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Supplementary Figure 3 from Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts

Supplementary Table 1 from Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts

Supplementary Figure 1 from Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts

Supplementary Figure 2 from Global Hypomethylation of Genomic DNA in Cancer-Associated Myofibroblasts

Emphysematous gastritis (EG) is a rare and potentially lethal process caused by invasive, gas-producing bacteria leading to inflammation and gas dissection of the stomach. The most common etiologic agents are Clostridium infections, but other organisms, including enterobacteria, staphylococcus, and fungi have also been identified. We report the first case of EG due to Sarcina ventriculi in a solid organ transplant recipient, who presented with epigastric pain and vomiting. The patient had a history of type 1 diabetes mellitus (DM) with recurrent episodes of ketoacidosis and systemic diabetic complications, including severe gastroparesis. CT scan studies demonstrated EG with venous air, and endoscopy showed severe gastritis and ulcerations. In the gastric biopsies, abundant Sarcina ventriculi were noted in areas of mucosal/submucosal necrosis. Antibiotic treatment was instituted at admission, and subsequent endoscopy demonstrated the disappearance of Sarcina, with some improvement of the gastric inflammation; however, the patient developed septic shock with multiorgan failure and expired. This case highlights the need to consider other infectious etiologies in transplant patients, in addition to the well-known opportunistic infections.

Highlights • Desmoid tumors are rare neoplasms with unpredictable clinical behavior. • Intra-abdominal desmoid tumors are uncommon in pregnancy. • Spontaneous regression of pregnancy-associated desmoid tumors may occur post-partum. • Desmoid tumors may be initially managed with observation alone. • Surgical resection may be required for large, symptomatic tumors., Introduction The clinical behavior of desmoid tumors can be unpredictable, particularly when they arise in the ante-partum or post-partum period. We present a case of an intra-abdominal desmoid tumor that was identified in the ante-partum period, progressed rapidly in the post-partum period, and was subsequently resected. Presentation of case The patient is a 19 year-old female who was found to have a 12 cm intra-abdominal mass on a fetal assessment ultrasound. The decision was made to observe the patient and monitor the mass for growth. However, the mass rapidly grew in the post-partum period. The patient was transferred to our institution after an exploratory laparotomy revealed a large intra-abdominal mass emanating from the small bowel mesentery. The 30 cm × 24 cm × 16 cm mass was successfully resected with negative margins, and the pathologic diagnosis of desmoid tumor was confirmed. The patient had an uncomplicated post-operative course and was discharged on post-operative day 6. Discussion The majority of pregnancy-associated desmoid tumors are in the abdominal wall, arising from the rectus abdominus muscle or from previous Cesarean section scars. These tumors may spontaneously regress in the post-partum period and therefore, patients with these tumors are often observed. Close follow-up is important so that rapid tumor progression, which may lead to unresectability, can be identified and managed appropriately. Conclusion A patient with a rare case of a giant pregnancy-associated, intra-abdominal desmoid tumor that rapidly progressed in the post-partum period and was successfully treated with surgical resection with negative margins.

Clostridium ventriculi is a rare infection in poorly controlled diabetic patients with a history of gastroparesis. We present the first documented case in a transplant recipient, who underwent a simultaneous liver kidney transplant. Computed tomography showed emphysematous gastritis, endoscopy revealed gastric necrosis, and microscopy confirmed the diagnosis. Operative intervention was high risk, given the previous liver transplant. Antibiotics and proton pump inhibitor treatment with repeat endoscopy at 4 days showed resolution of gastric necrosis and elimination of microscopic evidence of infection. Combination antibiotic and proton pump inhibitor therapy may be an effective treatment for this rare, life-threatening infection.

Introduction Transplant surgeons conventionally select against livers displaying high degrees (>30%) of macrosteatosis (MaS), out of concern for primary non-function or severe graft dysfunction. As such, there is relatively limited experience with such livers, and the natural history remains incompletely characterized. We present our experience of transplanted livers with high degrees of MaS and microsteatosis (MiS), with a focus on the histopathologic and clinical outcomes. Methods 29 cases were identified with liver biopsies available from both the donor and the corresponding liver transplant recipient. Donor liver biopsies displayed either MaS or MiS ≥ 15%, while all recipients received postoperative liver biopsies for cause. Results The mean donor MaS and MiS were 15.6% (range 0-60%) and 41.3% (7.5-97.5%) respectively. MaS decreased significantly from donor (M=15.6%) to recipient postoperative biopsies (M=0.86%), p < 0.001. Similarly, MiS decreased significantly from donor biopsies (M=41.3%) to recipient postoperative biopsies (M=1.8%), p < 0.001. At a median of 68 days postoperatively (range 4-384), full resolution of MaS and MiS was observed in 27/29 recipients. Conclusions High degrees of MaS and MiS in donor livers resolve in recipients following liver transplantation. Further insight into the mechanisms responsible could translate to therapeutic targets for treating fatty liver diseases. This article is protected by copyright. All rights reserved.

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

The management and diagnosis of drug-induced liver injury (DILI) is often challenging, particularly when patients are taking multiple medications. We present a 29-year-old African American man who presented with jaundice and malaise after starting bupropion and doxycycline 2 weeks prior. He was found to have acute hepatocellular drug-induced liver injury with autoimmune features, and made a complete recovery with prednisone. Although bupropion and doxycycline are both known to cause liver toxicity, a closer inspection of the signature of liver injury and a review of prior related DILI cases assigns causality more to bupropion than doxycycline.

Background Liquid nitrogen endoscopic spray cryotherapy can safely and effectively eradicate high-grade dysplasia in Barrett's esophagus (BE-HGD). Long-term data on treatment success and safety are lacking. Objective To assess the long-term safety and efficacy of spray cryotherapy in patients with BE-HGD. Design Single-center, retrospective study. Setting Tertiary-care referral center. Patients A total of 32 patients with BE-HGD of any length. Intervention Patients were treated with liquid nitrogen spray cryotherapy every 8 weeks until complete eradication of HGD (CE-HGD) and intestinal metaplasia (CE-IM) was found by endoscopic biopsy. Surveillance endoscopy with biopsies was performed for at least 2 years. Main Outcome Measurements CE-HGD, CE-IM, durability of response, disease progression, and adverse events. Results CE-HGD was 100% (32/32), and CE-IM was 84% (27/32) at 2-year follow-up. At last follow-up (range 24-57 months), CE-HGD was 31/32 (97%), and CE-IM was 26/32 (81%). Recurrent HGD was found in 6 (18%), with CE-HGD in 5 after repeat treatment. One patient progressed to adenocarcinoma, downgraded to HGD after repeat cryotherapy. BE segment length ≥3 cm was associated with a higher recurrence of IM ( P = .004; odds ratio 22.6) but not HGD. No serious adverse events occurred. Stricture was seen in 3 patients (9%), all successfully dilated. Limitations Retrospective study design, small sample size. Conclusion In patients with BE-HGD, liquid nitrogen spray cryotherapy has an acceptable safety profile and success rate for eliminating HGD and IM and is associated with a low rate of recurrence or progression to cancer with long-term follow-up.

Previously, we reported that azoxymethane (AOM)-induced liver injury is robustly exacerbated in M3 muscarinic receptor (M3R)-deficient mice. We used the same mouse model to test the hypothesis that selective pharmacological modulation of M3R activity regulates the liver injury response. Initial experiments confirmed that giving a selective M3R antagonist, darifenacin, to AOM-treated mice mimicked M3R gene ablation. Compared to vehicle controls, mice treated with the M3R antagonist had reduced survival and increased liver nodularity and fibrosis. We next assessed AOM-induced liver injury in mice treated with a selective M3R agonist, pilocarpine. After pilocarpine treatment, stimulation of post-M3R signaling in the liver was evidenced by ERK and AKT activation. In contrast to the damaging effects of the M3R antagonist, administering pilocarpine to AOM-treated mice significantly attenuated hepatic stellate cell activation, collagen deposition, bile ductule proliferation, and liver fibrosis and nodularity. As anticipated from these findings, livers from pilocarpine-treated mice exhibited reduced expression of key players in fibrosis (α1 collagen, α-smooth muscle actin, TGF-β1, PGDF, TGF-β1R, PGDFR) and decreased mRNA levels for molecules that regulate extracellular matrix formation (TIMP-1, TIMP-2, MMP-2, MMP-13). Cleaved caspase-3, nitrotyrosine and BrdU immunostaining provided evidence that pilocarpine treatment reduced hepatocyte apoptosis and oxidative stress, while increasing hepatocyte proliferation. Collectively, these findings identify several downstream mechanisms whereby M3R activation ameliorates toxic liver injury. These novel observations provide a proof-of-principle that selectively stimulating M3R activation to prevent or diminish liver injury is a therapeutic strategy worthy of further investigation.

Background and Aims. EUS-guided liver biopsy is an emerging method of liver tissue acquisition which is safe and had been shown to produce excellent histological yield. There is limited data comparing the diagnostic yield of different FNA needles. We aimed to compare the diagnostic performance of four commercially available 19-gauge FNA needles. Methods. Four FNA needles and one percutaneous needle were used to perform liver biopsies on two human cadaveric livers: Cook Echotip Procore™, Olympus EZ Shot 2™, Boston Scientific Expect Slimline™, Covidien SharkCore™, and an 18-gauge percutaneous needle (TruCore™, Argon Medical Devices). Each needle obtained biopsies by three, six, and nine complete back-and-forth motions of the needle (“throw”) with a fanning technique. The combined lengths of specimen fragments and the total number of complete portal tracts (CPT) were measured by a blinded pathologist. One-way analysis of variance (ANOVA) and Bonferroni correction were used for statistical analysis. Results. A total of 52 liver biopsies were performed. The Covidien SharkCore needle had significantly greater number of CPT compared to other FNA needles. The number of “throws” did not impact the number of CPT significantly. There was no statistically significant difference in mean total specimen length between each FNA needle type. Conclusion. The Covidien SharkCore needle produced superior histological specimen by capturing more CPT, possibly due to its unique needle design.

Esophageal cancer ranks sixth in cancer death. To explore its genetic origins, we conducted exomic sequencing on 11 esophageal adenocarcinomas (EAC) and 12 esophageal squamous cell carcinomas (ESCC) from the United States. Interestingly, inactivating mutations of NOTCH1 were identified in 21% of ESCCs but not in EACs. There was a substantial disparity in the spectrum of mutations, with more indels in ESCCs, A:T>C:G transversions in EACs, and C:G>G:C transversions in ESCCs (P < 0.0001). Notably, NOTCH1 mutations were more frequent in North American ESCCs (11 of 53 cases) than in ESCCs from China (1 of 48 cases). A parallel analysis found that most mutations in EACs were already present in matched Barrett esophagus. These discoveries highlight key genetic differences between EACs and ESCCs and between American and Chinese ESCCs, and suggest that NOTCH1 is a tumor suppressor gene in the esophagus. Finally, we provide a genetic basis for the evolution of EACs from Barrett esophagus. Significance: This is the first genome-wide study of mutations in esophageal cancer. It identifies key genetic differences between EACs and ESCCs including general mutation spectra and NOTCH1 loss-of-function mutations specific to ESCCs, shows geographic disparities between North American and Chinese ESCCs, and shows that most mutations in EACs are already present in matched Barrett esophagus. Cancer Discov; 2(10); 899–905. ©2012 AACR. Read the Commentary on this article by Collisson and Cho, p. 870. This article is highlighted in the In This Issue feature, p. 857.

Survivin, a member of the IAP (inhibitor of apoptosis protein) family, plays important roles in maintaining cellular homoeostasis and regulating cell-cycle progression. This IAP is overexpressed in oesophageal cancer cells, leading to uncontrolled cell growth and resistance to apoptosis. CUG-BP1 (CUG-binding protein 1) is an RNA-binding protein that regulates the stability and translational efficiency of target mRNAs. In the present paper, we report that CUG-BP1 is overexpressed in oesophageal cancer cell lines and human oesophageal cancer specimens. CUG-BP1 associates with the 3′-untranslated region of survivin mRNA, thereby stabilizing the transcript and elevating its expression in oesophageal cancer cells. Our results show that overexpression of CUG-BP1 in oesophageal epithelial cells results in increased survivin mRNA stability and consequently survivin protein expression. Conversely, silencing CUG-BP1 in oesophageal cancer cells destabilizes survivin mRNA, lowering the level of survivin protein. In addition, we have found that altering CUG-BP1 expression modulates susceptibility to chemotherapy-induced apoptosis. Overexpression of CUG-BP1 in oesophageal epithelial cells increases resistance to apoptosis, whereas silencing CUG-BP1 makes oesophageal cancer cells more susceptible to chemotherapy-induced apoptosis. Co-transfection experiments with small interfering RNA directed against survivin suggest that the anti-apoptotic role for CUG-BP1 is not entirely dependent on its effect on survivin expression.

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6–1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7–1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38–56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

Refractory celiac disease (RCD) is a rare but life-threatening complication of celiac disease (CD), and only 1 pediatric case has been reported. We report a case of a 14-year-old girl with CD presenting with persistent symptoms and positive tissue celiac-specific antibodies despite a gluten-free diet. Push enteroscopy showed jejunal scalloping and partial villous atrophy on histology. She was diagnosed with RCD and treated with infliximab with subsequent complete serological and histological remission.

Adenomatoid tumor of the male genitourinary tract is a rare benign neoplasm thought to be of mesothelial origin. In exceptional cases, these lesions may involve the testicular parenchyma, of which there are only 9 published cases in the literature. The authors describe a rare case of a testicular tumor in a 41-year-old male with normal tumor markers. Histopathology and immunohistochemical studies revealed an adenomatoid tumor with intratesticular growth. No involvement of the epididymis or testicular membranes was identified. The morphological clues leading to the correct diagnosis of adenomatoid tumor and the possible histogenesis and differential diagnosis are discussed.

The purpose of this study was to characterize changes in hepatic venous pressures in patients with chronic hepatitis C. The histology and laboratory data from patients with chronic hepatitis C who underwent a transjugular liver biopsy (TJLB) and hepatic venous pressure gradient measurement were analyzed. Portal hypertension was defined as hepatic venous pressure gradient ≥6 mm Hg. A single pathologist masked to hepatic venous pressure gradient scored liver sections for inflammation and fibrosis. The patients with high-grade inflammation (relative risk [RR] 2.82, P = 0.027, multivariate analysis) and late-stage fibrosis (RR 2.81, P = 0.022) were more likely to have a hepatic venous pressure gradient ≥6 mm Hg, while the patients on dialysis (RR 0.32, P = 0.01) were less likely to have a hepatic venous pressure gradient ≥6 mm Hg. The patients on dialysis (n = 58) had an elevated serum blood urea nitrogen and creatinine when compared with those who were not (n = 75) (47.6 ± 3.3 and 7.98 ± 0.4 vs. 25.9 ± 2.0 and 1.66 ± 0.22 mg/dL, respectively; P < 0.001). While the hepatic venous pressure gradient increased with the rising levels of liver fibrosis in the latter group (P < 0.01), it did not change in the patients on dialysis (P = 0.41). The median hepatic venous pressure gradient was especially low in late-stage fibrosis patients on dialysis when compared with the latter group (5 vs. 10 mm Hg, P = 0.017). In patients on dialysis, serum transaminases were low across all levels of fibrosis. Twenty-three of the 92 patients with early fibrosis had a hepatic venous pressure gradient ≥6 mm Hg. In patients with chronic hepatitis C, concomitant TJLB and hepatic venous pressure gradient measurement identify those who have early fibrosis and portal hypertension. Long-term hemodialysis may reduce portal pressure in these patients.

Global hypomethylation has long been recognized as a feature of the malignant epithelial component in human carcinomas. Here we show evidence for this same type of epigenetic alteration in cancer-associated stromal myofibroblasts. We used methylation-sensitive SNP array analysis (MSNP) to profile DNA methylation in early-passage cultures of stromal myofibroblasts isolated from human gastric cancers. The MSNP data indicated widespread hypomethylation in these cells, with rare focal gains of methylation, conclusions that were independently validated by bisulfite sequencing and by a methylation-sensitive cytosine incorporation assay. Immunohistochemistry with anti–5-methylcytosine (anti–5-methyl-C) in a series of gastrectomy specimens showed frequent loss of methylation in nuclei of both the malignant epithelial cells and α-smooth muscle actin (ASMA)–positive stromal myofibroblasts of both intestinal-type and diffuse carcinomas. We confirmed this phenomenon and established its onset at the stage of noninvasive dysplastic lesions by immunohistochemistry for anti–5-methyl-C in a transgenic mouse model of multistage gastric carcinogenesis. These findings indicate similar general classes of epigenetic alterations in carcinoma cells and their accompanying reactive stromal cells and add to accumulating evidence for biological differences between normal and cancer-associated myofibroblasts. [Cancer Res 2008;68(23):9900–8]

An asymptomatic 70-year-old Hispanic woman with type 2 diabetes was found in 2004 to have an AST of 132 U/L, ALT 146 U/L, alkaline phosphatase 1107 U/L, total serum bilirubin 3.5 mg/dL, and albumin 2.9 g/dL. Viral hepatitis testing was negative. Serum IgG, IgA, and IgM were all elevated, antimitochondrial antibody was weakly positive, and antinuclear antibody was negative. Liver biopsy was reported to show "evolving cirrhosis with marked lymphoid hyperplasia." Although the indication was nowhere stated, she was prescribed ursodeoxycholic acid 500 mg b.i.d, on which her biochemical tests initially improved. One year later she developed itching and jaundice. Imaging studies revealed multiple gallstones. An MRCP was suggestive of cirrhosis with a questionable common bile duct stricture, and she underwent ERCP with removal of gallbladder and common bile duct stones and placement of a biliary stent. A periampullary mass, which proved to be a somatostatinoma, was excised in 2006 via an open laparotomy, at which the stent was removed and a second liver biopsy performed. It was reported as showing chronic active hepatitis, activity stage 2, and fibrosis grade 3 with bridging. Her subsequent course was complicated by recurrent bleeding from small bowel arteriovenous malformations. Seen for the first time at Columbia University Medical Center in January 2007, she complained of continuing pruritus. AST was 69 U/L, ALT 43 U/L, alkaline phosphatase 491 U/L, and total bilirubin 3.3 mg/dL. Serum albumin was 2.6 g/dL. Antinuclear antibodies, negative in 2004, were now positive at 1:320, and antimitochondrial M2 antibodies were strongly positive. Serum IgG and IgA, but NOT IgM, were elevated. Review of her outside liver biopsies revealed features of primary biliary cirrhosis (PBC) in the first, and of both PBC and autoimmune hepatitis (AIH) in the second. The patient exhibits an overlap syndrome, in which both histologic and serologic features of AIH evolved in a setting initially most suggestive of PBC alone. The phenomenon of autoimmune overlap syndromes is discussed.

Cholinergic nervous system regulates liver injury. However, the role of M1 muscarinic receptors (M1R) in modulating chronic liver injury is uncertain. To address this gap in knowledge we treated M1R-deficient and WT mice with azoxymethane (AOM) for six weeks and assessed liver injury responses 14 weeks after the last dose of AOM. Compared to AOM-treated WT mice, M1R-deficient mice had attenuated liver nodularity, fibrosis and ductular proliferation, α-SMA staining and expression of α1 collagen, Tgfβ-R, Pdgf-R, Mmp-2, Timp-1 and Timp-2. In hepatocytes, these findings were associated with reductions of cleaved caspase-3 staining and Tnf-α expression. In response to AOM treatment, M1R-deficient mice mounted a vigorous anti-oxidant response by upregulating Gclc and Nqo1 expression and attenuating peroxynitrite generation. M1R-deficient mouse livers had increased expression of Trail-R2, a promotor of stellate cell apoptosis; dual staining for TUNNEL and α-SMA revealed increased stellate cells apoptosis in livers from M1R-deficient mice compared to those from WT. Finally, pharmacological inhibition of M1R reduced H2O2-induced hepatocyte apoptosis in vitro. These results indicate that following liver injury, anti-oxidant response in M1R-deficient mice attenuates hepatocyte apoptosis and reduces stellate cell activation, thereby diminishing fibrosis. Therefore, targeting M1R expression and activation in chronic liver injury may provide therapeutic benefit.

Gastrointestinal stromal tumors (GISTs) account for approximately 2% of gastric cancers and can be challenging to diagnose due to the difficulty sampling tissue from these lesions. Current biopsy methods are inadequate for determining the mitotic index, a major prognostic factor of these tumors. We sought to evaluate the ability of a prototype submucosal biopsy forceps to make a histologic diagnosis and mitotic index determination in cases of resected gastric GISTs. After obtaining informed consent and surgical resection of three gastric GISTs, an investigational submucosal biopsy forceps and sheath was passed under direct visualization into the central portion of the tumors (in the ex vivo setting) and biopsies were obtained. The tumor was then processed for standard histology. A gastrointestinal pathologist, blinded to the mitotic index of the full specimen, evaluated the research biopsies. The results from the submucosal forceps biopsies were then compared with the standard histology results. n all 3 cases, the submucosal forceps biopsies confirmed the diagnosis of a GIST. In two out of three cases, the submucosal forceps biopsies accurately classified the risk for progressive disease (based on the mitotic index) when compared to standard histology. In one case, the research biopsy mitotic index would have resulted in an underestimation of tumor risk (biopsies categorized the lesion as “very low” rather than “moderate” risk).

The role of muscarinic receptor subtypes in modulating acute liver injury is unknown. We detected M1 muscarinic receptor (M1R) expression in human and murine hepatocytes, and investigated the consequences of M1R deficiency on acute liver injury in vivo and inhibiting M1R activation on hepatocyte injury in vitro. Age-matched wild-type (WT) and M1R-deficient (Chrm1(-/-)) male mice were injected intraperitoneally with 200mg/kg acetaminophen (APAP) and euthanized 0, 2, 4, 16, 24, and 36h later. Biochemical and histological parameters indicated that liver injury peaked within 16h after APAP treatment and resolved by 24h. Compared to WT, M1R-deficient mice had reduced intrahepatic hemorrhage and hepatocyte necrosis, reflected by an attenuated rise in serum alanine aminotransferase levels. Livers of M1R-deficient mice showed reduced hepatocyte DNA fragmentation and attenuated expression of injury cytokines (Il-1α, Il-1β, Il-6, and Fasl). In all mice hepatic glutathione levels decreased after APAP injection, but they recovered more quickly in M1R-deficient mice. During the course of APAP-induced liver injury in M1R-deficient compared to WT mice, hepatic Nrf-2, Gclc, and Nqo1 expressions increased and nitrotyrosine generation decreased. APAP metabolic pathways were not altered by M1R deficiency; expression of hepatic Cyp2e1, Cyp1a2, Cyp3a11, Cyp3a13, Car, and Pxr was similar in Chrm1(-/-) and WT mice. Finally, treatment of murine AML12 hepatocytes with a novel M1R antagonist, VU0255035, attenuated H2O2-induced oxidative stress, prevented GSH depletion, and enhanced viability. We conclude that M1R modify hepatocyte responses to oxidative stress and that targeting M1R has therapeutic potential for toxic liver injury.

The differential diagnoses for anterior wall vaginal prolapse (AWVP) include cystocele, enterocele, urethral diverticulum, and Gartner duct cyst. We present a case of a patient with a known solitary right kidney (congenital absence of a left kidney) presenting with lower urinary tract symptoms, absence of urinary incontinence, and feeling of bulge in the vagina. Physical examination revealed grade II AWVP. Because congenital solitary kidney can be associated with other possible genitourinary abnormalities, a pelvic magnetic resonance imaging was obtained. Magnetic resonance imaging demonstrated a tubular structure spanning the left retroperitoneum to the region of the AWVP. Urodynamics revealed an obstructive voiding pattern during pressure-flow phase. The combination of transvaginal and transabdominal surgical excision of the tubular structure resolved the patient's lower urinary tract and prolapse symptoms. Anatomically, her AWVP was corrected. Histopathologic examination of the tubular structure revealed presence of urothelium lining the lumen of the tubular structure consistent with a ureter. This case represents the rare situation in which an ectopic ureter presented as an AWVP. However, patients with congenital solitary kidney presenting with vaginal prolapse should raise the suspicion for other associated genitourinary anomalies, whether these anomalies are related to the prolapse or not. Cross-sectional imaging should be performed in these situations to delineate precise anatomy.

A 29-year-old woman with acute iliofemorocaval thrombosis was discovered to have suprarenal caval agenesis with azygous continuation, hepatic congestion, and fibrosis as a result of chronic Budd–Chiari syndrome. Three staged procedures were performed: pharmacomechanical thrombolysis of acute thromboses, transfemoral liver biopsy and hemodynamic assessment, and percutaneous endovascular creation of a "neocava" lined with endografts. Symptomatic improvement and patency were maintained at 12-week follow-up.

Green tea (Camellia sinensis)-associated hepatotoxicity is reported. However, the presence of alpha-1 antitrypsin MZ phenotype as a predisposing factor to green tea-associated drug-induced liver injury (DILI) is unknown. A previously healthy woman with alpha-1 antitrypsin MZ phenotype who took SlimQuick™, an herbal supplement containing green tea extract, developed severe hepatotoxicity requiring corticosteroid treatment. Green tea-associated hepatotoxicity is reviewed and alpha-1 antitrypsin MZ phenotype as a predisposing factor to green tea-associated DILI is discussed. Liver biopsy demonstrated marked inflammation with necrosis suggestive of toxic injury with diffuse alpha-1 antitrypsin globule deposition on immunostaining. Corticosteroid therapy resulted in rapid clinical improvement. Alpha-1 antitrypsin MZ phenotype may increase vulnerability to herbal hepatotoxicity.

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by65 and17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

Helicobacter pylori (H. pylori) is a bacterial pathogen that resides at the gastric mucosa and has a world-wide prevalence of over 50%. Infection usually lasts for the life of the host, and although all infected individuals will develop histologic gastritis only a subset will develop symptomatic gastritis, peptic ulcer disease, gastric MALT lymphoma, or gastric adenocarcinoma. The bacterial and host factors that determine clinical outcome and influence the development of widely varying diseases have not been elucidated. We compared disease in Helicobacter-infected severe combined immunodeficient (SCID) mice on different genetic backgrounds with their corresponding immunocompetent partners to determine if the genetics of the host significantly impacts the innate inflammatory outcome, independent of variations in bacterial virulence factors. BALB/c SCID and C57BL/6 SCID mice developed equivalent histologic gastritis by 8 weeks of infection. Immunocompetent BALB/c mice and C57BL/6 mice developed significantly lower or higher degrees of inflammation respectively. Innate inflammation in immunodeficient mice on the C57BL/6 background remained low even in the absence of the regulatory cytokine IL-10. These results demonstrate that adaptive immunity is not required for the generation of low level inflammation in response to Helicobacter infection and that the degree of inflammation is consistent among different genetic backgrounds. Additionally, this inflammation is limited even in the absence of regulatory T cells.

Interleukin (IL)-8-driven neutrophil infiltration of the gastric mucosa is pathognomonic of persistent Helicobacter pylori infection. Our prior study showed that ectopic over-expression of MUC1 in human AGS gastric epithelial cells reduced H. pylori-stimulated IL-8 production compared with cells expressing MUC1 endogenously. Conversely, Muc1 knockout (Muc1-/-) mice displayed an increased level of transcripts encoding the keratinocyte chemoattractant (KC), the murine equivalent of human IL-8, in gastric mucosa compared with Muc1(+/+) mice during experimental H. pylori infection. The current study tested the hypothesis that a decreased IL-8 level observed following MUC1 over-expression is mediated through the ability of MUC1 to associate with β-catenin, thereby inhibiting H. pylori-induced β-catenin nuclear translocation. Increased neutrophil infiltration of the gastric mucosa of H. pylori-infected Muc1(-/-) mice was observed compared with Muc1(+/+) wild type littermates, thus defining the functional consequences of increased KC expression in the Muc1-null animals. Protein co-immunoprecipitation (coIP) studies using lysates of untreated or H. pylori-treated AGS cells demonstrated that (a) MUC1 formed a coIP complex with β-catenin and CagA, (b) MUC1 over-expression reduced CagA/β-catenin coIP, and (c) in the absence of MUC1 over-expression, H. pylori infection increased the nuclear level of β-catenin, (d) whereas MUC1 over-expression decreased bacteria-driven β-catenin nuclear localization. These results suggest that manipulation of MUC1 expression in gastric epithelia may be an effective therapeutic strategy to inhibit H. pylori-dependent IL-8 production, neutrophil infiltration, and stomach inflammation.

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor–positive (ER+) MCF7 breast cancer cells and progesterone receptor–positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA–protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER−/PR− cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER−/PR− cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER−/PR− tumors had elevated levels of ALT NHEJ and reduced levels of DNA–PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. Mol Cancer Res; 10(1); 96–107. ©2011 AACR.

Objective The receptor for advanced glycation end products (RAGE) is expressed at high levels in the lung, particularly in type 1 alveolar cells, and has been shown to amplify injury triggered by acute stress. Previous studies suggest serum concentrations of soluble RAGE increase during pulmonary reperfusion injury after transplantation. RAGE blockade has been shown to suppress hepatic and cardiac ischemia and reperfusion injury in mice. Thus we tested the hypothesis that RAGE mediates tissue-injury mechanisms in ischemia and reperfusion injury in the lung. Methods C57BL/6 mice were subjected to 30 minutes of pulmonary ischemia by clamping the left hilum, followed by 60 minutes of reperfusion. Lung function was assessed by means of blood gas analysis, and capillary leak was assessed by injecting fluorescein isothiocyanate–labeled albumin and comparing fluorescence in bronchial lavage fluid with that in serum. Histologic analysis of the lung was performed by a pathologist naive to the experimental conditions. Results In animals subjected to RAGE blockade, significant increases in P o 2 (108 vs 73 mm Hg, P = .0094) and more than 3-fold decrease in capillary leak Relative Fluorescent Units (RFU, 6.12 vs 1.75; P = .001) were observed. Histologic examination revealed significant injury reduction in soluble RAGE-treated animals versus control animals. RAGE knockout mice exhibited a protected phenotype when exposed to pulmonary ischemia and reperfusion. Additionally, interleukin 8 production and nuclear factor κB activation were increased in control mice. Conclusion Abrogation of RAGE signaling attenuates pulmonary ischemia and reperfusion injury. This study suggests that RAGE might play a central role in pulmonary reperfusion injury and in transplantation and that blockade of RAGE might offer a potential target to abrogate pulmonary reperfusion injury in clinical transplantation.

BACKGROUND Although the majority of melanomas demonstrate high rates of mutations in B-RAF or N-RAS that result in constitutive activation of the mitogen-activated protein kinase-signaling pathway, emerging data suggest molecular differences among melanoma subtypes. In this study, the authors evaluated the contribution of B-RAF and N-RAS mutations to the pathogenesis of Spitzoid melanomas. METHODS In total, 33 Spitzoid melanomas were analyzed for clinical and pathologic characteristics as well as for hot-spot mutations in the B-RAF and N-RAS genes. In the majority of patients (28 of 33 melanomas), the tumors were confined to the skin with no evidence of metastasis (average follow-up, 32.5 mos). There were five metastasizing melanomas (5 of 33 tumors) with regional or systemic spread. RESULTS Of 33 Spitzoid melanomas, only 1 showed the V600E mutation in the B-RAF gene (1 of 33 tumors; 3%). It was noteworthy that none of the metastatic Spitzoid melanomas (0 of 5 tumors; 0%), of which 2 resulted in fatal outcomes, demonstrated mutations in B-RAF or N-RAS. CONCLUSIONS In contrast to the majority of cutaneous melanomas, activating hot-spot mutations in B-RAF or N-RAS were not involved in the pathogenesis of Spitzoid melanoma. These data suggested that Spitzoid melanoma is a distinct form of melanoma with unknown genes and/or signaling pathways involved in its development. Cancer 2006. © 2006 American Cancer Society.