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Background Ghrelin (GHRL), a gastric peptide encoded by the GHRL gene, is known to be involved in energy homeostasis via its G protein receptor, encoded by the growth hormone secretagogue receptor (GHSR) gene. Some studies have shown associations between plasma GHRL levels and GHRL single-nucleotide polymorphisms (SNPs), namely the Leu72Met polymorphism (rs696217 TG), with type 2 diabetes mellitus (T2DM) and insulin resistance (IR), while others have not. The controversies in these associations raise the issue of 'which SNPs in which populations.' The aim of this study was to investigate whether SNPs in GHRL and/or GHSR genes were associated with T2DM, IR, or plasma GHRL levels among Arab Saudis. Methods Blood was collected from 208 Saudi subjects with (n=107) and without (n=101) T2DM. DNA samples from these subjects were analyzed by real-time polymerase chain reaction to genotype five intronic SNPs in the GHRL (rs696217 TG, rs27647 CT, rs2075356 CT, and rs4684677 AT) and GHSR (rs509030 GC) genes. In addition, plasma GHRL levels were measured by a radioimmunoassay. Results None of the SNPs were associated with T2DM, IR, or plasma GHRL levels. The frequencies of the alleles, genotypes, and haplotypes of the five SNPs were comparable between the T2DM patients and the non-diabetic subjects. A large number of the GHRL haplotypes indicates the molecular heterogeneity of the preproghrelin gene in this region. Conclusion Neither the Leu72Met polymorphism nor the other intronic GHRL and GHSR SNPs were associated with T2DM, IR, or GHRL levels. Further investigations should be carried out to explain the molecular basis of the association of the GHRL peptide with T2DM and IR.

Background Although the exact mechanism of insulin resistance (IR) has not yet been established, IR is the hallmark characteristic of type 2 diabetes mellitus (T2DM). The aim of this study was to examine the relationship between plasma ghrelin levels and IR in Saudi subjects with T2DM. Methods Patients with T2DM (n=107, cases) and non-diabetic apparently healthy subjects (n=101, controls) from Saudi Arabia were included in this study. The biochemical profiles and plasma insulin levels of all subjects were analyzed, and IR was estimated using the homeostatic model assessment of insulin resistance (HOMA-IR) index. Active ghrelin levels in plasma were measured using the radioimmunoassay technique. Results Only 46.7% (50 of 107) of the T2DM subjects had IR, including 26% (28 of 107) with severe IR (HOMA-IR ≥5), while 5.9% (six of 101) of the controls had moderate IR (3 ≤HOMA-IR

To study the frequencies of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, and their associations with hepatocellular carcinoma (HCC) in immigrant Somalis seen at Mayo Clinic in Rochester, Minnesota.We determined the frequencies of HBV and HCV infection and HCC in immigrant Somalis seen at Mayo Clinic from July 1, 1996, through October 31, 2009. Non-Somali Olmsted County residents served as controls.For Somali males and females, age-adjusted proportions (per 1000 population) were 209 and 123 for hepatitis B surface antigen (HBsAg), 644 and 541 for hepatitis B core antibody (HBcAb), and 99 and 66 for anti-HCV. The comparative proportions in non-Somalis were 20 and 9 for HBsAg, 126 and 97 for HBcAb, and 32 and 17 for anti-HCV. Hepatitis C virus RNA confirmed that 68 of 73 Somalis (93.2%) and 261 of 282 non-Somalis (92.6%) with positive anti-HCV test results had active HCV infection. Of 30 Somali patients with HCC, 22 (73.3%) tested anti-HCV positive (odds ratio [OR], 31.3; 95% confidence interval [CI], 13.0-75.5; P.001; compared with anti-HCV-negative Somalis), 5 (16.7%) were HBsAg positive (OR, 1.4; 95% CI, 0.5-3.7; P=.53), and 18 (60.0%) were HBcAb positive (OR, 1.8; 95% CI, 0.8-4.2; P=.16). Viral hepatitis was diagnosed coincident with HCC in 9 of 20 patients (45.0%) with HCV-associated HCCs. Only 4 of 24 cases of HCC (16.7%) were detected during surveillance.Both HBV and HCV occurred frequently in this sample of Somali immigrants. However, HCV was the major risk factor for HCC. Screening Somali immigrants for HCV infection may enhance the prevention, early detection, and optimal treatment of HCC.

Elevated serum immunoglobulin G4 (sIgG4) is a feature of autoimmune pancreatitis (AIP) and IgG4-associated cholangitis (IAC); a >2-fold increase in sIgG4 is considered highly specific for these disorders. Many patients with IAC present with biliary strictures and obstructive jaundice, making cholangiocarcinoma (CCA) an important differential diagnosis. We determined the value of sIgG4 in distinguishing IAC from CCA. sIgG4 levels were measured in a test cohort of 126 CCA and 50 IAC patients. The results were confirmed in a validation cohort of 161 CCA and 47 IAC patients. Of the 126 CCA patients in the test cohort, 17 (13.5%) had elevated sIgG4 (>140 mg/dL) and four (3.2%) had a >2-fold (>280 mg/dL) increase. Primary sclerosing cholangitis (PSC) was present in 31/126 CCA patients, of whom seven (22.6%) had elevated sIgG4 and two (6.5%) had a >2-fold elevation. Of the 50 IAC patients, 39 (78.0%) had elevated sIgG4 and 25 (50.0%) had a >2-fold increase. The results in the validation cohort were consistent with those of the test cohort. Conclusion: Although elevated sIgG4 levels are characteristic of IAC, some patients with CCA, particularly with PSC, have elevated sIgG4 levels, including a small percentage with a more than a 2-fold increase in sIgG4. Therefore, sIgG4 elevation alone does not exclude the diagnosis of CCA. Depending on the prevalence of the two diagnoses, the use of a 2-fold cutoff for sIgG4 may not reliably distinguish IAC from CCA. At a cutoff of 4 times the upper limit of normal, sIgG4 is 100% specific for IAC. (HEPATOLOGY 2011;)

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide (1). Because of frequent de novo and acquired resistance of HCCs to chemotherapy, there are limited options for therapy of HCC (2, 3). There is therefore an urgent need for improved therapy of HCC. Consequently there is strong interest in identifying novel molecular targets for therapy of advanced HCC. The role of the extracellular heparan sulphate 6-O-endosulphatases, sulfatase 1 (SULF1) and sulfatase 2 (SULF2) in human carcinogenesis has not been completely elucidated (4, 5). SULF1 has been shown to function as a tumour suppressor in HCC, head and neck cancer, ovarian cancer and pancreatic cancer (5–10). SULF1 and SULF2 have also been reported to inhibit tumour growth in multiple myeloma (11). In contrast, SULF2 is upregulated in breast cancer and functions as an oncogene in HCC, pancreas cancer, lung cancer and chronic lymphocytic leukemia (12–16). Gene expression microarray analysis of 139 pairs of HCC tumour and adjacent benign tissue showed upregulation of SULF2 in 57% of HCCs (13). The 5-year survival rate for patients with HCCs with upregulated SULF2 was significantly worse than for those with down-regulated SULF2. Patients with upregulated SULF2 also had earlier recurrence of HCC after surgery. Immunohistochemical analysis of cell proliferation and apoptosis was performed in 30 of the HCCs (13). Tumours were classified into subclass A (poor prognosis) or subclass B (good prognosis) based on the prior gene expression profiling study by Lee et al. (17). Subclass A tumours were more frequent in the high SULF2 group (13 of 14 tumours) and less frequent in the low SULF2 group (two of 16 tumours; P=0.00001). HCCs with high SULF2 expression also had a significantly higher Ki-67 proliferation index (P= 0.007) and a significantly lower apoptosis index (P = 0.0001) than those with low SULF2 expression. SULF2 expression therefore correlated with increased proliferation and decreased apoptosis (13). In experiments to validate these results, we showed that SULF2 promoted proliferation and migration of HCC cells in vitro (13, 18). Mechanistically, SULF2 upregulated cell surface glypican 3 and promoted FGF signalling. Expression of SULF2 increased phosphorylation of Erk and Akt (13). SULF2 expression also increased phosphorylation of the anti-apoptotic Akt substrate GSK3β and stimulated Wnt/β-catenin signalling(19). Other investigators have also demonstrated that SULF2 promotes signalling by receptor tyrosine kinase ligands, Wnts and other growth factors (14, 20, 21). In terms of associations with other known pro-apoptotic molecules, SULF2 has been shown to be a transcriptional target of p53 in colon cancer, lung cancer, ovarian cancer and HCC cells, but the direct or indirect effects of SULF2 on apoptosis and apoptosis-related pathways in HCC have not been reported (22, 23). ERK, PI3K/Akt and JNK pathway inhibitors and histone deacetylase (HDAC) inhibitors induce apoptosis and are currently in clinical trials for cancer therapy (24–26). We studied the expression of SULF2 in HCCs and determined the role of SULF2 in modulating apoptosis induced by these kinase and HDAC inhibitors in HCC cells. The questions addressed in this study were: Is SULF2 mRNA expression correlated to protein expression in HCCs? Do changes in SULF2 expression affect cell viability, caspase activation and induction of apoptosis of HCC cells by ERK, PI3K, JNK or HDAC inhibitors? Does knockdown of SULF2 inactivate the Akt pathway? Does knockdown of SULF2 inhibit cell cycle progression as measured by cyclin D1 expression? Does SULF2 mediate its effects by regulating apoptosis-related Bcl-2, Bcl-XL and BAD protein expression?

Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/β-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. β-catenin–dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized β-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/β-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and β-catenin levels. Conclusion: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis. (HEPATOLOGY 2010;)

Background/Aims The FRA16D fragile site gene WWOX is a tumor suppressor that participates in p53-mediated apoptosis. The c-jun N-terminal kinase JNK1 interacts with WWOX and inhibits apoptosis. We investigated the function of WWOX in human hepatocellular carcinoma (HCC) and the effect of JNK inhibition on WWOX-mediated apoptosis. Methods Allelic imbalance on chromosome 16 was analyzed in 73 HCCs using 53 microsatellite markers. WWOX mRNA in HCC cell lines and primary HCCs was measured by real-time RT-PCR. Effects of WWOX on proliferation and apoptosis and the interaction between WWOX and JNK inhibition were examined. Results Loss on chromosome 16 occurred in 34 of 73 HCCs. Of 11 HCC cell lines, 2 had low, 7 intermediate, and 2 had high WWOX mRNA. Of 51 primary tumors, 23 had low WWOX mRNA. Forced expression of WWOX in SNU387 cells decreased FGF2-mediated proliferation and enhanced apoptosis induced by staurosporine and the JNK inhibitor SP600129. Conversely, knockdown of WWOX in SNU449 cells using shRNA targeting WWOX increased proliferation and resistance to SP600129-induced apoptosis. Conclusions WWOX induces apoptosis and inhibits human HCC cell growth through a mechanism enhanced by JNK inhibition.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death world-wide. 1 Risk factors for HCC include chronic hepatitis and cirrhosis secondary to hepatitis B virus or hepatitis C virus infection, alcoholic cirrhosis, nonalcoholic steatohepatitis, hemochromatosis, alpha-1-antitrypsin deficiency, and dietary aflatoxin exposure.2 Survival of patients diagnosed with advanced HCC is dismal. Only a small proportion of HCCs are detected at an early stage at which potentially curative therapies are feasible. Because of frequent de novo and acquired resistance of HCCs to chemotherapy, standard chemotherapeutic agents are not effective against HCC.3 Recent trials have proven the efficacy of the multi-targeting Raf and receptor tyrosine kinase inhibitor sorafenib in advanced HCC.4 Therefore, there is considerable interest in identifying additional novel gene targets for therapy against HCC. The recently identified human sulfatase 2 (SULF2) gene at chromosome 20q13 encodes a protein with heparin-degrading endosulfatase activity.5 Cell-surface signaling by several heparin-binding growth factors, including fibroblast growth factor (FGF), heparin-binding epidermal growth factor, hepatocyte growth factor, and vascular endothelial growth factor, requires the binding of both the growth factor and its cognate cell-surface receptor tyrosine kinase to sulfated heparan sulfate glycosaminoglycans. 6,7 For these growth factors, sulfation of particular saccharide moieties of heparan sulfate proteoglycans (HSPGs) is required for growth factor signaling. It has been shown that desulfation of HSPGs by the related protein, sulfatase 1 (SULF1), inhibits binding of the growth factor to its receptor, abrogates growth factor signaling, and has a tumor suppressor effect.8–15 In contrast to SULF1, it has been reported that SULF2 messenger RNA (mRNA) is up-regulated in human breast cancers,16 and suppression of SULF2 expression decreases tumorigenesis of pancreatic cancer cell lines.17 Thus, although SULF2 and SULF1 share the same HSPG substrate, it appears that they have different functional effects.18 This model is supported by recent evidence that human SULF2 mobilizes growth factors and chemokines from heparin and consequently facilitates growth factor signaling. 19 Because our initial studies suggested that SULF2 has an oncogenic effect, we hypothesized that SULF2 increases HCC bcell growth by release of growth factors from cell surface or extracellular matrix HSPG storage sites, thereby enhancing binding of FGF2 and other receptor tyrosine kinase ligands to their corresponding receptors and leading to cell growth. Therefore, we studied the expression of SULF2 in HCCs and examined the role of SULF2 in modulating growth of HCCs. Glypicans are a family of HSPGs that are bound to the cell surface by a lipid glycosyl-phosphatidylinositol anchor. Glypican 3 (GPC3) is markedly overexpressed in a high proportion of HCCs and promotes the growth of HCCs.20–22 We therefore also investigated the potential interactions between SULF2, FGF2 signaling, and GPC3. The following questions were addressed: Is SULF2 expression increased in HCC? Does SULF2 expression affect growth and migration of HCC cells? Does SULF2 affect FGF2 binding to the cell surface? Does SULF2 modulate expression of GPC3? Does SULF2 predict prognosis of HCC patients?

Age-related macular degeneration (AMD) is the most common cause of irreversible visual loss in the developed world. Previous studies have demonstrated that the c.1204T>C, p.Tyr402His allelic variant in the complement factor H (CFH) gene is associated with an approximately three-fold increased risk for AMD in Caucasians of predominantly European descent. Both the prevalence as well as the phenotypic spectrum of AMD varies widely among persons of different ethnicities. We hypothesized that populations with a lower prevalence of AMD might also have a lower prevalence of the CFH risk allele. In this study we sought to determine the frequency of this sequence variant in control populations of Caucasians, African Americans, Hispanics, Somalis, and Japanese. Normal control populations were assembled for each ethnic group: Caucasian (n=148), Somali (n=128), African American (n=75), Hispanic (n=81), and Japanese (n=82). Individuals were genotyped using a restriction digest assay and the frequency of the C allele at nucleotide position 1204 of the CFH gene was determined. A bioinformatic approach was used to identify SNPs in linkage disequilibrium with rs1061170 (c.1204T>C, p.Tyr402His) from the human haplotype map project database (HapMap) in order to validate the findings. We found widely discordant frequencies of the risk allele between some of the different ethnic groups: Japanese 0.07±0.02, Hispanics 0.17±0.03, African-Americans 0.35±0.04, Caucasians 0.34±0.03, and Somalis 0.34±0.03. Allele frequencies generated by analysis of the HapMap database were consistent with these findings. This study suggests that there are other yet unidentified genetic factors important in the pathogenesis of AMD that may mitigate the effects of c.1204T>C, p.Tyr402His variant. Hum Mutat 27(9), 921–925, 2006. © 2006 Wiley-Liss, Inc.

Objective It is unknown whether the Somali population in the United States is likely to participate in human papillomavirus (HPV) vaccination. We aimed to determine whether Somali girls living in a US community are following the recommendations for HPV vaccination. Materials and methods We conducted a study of HPV vaccination among Somali girls seen at Mayo Clinic, Rochester, MN. Each Somali subject was matched by year of birth to white/non-Hispanic subjects in a 1:3 ratio. We abstracted information between August 1, 2006, and December 31, 2009, related to HPV vaccine series initiation and completion. Initiation and completion frequencies were compared between study groups using the χ(2) test. Results A total of 251 Somali and 727 white/non-Hispanic girls were identified, using the Rochester Epidemiology Project, who met all inclusion criteria for final analysis. A total of 114 Somali girls (45%) and 334 white/non-Hispanic girls (46%) initiated the series (odds ratio = 0.98; 95% confidence interval = 0.73-1.31), but only 59 Somali girls (52%) completed the vaccination series, compared with 240 (72%) of the white/non-Hispanic girls (odds ratio = 0.42; 95% confidence interval = 0.27-0.65). Conclusions We found Somali girls to be generally accepting of initiating the HPV vaccine series but less likely to complete the series as compared with white non-Hispanic girls of the same age.

Sulfatase 2 (SULF2), an extracellular heparan sulphate 6-O-endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin-binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti-apoptotic Akt kinase substrate GSK3β and SULF2 expression is associated with a decreased apoptotic index in human HCCs.We investigated the functional and mechanistic effects of SULF2 on drug-induced apoptosis of HCC cells using immunohistochemistry, Western immunoblotting, gene transfection, real-time quantitative polymerase chain reaction, MTT and apoptosis assays and immunocytochemistry.The increased expression of SULF2 in human HCCs was confirmed by immunohistochemistry and immunoblotting. Treatment with inhibitors of MEK, JNK and PI3 kinases decreased the viability of SULF2-negative Hep3B HCC cells and induced apoptotic caspase 3 and 7 activity, which was most strongly induced by the PI3K inhibitor LY294002. Forced expression of SULF2 in Hep3B cells significantly decreased activity of the apoptotic caspases 3 and 7 and induced resistance to LY294002-induced apoptosis. As expected, LY294002 inhibited activation of Akt kinase by PI3K. Conversely, knockdown of SULF2 using an shRNA construct targeting the SULF2 mRNA induced profound cell growth arrest and sensitized the endogenously SULF2-expressing HCC cell lines Huh7 and SNU182 to drug-induced apoptosis. The effects of knockdown of SULF2 on HCC cells were mediated by decreased Akt phosphorylation, downregulation of cyclin D1 and the anti-apoptotic molecule Bcl-2, and upregulation of the pro-apoptotic molecule BAD.The prosurvival, anti-apoptotic effect of SULF2 in HCC is mediated through activation of the PI3K/Akt pathway.