ROSE assessments by cytopathologists were more concordant with the final diagnosis (87%) than those by cytotechnologists (82%) or cytopathology fellows (79%); this difference was not statistically significant ( P = 0.28). Conclusions ROSE procedural and diagnostic outcomes for transthoracic CT-guided lung biopsies were similar among cytopathologists, cytotechnologists, and cytopathology fellows.

Obesity is rapidly increasing and has reached epidemic features worldwide. Obesity is linked to insulin resistance, systemic low-grade inflammation and common pathogenic pathways with a number of comorbidities (including cancer), leading to high mortality rates. Besides change of lifestyles (diet and physical exercise) and pharmacological therapy, bariatric surgery is able to rapidly improve several metabolic and morphologic features associated with excessive fat storage, and currently represents an in vivo model to study the pathogenic mechanisms underlying obesity and obesity-related complications. Studies on obese subjects undergoing bariatric surgery find that the effects of surgery are not simply secondary to gastric mechanical restriction and malabsorption which induce body weight loss. In fact, some surgical procedures positively modify key pathways involving the intestine, bile acids, receptor signaling, gut microbiota, hormones and thermogenesis, leading to systemic metabolic changes. Furthermore, bariatric surgery represents a suitable model to evaluate the gene-environment interaction and some epigenetic mechanisms linking obesity and insulin resistance to metabolic diseases.

Background The Bethesda System for Reporting Thyroid Cytopathology was published in 2008 and was implemented at Beth Israel Deaconess Medical Center (BIDMC) in June, 2010. Prior to this date, our diagnostic scheme was similar to the Bethesda System, except for the category of “Atypia/Follicular Lesion of Undetermined Significance” (AUS). This study evaluates the impact of the Bethesda System on the rate and the positive predictive value (PPV) of the diagnostic categories at BIDMC. Methods We performed a retrospective review of all thyroid fine-needle aspirations (FNAs) during the time periods January, 2006 to November, 2008 and June, 2010 to July, 2011 and the subsequent thyroidectomy specimens. Results Post-Bethesda System, diagnoses that are equivocal for diverse reasons and which have wide-ranging PPVs are now all grouped into the AUS category, and the proportion of cases that are in the atypical/AUS category rose from 3.7% in the pre-Bethesda period to 12% in the post-Bethesda period. Conclusion The AUS category has a PPV approaching 50% in our lab. This creates uncertainty regarding the appropriate management for this category and may cause unnecessary overuse of molecular testing for cases in the AUS category. Diagn. Cytopathol. 2015;43:28–31. © 2014 Wiley Periodicals, Inc.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as a minimally invasive technique for evaluating the mediastinum and staging patients with lung cancer. In the hands of an experienced operator, the procedure is safe and provides excellent sensitivity, specificity, and predictive diagnostic values. In conjunction with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), a nearly complete mediastinal evaluation can be performed in a minimally invasive fashion. This strategy results in improved lymph node staging, markedly reduced need for mediastinoscopy, and fewer futile thoracotomies compared with a traditional surgical staging procedure. The procedure is cost effective and provides excellent cytologic specimens that have proven well suited for ancillary testing, such as immunohistochemistry and tumor genotyping. EBUS-TBNA, initially used as a tool to sample the lymph nodes adjacent to the airway walls, has now become instrumental in sampling lesions in the mediastinum, hilum, and lung parenchyma, where previously more than 1 procedure would have been necessary. Looking forward, expanded use of this procedure is likely to revolutionize the access to cytology-proven staging and restaging of lung cancer and other thoracic malignancies in a minimally invasive fashion.

This paper presents commentaries on the microscopic morphology of esophageal squamous epithelium; the frequency of duplication of the muscularis mucosae (MM) in Barrett's esophagus (BE); the significance of multilayered epithelium; whether cells in the lamina propria reflect those in the epithelium; how stem cells are identified in the squamous esophagus; dilated intercellular spaces; the metastasizing potential of early carcinoma-dependent, molecular or immunohistochemical tests that improve diagnosis; the role of immunohistochemistry IHC in grading of neoplasia in Barrett's esophagus and defining the risk of progression to adenocarcinoma; the roles of CDX1 and CDX2 in squamous and cardiac mucosa; and the role of desmosomal cadherins and lectins in squamous and cardiac mucosa.

Summary Esophageal symptoms are common during radiation and chemotherapy. It is unclear how often these symptoms persist after therapy. We retrospectively reviewed medical records of 320 adults treated for nonmetastatic breast cancer (84), lung cancer (109), or Hodgkin and non-Hodgkin lymphoma (127) who were disease-free at 10–14 months after therapy. Treatment included chemotherapy with or without nonmediastinal radiation therapy (150 patients), chemotherapy plus sequential mediastinal radiation therapy (MRT) (48 patients), chemotherapy plus concurrent MRT (61 patients), or non-MRT only (61 patients). Proton pump inhibitor use was documented. All treatment groups had similar prevalence of the esophageal symptom of heartburn before therapy. Rates were higher during treatment in those who received MRT with or without chemotherapy, but declined by 10–14 months after treatment. However, low baseline rates of dysphagia (4%) and odynophagia (2%) increased significantly after combined chemotherapy and MRT to 72% for dysphagia and 62% for odynophagia (P