Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study.

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

Acute acalculous cholecystitis in children.

Acute acalculous cholecystitis (AAC) is the inflammatory disease of the gallbladder in the absence of gallstones. AAC is estimated to represent at least 50% to 70% of all cases of acute cholecystitis during childhood. Although this pathology was originally described in critically ill or post-surgical patients, most pediatric cases have been observed during several infectious diseases. In addition to cases caused by bacterial and parasitic infections, most pediatric reports after 2000 described children developing AAC during viral illnesses (such as Epstein-Barr virus and hepatitis A virus infections). Moreover, some pediatric cases have been associated with several underlying chronic diseases and, in particular, with immune-mediated disorders. Here, we review the epidemiological aspects of pediatric AAC, and we discuss etiology, pathophysiology and clinical management, according to the cases reported in the medical literature.

Epithelial Inclusions in Gallbladder Specimens Mimic Parasite Infection: Histologic and Molecular Examination of Reported Cystoisospora belli Infection in Gallbladders of Immunocompetent Patients.

Recent publications have described epithelial cytoplasmic vacuoles and inclusions incidentally noted within gallbladder epithelium and concluded that they represent coccidian parasite infection, in particular, Cystoisospora belli. We identified 8 gallbladder specimens from our institution in the past 3 years in which this diagnosis was suggested or in which similar epithelial alterations were prominent. Molecular analysis was performed on the 8 gallbladder specimens and on 3 positive control specimens: small bowel biopsies from acquired immunodeficiency syndrome patients with diarrhea. Polymerase chain reaction using primers designed to amplify an internal transcribed spacer (ITS2) in the C. belli ribosomal gene cluster was performed on the DNA samples. All 8 gallbladder specimens were negative for amplification, while a product consistent with C. belli was amplified from all 3 positive controls. Histologically, the gallbladder cytoplasmic inclusions stained diffusely positive for Grocott-Gomori's methenamine silver and Periodic acid-Schiff with diastase. In contrast, sections from a positive control small bowel biopsy demonstrated organisms that were negative for Grocott-Gomori's methenamine silver and showed a distinct capsular and punctate internal staining on Periodic acid-Schiff with diastase in various parasite forms. Together, the lack of molecular evidence of C. belli and the distinct morphologic and special staining patterns in these gallbladders compared with positive control small bowel suggest that these epithelial changes do not represent true C. belli infection. Our results suggest that gallbladders of immunocompetent patients may occasionally show epithelial changes that can morphologically mimic C. belli infection. Pathologists should be aware of this histologic variant to minimize unnecessary treatment, testing, and patient anxiety.

Deciphering the landscape of host barriers to Listeria monocytogenes infection.

Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate Lmonocytogenes population dynamics during infection. We created a genetically barcoded library of murinized Lmonocytogenes and then used deep sequencing to track the pathogen's dissemination routes and quantify its founding population (Nb) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that Nb sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen's capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection.

Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-κB.

AIM: Gemcitabine has been increasingly prescribed for the treatment of gallbladder cancer. However, the response rate is low. The aim of this study is to determine whether icariin, a flavonoid isolated from Epimedi herba, could potentiate the antitumor activity of gemcitabine in gallbladder cancer. METHODS: Human gallbladder carcinoma cell lines GBC-SD and SGC-996 were tested. Cell proliferation and apoptosis were analyzed using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related molecules was detected with Western blotting. Caspase-3 activity was analyzed using colorimetric assay, and NF-κB activity was measured with ELISA. A gallbladder cancer xenograft model was established in female BALB/c (nu/nu) mice. The mice were intraperitoneally administered gemcitabine (125 mg/kg) in combination with icariin (40 mg/kg) for 2 weeks. RESULTS: Icariin (40-160 µg/mL) dose-dependently suppressed cell proliferation and induced apoptosis in both GBC-SD and SGC-996 cells, with SGC-996 cells being less sensitive to the drug. Icariin (40 µg/mL) significantly enhanced the antitumor activity of gemcitabine (0.5 µmol/L) in both GBC-SD and SGC-996 cells. The mice bearing gallbladder cancer xenograft treated with gemcitabine in combination with icariin exhibited significantly smaller tumor size than the mice treated with either drug alone. In GBC-SD cells, icariin significantly inhibited both the constitutive and gemcitabine-induced NF-κB activity, enhanced caspase-3 activity, induced G(0)-G(1) phase arrest, and suppressed the expression of Bcl-2, Bcl-xL and surviving proteins. CONCLUSION: Icariin, by suppressing NF-κB activity, exerts antitumor activity, and potentiates the antitumor activity of gemcitabine in gallbladder cancer. Combined administration of gemcitabine and icariin may offer a better therapeutic option for the patients with gallbladder cancer.

ATP-binding cassette sterol transporters are differentially expressed in normal and diseased human gallbladder.

BACKGROUND AND AIMS: Gallbladder epithelial cells (GBEC) are exposed to high cholesterol concentrations in bile, and export cholesterol via an ATP-binding cassette (ABC) transporter-mediated pathway in vitro. These findings suggest that aberrant expression and/or function of ABC sterol transporters may be associated with cholesterol-related gallbladder diseases (CAGD). In this study, we investigated the relative levels of the sterol transporters ABCA1, ABCG5, and ABCG8 in human gallbladders in CAGD, and the relationship between ABCA1 and inflammation. METHODS: Expression of ABCA1, ABCG5, and ABCG8 was evaluated in 31 gallbladders with CAGD and 6 normal gallbladders by western blotting and immunohistochemistry. RT-PCR was used to measure ABCA1 mRNA expression. To investigate the relationship between ABCA1 and inflammation, wWestern blots were performed on cultured dog GBEC treated with lipopolysaccharide (LPS) using an anti-ABCA1 antibody. RESULTS: Immunohistochemistry showed ABCA1 to be localized predominantly to the basolateral membrane, while ABCG8 formed a diffuse intracellular pattern at the apical pole of human GBEC. ABCA1 and ABCG8 expression was more prominent in GBEC that were surrounded by cholesterol-laden macrophages. ABCA1 and ABCG8 expression was increased in gallbladders with CAGD. Western blots showed increased ABCA1, ABCG5, and ABCG8 expression in CAGD. ABCA1 mRNA levels were increased in all gallbladders with CAGD. LPS treatment of cultured dog GBEC enhanced ABCA1 expression. CONCLUSIONS: The sterol transporters ABCA1, ABCG5, and ABCG8 may play a role in the pathogenesis of human CAGD. Inflammation appears to be a key factor that increases ABCA1 expression and activity in the human gallbladder.

TLR4 expression in normal gallbladder, chronic cholecystitis and gallbladder carcinoma.

BACKGROUND/AIMS: Chronic inflammation is a risk factor for gallbladder carcinoma. The molecular mechanisms linking inflammation and gallbladder carcinogenesis are incompletely understood. Toll-like receptors are involved in inflammatory response and play an important role in the innate immune system by initiating and directing immune response to pathogens. We tested the hypothesis that TLR4 participated in the development of gallbladder carcinoma through investigating the expression of TLR4 in chronic cholecystitis, gallbladder carcinoma and normal gallbladder. METHODOLOGY: The expression of TLR4 in 30 specimens of chronic calculous cholecystitis, 13 specimens of gallbladder adenocarcinoma and 10 specimens of normal gallbladder tissue was determined by immunohistochemistry, western blotting analysis and quantitative RT-PCR. RESULTS: We showed that TLR4 was mostly localized to the glandular and luminal epithelium of gallbladder. TLR4 expression was lower in gallbladder carcinoma tissue than in chronic cholecystitis and normal gallbladder tissue, whereas the difference between chronic cholecystitis tissue and normal gallbladder tissue was not statistically significant. CONCLUSIONS: The expression of TLR4 may be closely associated with the course of gallbladder carcinoma.

An immunohistochemical study of osteopontin in pigment gallstone formation.

Mucin glycoproteins from the gallbladder epithelium are thought to contribute to the matrix or nucleus of gallstones and other biomineralization systems. The involved acidic glycoproteins have been reported in bile and gallstones. In addition, osteopontin (Opn) is a noncollagenous acidic bone matrix glycoprotein that possesses calcium-binding properties. To investigate the role of Opn in pigment gallstone formation, the involvement of Opn in pigment gallstone formation was studied immunohistochemically in the gallbladder wall and in the stones. Staining for Opn was strongly positive in the epithelium of stone-laden gallbladders and in their stones. The stone-laden gallbladders were infiltrated by macrophages, which intensely stained for Opn. Sections of the pigment stones, under low magnification, showed a lamellar pattern of Opn immunolabeling and showed a reticular pattern under high magnification. Our results indicate that Opn, an acidic glycoprotein from the gallbladder epithelium, seems to be involved in lithiasis. Opn from macrophages and/or the epithelium seems to help form the matrix protein.

Autoimmune pancreatitis-related cholecystitis: a morphologically and immunologically distinctive form of lymphoplasmacytic sclerosing cholecystitis.

AIMS: Gallbladder involvement in autoimmune pancreatitis (AIP) is reported to be histologically similar to that seen in primary sclerosing cholangitis (PSC) and biliary obstruction secondary to pancreatic ductal adenocarcinoma (PDAC). The aim was to identify unique morphological and immunological features that could help distinguish gallbladders of AIP from those associated with PSC and PDACs. METHODS AND RESULTS: Archival gallbladders from well-characterized individuals with AIP (n = 22), PSC (n = 13) and PDAC (n = 23) were examined. Quantitative immunohistochemical analysis for IgG and IgG4 plasma cells was performed and an IgG4/IgG ratio was derived. Dense extramural infiltrates were almost exclusively seen in AIP cases (41%), but seen in only 4% of PDAC-associated cases and 0% of PSC cases (P = 0.001). Phlebitis was more frequently noted in AIP cases (41%) (P = 0.03). Inflammatory nodules were almost exclusively seen in AIP (27%) (P = 0.04). AIP gallbladders showed higher IgG4/IgG ratios (P = 0.0001) than PDAC-associated and PSC gallbladders. CONCLUSIONS: The findings support our hypothesis that gallbladder involvement in AIP is a primary manifestation of this disease and not a secondary phenomenon related to biliary obstruction. In conjunction with imaging and serology, examination of the gallbladder could provide collaborative evidence of AIP. Evaluation of the gallbladder could also distinguish PSC from AIP-related cholangitis.

T-cell function is critical for murine cholesterol gallstone formation.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Distribution of natural killer cells and T-lymphocyte subsets in peripheral blood, gallbladder cancer and surrounding tissue.

BACKGROUND: The patient with malignant tumor always show immunologic function drawback and ingravescent with tumor development, especially in the aspect of cell-mediated immunity. This study was undertaken to define the relationship between the immune function of local cells and cancer development by investigating the distribution of natural killer (NK) cells and T-lymphocyte subsets in peripheral blood, the cancer tissue and the tissue surrounding gallbladder carcinoma. METHODS: The numbers of CD4+ and CD8+ T-lymphocytes and NK cells were measured by flow cytometry in samples taken from gallbladder cancer tissue, the surrounding tissues and peripheral blood of 38 patients, and compared with the numbers in the peripheral blood and gallbladder tissue of 30 patients with cholecystitis as controls. RESULTS: The numbers of CD4+ and CD8+ T-cells and NK cells in gallbladder cancer tissues were significantly higher than those in the surrounding tissue and gallbladder with gallstone. However, the ratio of CD4+/CD8+ was lower in the cancer tissue than that in the surrounding tissue and tissue from gallbladders with gallstones. The distribution of CD4+ and CD8+ T-cells and NK cells in mucous membrane of cholecystitis gallbladder and that in the tissue surrounding gallbladder cancer were significantly different. CONCLUSIONS: Disproportionate and imbalanced distribution of NK cells and subsets of T-lymphocytes occurs in the mucous membrane proper of gallbladder cancer and surrounding tissue. Although gallbladder cancer tissue has higher expressions of CD4+, CD8+ and NK cells, the immune function is low or in an inhibited state. In gallbladder cancer immunization therapy, local cellular immunological function should be enhanced and the protective barrier improved.

Gallbladder wall inflammatory cells in pediatric patients with biliary dyskinesia and cholelithiasis: a pilot study.

BACKGROUND/PURPOSE: Inflammation has been implicated in functional gastrointestinal disorders, including functional dyspepsia and irritable bowel syndrome. This study was undertaken to evaluate gallbladder wall inflammatory cells in children with abdominal pain related to gallstones and biliary dyskinesia to determine the candidate cell types that may be contributing to the pathophysiology of these entities. METHODS: Gallbladder specimens from 20 patients with cholelithiasis, 20 biliary patients with dyskinesia, and 12 autopsy controls were evaluated in a blinded fashion. Eosinophil, tryptase-positive, and CD3+ cell densities were determined for the lamina propria and muscularis mucosa layers and compared between groups. RESULTS: Patients with biliary dyskinesia and cholelithiasis had a 9- to 12-fold increase in mean and peak mast cell densities, respectively, in both layers as compared with controls. Peak (13.7 vs 8.4) and mean (9.2 vs 5.2) CD3+ cell densities were increased in the muscularis mucosae of cholelithiasis specimens as compared with biliary dyskinesia specimens. CONCLUSION: Gallbladder wall inflammatory cell densities, particularly mast cells, differ between children with cholelithiasis, children with biliary dyskinesia, and controls. Future studies are warranted to define the roles for specific inflammatory cell types.

Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures.

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.

Biliary tract involvement in autoimmune pancreatitis.

OBJECTIVES: Autoimmune pancreatitis (AIP) is a unique clinical entity that has been recently proposed, and it is frequently associated with bile duct stricture. The aim of this study was to investigate the pathophysiology of the biliary tract involvement in patients with AIP. METHODS: We evaluated the clinicopathologic findings in 16 patients with AIP. Surgical resection was performed in 7 of the patients because of suspicion of a pancreatic tumor; 8 of the other patients were treated with oral prednisolone (PSL) therapy, and the remaining patient was observed clinically and not treated. The pancreas, bile duct, and gallbladder in the surgical cases were examined histologically and immunohistochemically. We also assessed the clinical manifestations and diagnostic imaging findings before and after oral PSL therapy in the 8 patients treated with PSL. RESULTS: Stricture of the extrahepatic bile duct was detected in 88% (14/16) of the patients. Thickening of the bile duct wall was detected in 94% (15/16), and thickening of the gallbladder wall was observed in 56% (9/16). Histologically, the bile duct and gallbladder wall were characterized by diffuse lymphoplasmacytic infiltration and marked interstitial fibrosis. Immunohistochemically, the diffusely infiltrating cells consisted of predominantly CD8- or CD4-positive T lymphocytes and IgG4-positive plasma cells. These findings were the same as in the inflammatory process that was observed in the pancreas. After oral PSL therapy, the pancreatic enlargement and irregular narrowing of the main pancreatic duct improved to almost their normal size in all 8 patients; however, stricture of the extrahepatic bile duct persisted in 4 of the patients (57%, 4/7) in whom it was detected before PSL therapy. CONCLUSIONS: Based on the pathophysiologic and histologic findings and the response to PSL therapy, the biliary involvement in AIP developed by the same mechanism as the pancreatitis. CD8- and CD4-positive lymphocytes and IgG4-positive plasma cells may play an important role in the pathogenesis of AIP.