Skin advanced glycation end-products as indicators of the metabolic profile in diabetes mellitus: correlations with glycemic control, liver phenotypes and metabolic biomarkers.

INTRODUCTION: The production of advanced glycation end-products (AGEs) is a key pathomechanism related to the complications of diabetes mellitus. The measurement of HbA1c as one of the AGEs is widely used in the clinic, but also other proteins undergo glycation in the course of diabetes. Here, we measure skin AGEs (SAGEs) in patients with diabetes type 1 (DM1) and type 2 (DM2) and correlate them with metabolic markers as well as non-invasively measured liver fibrosis and steatosis. PATIENTS AND METHODS: In this cross-sectional study, a total of 64 patients with either DM1 or DM2 and 28 healthy controls were recruited. SAGEs were measured using autofluorescence (AGE Reader). Liver fibrosis and steatosis were quantified using transient elastography, which determines liver stiffness measurement (LSM) and controlled attenuation parameter (CAP). FGF19, FGF21 and GDF-15 were measured in blood samples using ELISA. RESULTS: SAGEs were elevated in both groups of patients with diabetes as compared to healthy controls (both p < 0.001) and were higher in patients with DM2 in comparison to DM1 (p = 0.006). SAGEs correlated positively with HbA1c (r = 0.404, p < 0.001), CAP (r = 0.260, p = 0.016) and LSM (r = 0.356, p < 0.001), and negatively with insulin growth factor binding protein 3 (p < 0.001). We also detected a positive correlation between GDF15 and SAGEs (r = 0.469, p < 0.001). CONCLUSIONS: SAGEs are significantly elevated in patients with both DM types 1 and 2 and correlate with metabolic markers, including HbA1c and GDF15. They might also help to detect patients with advanced liver injury in the setting of diabetes.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Fibroblast Growth Factor 21 Response in a Preclinical Alcohol Model of Acute-on-Chronic Liver Injury.

BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4-/- mice with deficiency of the hepatobiliary phospholipid transporter. METHODS: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4-/- (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. RESULTS: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. CONCLUSIONS: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.

The role of PNPLA3 (rs738409) c>g variant on histological progression of non-alcoholic fatty liver disease.

Background and Aim: The present study aims to investigate the role of genetic variability of the PNPLA3 gene (adiponectin) in predisposition to non-alcoholic fatty liver disease (NAFLD) by comparing NAFLD patients to healthy controls and to investigate any impact of the PNPLA3 genetic variability on the natural course of the disease. Materials and Methods: This cohort consisted of 174 patients with biopsy-proven NAFLD and 151 healthy controls. DNA was extracted from peripheral blood and the rs738409 C>G single nucleotide polymorphism was assessed using PCR-DNA sequencing. Results: The frequency distribution of the GG genotype was significantly higher in NAFLD patients than in controls (p=0.01). In patients with NAFLD, the GG genotype was associated with lower platelet counts (p=0.001), the presence of steatohepatitis (p=0.04) and hepatic fibrosis (p=0.016). After adjustment for age, gender, obesity, and diabetes mellitus, the GG genotype was an independent predictor of significant hepatic fibrosis (adjusted odds ratio =3.031 p=0.012). From the baseline to sequential liver biopsies, the progression of NAS in NAFLD patients was slightly higher in the GG genotype than that of CC and GG genotypes (p=0.18). Conclusion: The PNPLA3 GG genotype is a predisposing factor for the development of hepatic steatosis in NAFLD patients and related to a more severe liver disease.

Evaluation of the results of MOTAKK hepatitis C virus RNA genotyping and hepatitis delta virus external quality assessment programs during 2015-2016.

BACKGROUND/AIMS: To evaluate the HCV RNA genotyping and HDV RNA tests that are performed in molecular microbiology laboratories in Turkey as part of a national external quality assessment programme, MOTAKK (Moleküler Tanida Kalite Kontrol) (English translation: Quality control in molecular diagnostics). MATERIALS AND METHODS: Plasmas having different HCV RNA genotypes were used to prepare HCV genotype control sera. The HDV RNA main stock was prepared from patients with chronic delta hepatitis who had a significant amount of viral load detected, as per the WHO reference materials on viral load studies that were compiled for the purpose of developing HDV RNA control sera. Samples with different viral loads were prepared from this main stock by dilution. The prepared controls were delivered to the registered laboratories. The laboratories carried out the relevant tests and entered their results via the MOTAKK web page. External quality assessment (EQA) reports of the participants were uploaded to the website as well. RESULTS: In total, there were 23 participating laboratories, out of which 20 exclusively performed HCV genotyping, and 15 and 16 only performed HDV RNA in 2015 and 2016, respectively. The success rate of the results of the HCV genotype was 56-96% in 2015 and 30-95% in 2016. The tube with a 30% success rate had a recombinant type of HCV, therefore, it could not be detected in most of the laboratories. The HDV RNA results were evaluated qualitatively. Accordingly, HDV RNA detection rates of participant laboratories were 71-100% in 2015 and 50-100% in 2016. CONCLUSION: This study was the first national external quality control program in Turkey regarding HCV RNA genotyping and HDV RNA in the field of molecular microbiology, and it was implemented successfully.

HEV seroprevalence in blood donors in Turkey by two commercial total anti-HEV Ab ELISA kits.

Previous hepatitis E virus (HEV) seroprevalence studies in Turkey have shown high variabilities, leading to conflicting results. We aimed to re-evaluate HEV seroprevalence among blood donors in Turkey using the Wantai (Beijing, China) and the Dia.Pro (Milan, Italy) total anti-HEV antibody (Ab) enzyme-linked immunosorbent assay (ELISA) kits and compare their performances and to investigate the presence of HEV RNA in blood donors. Serum total anti-HEV antibodies were determined in a total of 2011 volunteer blood donor samples collected from different regions of Turkey (807 from Ankara, 243 from Kayseri, 284 from Izmir, 200 from Malatya, 200 from Kahramanmaras, and 277 from Van). HEV RNA was evaluated by a real-time polymerase chain reaction in a total of 272 anti-HEV seropositive samples. The country-wide HEV seroprevalence was calculated as 11.5% (Dia.Pro) and 12.2% (Wantai) with seropositivity rates of 12.0%-12.5% in Ankara, 7.4%-8.2% in Kayseri, 14.5%-15.5% in Malatya, 8.1%-8.8% in Izmir, 15.0%-16.0% in Kahramanmaras, and 12.6%-13.4% in Van by Dia.Pro and Wantai kits, respectively. The lowest detectable Ab concentrations were 0.16 and 0.14 units/mL WHO, for the Dia.Pro and the Wantai assays, respectively, showing no significant difference between assays. HEV RNA was not detected in any of the anti-HEV seropositive samples. Compared with previous studies, HEV was shown to have a higher overall seroprevalence in Turkey. Despite its limitation, the current study represents the most comprehensive HEV seroprevalence study in Turkey performed with two different commercial ELISA assays with high sensitivities so far. Further investigation is required to determine HEV genotypes in Turkey.

[Evaluation of 2015-2016 MOTAKK HBV DNA and HCV RNA external quality assessment national program results]. / MOTAKK HBV DNA ve HCV RNA dis kalite kontrol ulusal programi 2015-2016 sonuçlarinin degerlendirilmesi.

MOTAKK, as a national external quality control program has been launched to evaluate the molecular detection of viral infections including HBV DNA and HCV RNA in molecular microbiology diagnostic laboratories in Turkey. This program is prepared in compliance with ISO 17043:2010 (Conformity assessment general requirements for proficiency testing) standards, and aims to take the place of external quality control programs from abroad, contributing to standardization and accuracy of molecular diagnostic tests in our country. The aim of this study was to evaluate 2015 and 2016 results of the MOTAKK External Quality Control Program for HBV DNA and HCV RNA viral load . The calls were announced on the web page of MOTAKK (www.motakk.org). The quality control samples were sent to participating laboratories in 2015 and 2016. Main stocks were prepared from patients with chronic hepatitis B and C who had viral load detection with reference methods according to WHO reference materials for viral load studies to improve quality control sera. From these main stocks, samples with different viral loads were prepared from dilutions of plasma with HBV, HCV, HAV, HIV, Parvovirus B19 and CMV negative serologic markers. Quality control samples were sent to the participating laboratories along with the negative samples in the cold chain. The laboratories accomplished the related tests within 2-3 weeks and entered their results on the MOTAKK web page. These results were analysed according to ISO 13528 (Statistical methods for use in proficiency testing by interlaboratory comparison) and scoring reports were created by a software developed by MOTAKK and sent to participating labs. Each laboratory evaluated their own results in comparison with the other laboratory results, reassessed the tests via observing the distance from the mean result and the reference values. The number of laboratories participating in the HBV DNA and HCV RNA external quality control program was 70-73 in 2015-2016. Participants were able to comply with the program tools, registering, entering results and receiving the results reports without problem. In HBV panel, 72.6-89.1% and 84.7-90.3% of the participant laboratories were in 1 standard deviation (SD) in 2015-2016, respectively. In HCV panel, 70.8-89.1% and 84.7-90.3% of the participant laboratories were in 1 SD in 2015-2016, respectively. A national external quality control program for HBV DNA and HCV RNA in Turkey has been prepared for the first time with this project and implemented successfully. All the data provided in the MOTAKK external quality control program final report, compensate all the data provided by the quality control program final reports from abroad; additionally, the report allows comparison of used technologies and commercial products.

Hepatitis C virus genotype distribution in Turkey remains unchanged after a decade: performance of phylogenetic analysis of the NS5B, E1, and 5'UTR regions in genotyping efficiency.

BACKGROUND/AIMS: Hepatitis C virus (HCV) genotyping has a considerable effect on therapy. The aim was to determine the change in prevalence of HCV genotypes in Turkey during the last decade and to compare the performance of DNA sequencing of different targets in the HCV genome (NS5B, E1, and 5'UTR). MATERIALS AND METHODS: Five hundred HCV RNA-positive patients (226 males, 274 females) were included in the study. The NS5B, E1, and 5'UTR regions of the HCV genome were amplified by polymerase chain reaction (PCR) in patients where possible. Amplified PCR products were sequenced directly, and phylogenetic analysis was performed. A commonly used database, namely www.hcv.lanl.gov, was also used to determine the genotypes. RESULTS: Phylogenetic analysis of the NS5B, E1, and 5'UTR regions showed that 1b was the most frequent genotype, with percentages of 92.5%, 93.5%, and 87.7%, respectively. Genotype 1a was the second most prevalent genotype, with ratios of 6.7%, 5.6%, and 6.6%, whereas genotype 2a was detected in proportions of 0.4%, 0.2%, and 0.8%, respectively. Genotype 5 or 6 was not detected among patients. The phylogenetic analysis showed discordant results with 18 patients' genotypes for different targets. The phylogenetic analysis showed similar results with the hcv.lanl.gov database for the E1 and NS5B sequences. CONCLUSION: There has been no change in genotyping profiles of Turkey during the last decade, representing 1b as the most prevalent subtype, followed by 1a. Phylogenetic analysis of HCV indicated high performance compared with the hcv.lanl.gov database when sequences of E1 and NS5B regions were analyzed.

Free circulating nucleic acids in plasma and serum as a novel approach to the use of internal controls in real time PCR based detection.

Internal controls (ICs), are the main components of any real-time PCR based amplification methods, which are co-purified and co-amplified with the actual target. The existence of free circulating nucleic acids in plasma and serum (CNAPS) has been known for many years. The aim of this study was to verify whether CNAPS can be used as ICs in real-time PCR based detection and quantification of DNA or RNA targets in plasma and serum samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, was chosen at random as CNAPS to serve as an intrinsic internal control in two different real-time PCR based quantification models in plasma and serum. Viral loads of hepatitis B virus (HBV) DNA and hepatitis delta virus (HDV) RNA were quantified as actual targets in parallel to GAPDH as IC in a total of 519 serum or plasma samples including 21 healthy controls, 202 positive chronic hepatitis delta patients, 37 chronic hepatitis C patients, 168 chronic hepatitis B patients, 52 patients with hepatocellular carcinoma, and 39 patients with non-alcoholic steatohepatitis/non-alcoholic fatty liver disease. GAPDH levels did not show significant variance in different patient groups and yielded positive signals in all 519 patients with persistent cycle threshold (CT) values 27.85±1.57 (mean±standard deviation (SD)). Reproducibility of the GAPDH amplification in HDV RNA and HBV DNA quantifications was shown with a SD value of CT ranging from 0.42 to 2.14 (mean SD; 1.18) and 0.24 to 1.75 (mean SD; 1.03), respectively. In conclusion, the freely circulating nucleic acids can clearly be used as internal controls for real-time PCR based detection and quantification of any RNA and mainly DNA targets (pathogens) in serum or plasma and this simply excludes the compulsory external addition of any IC molecules into the reaction.

[Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies]. / Helicobacter pylori CagA antikorlarinin saptanmasi için rekombinant bir CagA proteininin üretilmesi.

At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H.pylori infections, the detection of anti-CagA antibodies done by non-invasive commercial ELISA or WB methods, is important in diagnosis. Recombinant CagA protein produced in this study could easily be used in the ELISA tests to be developed for screening anti-CagA antibodies in the patients' sera. However, since an ELISA method using this antigen has not been developed yet, its diagnostic performance could not be evaluated in this study. In conclusion, the construction of such a system for recombinant CagA antigen expression may be a pilot study for the development of our own ELISA tests in Turkey, and also will help the clinicians for the prediction of disease outcome and decision of the appropriate antimicrobial treatment by the help of anti-CagA antibody detection.

A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

BACKGROUND: Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. OBJECTIVES: Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. STUDY DESIGN: A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. RESULTS: The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. CONCLUSION: In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube.

Importance of serum SELDI-TOF-MS analysis in the diagnosis of early lung cancer.

BACKGROUND: Different methods of diagnosis have been found to be inefficient in terms of screening and early diagnosis of lung cancer. Cancer cells produce proteins whose serum levels may be elevated during the early stages of cancer development. Therefore, those proteins may be recognized as potential cancer markers. The aim of this study was to differentiate healthy individuals and lung cancer cases by analyzing their serum protein profiles and evaluate the efficacy of this method in the early diagnosis of lung cancer. MATERIALS AND METHODS: 170 patients with lung cancer, 53 under high risk of lung cancer, and 47 healthy people were included in our study. Proteomic analysis of the samples was performed with the SELDI-TOF-MS approach. RESULTS: The most discriminatory peak of the high risk group was 8141. When tree classification analysis was performed between lung cancer and the healthy control group, 11547 was determined as the most discriminatory peak, with a sensitivity of 85.5%, a specificity of 89.4%, a positive predictive value (PPV) of 96.7% and a negative predictive value (NPV) of 62.7%. CONCLUSIONS: We determined three different protein peaks 11480, 11547 and 11679 were only present in the lung cancer group. The 8141 peak was found in the high-risk group, but not in the lung cancer and control groups. These peaks may prove to be markers of lung cancer which suggests that they may be used in the early diagnosis of lung cancer.

Potential proteomic biomarkers in assessing liver fibrosis using SELDI-TOF MS.

BACKGROUND/AIMS: The accurate assessment of the severity of liver fibrosis is of paramount importance in determining treatment strategies, response to treatment and prognosis in patients with chronic liver disease. The aim of this study was to investigate potential proteomic biomarkers for assessing stages of hepatic fibrosis. METHODS: Serum samples of 83 patients with chronic liver disease (using METAVIR index, 17 F0, 30 F1, 6 F2, 9 F3, and 21 F4 patients) and 29 healthy controls were analyzed using surface-enhanced laser desorption/ionization time-of- flight mass spectrometry on IMAC30 ProteinChip arrays. Discriminatory peaks between groups were identified using Mann-Whitney U non-parametric test. Comparison of mild (F0, F1) and severe fibrosis (F2, F3, F4) was performed using tree classification (cross-validation) with the Biomarker Patterns Software, version 5.0 (Ciphergen Biosystems, US). RESULTS: No statistically significant discriminatory peak was evident between F0, F1 and F2 fibrosis. More than 30 peaks were found to be discriminatory between patients with cirrhosis (F4) and all other stages of liver fibrosis, including F2 and F3. Six surface-enhanced laser desorption/ionization proteomic features were found to be discriminative for mild (F0, F1) vs. advanced (F2, F3, F4) fibrosis (AUROC ≥0.8, p<0.05, Mann-Whitney test). The decision tree (m/z 4280, 10453 and 6376) yielded a sensitivity of 83.3% (30/36), a specificity of 85.1% (40/47), a positive predictive value of 81.1%, and a negative predictive value of 86.9%, with an AUROC of 0.94. CONCLUSIONS: The results of this study revealed discriminatory peaks between the protein profiles of patients with cirrhosis and other stages of liver fibrosis. Potential proteomic biomarkers can be notably determined for discriminating mild and advanced fibrosis using surface-enhanced laser desorption/ionization time-of- flight mass spectrometry.

Molecular characterization of hepatitis A virus isolated from acute infections in Turkey.

BACKGROUND/AIMS: Hepatitis A virus is a global public health problem, especially in developing countries, and the most common cause of hepatitis in childhood. Hepatitis A virus is a single- stranded positive RNA virus subdivided to 6 genotypes (3 human,3 simian). The aim of this study was to determine the prevalent genotype in Turkey using sera of acute hepatitis A virus-infected patients from different geographical regions of the country. MATERIALS AND METHODS: Sera of 137 patients with acute hepatitis A virus from different geographical regions were collected for phylogenetic analysis. The VP1-2A region of the hepatitis A virus genome was amplified by real-time-polymerase chain reaction in 76 patients where possible. Amplified polymerase chain reaction fragments were sequenced, and phylogenetic analysis was done together with other reference hepatitis A virus sequences obtained from GenBank database. RESULTS: Sequencing and phylogenetic analysis of the VP1-2A junction of hepatitis A virus showed that the most prevalent genotype in Turkey is IB (100%). Comparison of Turkish isolates and reference sequences of genotype IB showed a similarity of 94.9%. The same comparison was done between Turkish isolates and reference hepatitis A virus genotype IB and HM175, and it was found that similarity between them ranged from 93.0-95.9%. When Turkish isolates were compared according to Mean Percentage Nucleotide Distance analysis, similarity ranged between 95.3%-100%. CONCLUSIONS: Phylogenetic analysis pointed out that all Turkish isolates belong to genotype IB. Sequence analysis is a useful tool in revealing hepatitis A outbreaks, and allows us to detect and distinguish the presence of epidemic and small outbreaks.

Effect of Turkish propolis extracts on proteome of prostate cancer cell line.

BACKGROUND: Propolis is a natural, resinous hive product that has several pharmacological activities. Its composition varies depending on the vegetation, climate, season and environmental conditions of the area from where it was collected. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a proteomic approach which has been used in cancer proteomics studies. Prostate cancer is one of the most commonly diagnosed cancers in men. It has shown that nutritional supplements rich in polyphenolic compounds such as propolis play a significant role in prostate cancer chemoprevention. The aim of this study is to evaluate if protein expression profile in PC-3 prostate cancer cell lines could be differentiated when incubated with dimethyl sulfoxide and water extracts of Turkish propolis. RESULTS: The antioxidant potentials of dimethyl sulfoxide and water extracts of propolis were found in correlation with the amount of total phenolic compounds of them. Dimethyl sulfoxide and water extracts of propolis of 20 µg/mL reduced the cell viability to 24.5% and 17.7%, respectively. Statistically significant discriminatory peaks between control PC-3 cells and dimethyl sulfoxide extract of propolis-treated PC-3 cells were found to be the proteomic features at m/z 5143, 8703, 12661, 20184 and 32794, detected by CM10 ProteinChip, and the peak at m/z 3772, detected by Q10 ProteinChip. Between control PC-3 cells and water extract of propolis-treated PC-3 cells, statistically significant discriminatory peaks were found to be the proteomic features at m/z 15846, 16052 and 24658, detected by CM10 ProteinChip and the peaks at m/z 10348, 10899 and 11603, detected by Q10 ProteinChip. CONCLUSIONS: It was concluded that dimethyl sulfoxide and water extracts of Turkish propolis may have anti-proliferative activity through differentiating protein expression profile in PC-3 prostate cancer cell lines along with their antioxidant capacity.

Complete genome sequences and phylogenetic analysis of hepatitis delta viruses isolated from nine Turkish patients.

Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.

Serum connective tissue markers as predictors of advanced fibrosis in patients with chronic hepatitis B and D.

BACKGROUND/AIMS: Liver biopsy to assess fibrosis is invasive and prone to sampling error. While algorithms of serum markers to predict fibrosis stage have been described for chronic hepatitis C, these cannot be applied equally well to hepatitis B. METHODS: We therefore determined 9 serum fibrosis markers, liver biochemical tests and ultrasound parameters in 109 consecutive adult patients with chronic hepatitis B and D. All patients had compensated liver disease. Using the METAVIR score, advanced disease was defined as fibrosis stage ≥F2, and active inflammation as grade ≥A2. A gold standard was created considering splenomegaly and/or platelets <150,000 as indicators of advanced fibrosis irrespective of histology. Area under receiver operating characteristics curves was used for assessment of single markers and odds ratio for their combinations. RESULTS: Patients with advanced disease were older, had lower albumin, higher gamma glutamyl transferase and lower platelet. Levels of 6 of the 9 fibrosis markers, tissue inhibitor of metalloproteinases-1, procollagen type III aminoterminal propeptide, matrix metalloproteinase-2, laminin, hyaluronan and collagen IV correlated with advanced fibrosis. Markers useful for fibrosis prediction also predicted marked inflammation. Using the gold standard, age, prothrombin time, gamma glutamyl transferase and albumin were independent predictors of fibrosis with odds ratio's of 3.11, 4.18, 3.35 and 5.25, respectively. Their combined use predicted fibrosis with an odds ratio of 228.8. Tissue inhibitor of metalloproteinases-1 and hyaluronan were powerful predictors of fibrosis (Odds ratio's of 8.65 and 8.38). Their combined use revealed an odds ratio of 28.6, when compared with the gold standard. CONCLUSION: In conclusion, advanced liver fibrosis in chronic hepatitis B and D may be predicted with use of these two fibrosis markers.

Effects of polymorphism in G2677T/A triallelic region of MDR1 gene in Turkish patients with inflammatory bowel disease.

BACKGROUND/AIMS: Crohn's disease and ulcerative colitis are both chronic inflammatory disorders of the gastrointestinal tract, the main causes of which remain unknown. Crohn's disease and ulcerative colitis are characterized by cell-mediated immune response against the luminal bacteria. It is suggested that expression levels and function of P-glycoprotein, encoded by the MDR1 gene, are important for protection of the gut against xenobiotics and bacterial toxins. Therefore, the mutations of the MDR1 gene are thought to be related with the pathogenesis of inflammatory bowel disease. The aim of this study was to investigate the G2677T/A polymorphism in the MDR1 gene in Turkish patients with inflammatory bowel disease and a healthy control group. METHODS: In our study, the genotypes of endoscopically or histopathologically diagnosed Crohn's disease (n: 35; 14 F, 21 M) and ulcerative colitis (n: 82; 36 F, 46 M) patients and of 70 healthy individuals (39 F, 31 M) were compared. In the patient and control groups, polymerase chain reaction restriction fragment length polymorphism analysis was performed for two polymorphisms (G2677T and G2677A) of the MDR1 gene. RESULTS: In this study, the frequency of alleles at position 2677 of the MDR1 gene, which has a triallelic polymorphism, was not found to be significantly different between the patient and the healthy control groups. Moreover, the 2677A allele was not detected in either the patient group or the healthy control group. CONCLUSIONS: In this study, the G2677T/A polymorphism observed in the MDR1 gene was not found to be a risk factor for Crohn's disease or ulcerative colitis.