N‐terminus of hMLH1 confers interaction of hMutLα and hMutLβ with hMutSα

Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSα (hMSH2‐hMSH6) and hMutLα (hMLH1‐hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSα recruits hMutLα, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLα as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSα using a novel interaction assay. We show that, although the interaction requires ATP, hMutLα does not need to hydrolyse this nucleotide to join hMutSα on DNA, suggesting that ATP hydrolysis by hMutLα happens downstream of complex formation. The analysis of the individual subunits of hMutLα demonstrated that the hMutSα–hMutLα interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N‐terminus of hMLH1 confers this interaction. In contrast, only the C‐terminus stabilised and co‐immunoprecipitated hPMS2 when both proteins were co‐expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C‐terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLβ (hMLH1–hPMS1). We show that hMutLβ interacts as efficiently with hMutSα as hMutLα, and that it predominantly binds to hMutSα via hMLH1 as well

Serum microRNA-122 predicts survival in patients with liver cirrhosis

Background: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease. Aim: Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis. Methods: 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis. Results: Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age. Conclusions: Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis

Market uptake of pegylated interferons for the treatment of hepatitis C in Europe : meeting abstract

Introduction and Objectives Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease with life threatening sequelae such as end-stage liver cirrhosis and liver cancer. It is estimated that the infection annually causes about 86,000 deaths, 1.2 million disability adjusted life years (DALYs), and ¼ of the liver transplants in the WHO European region. Presently, only antiviral drugs can prevent the progression to severe liver disease. Pegylated interferons combined with ribavirin are considered as current state-of-the-art treatment. Objective of this investigation was to assess the market uptake of these drugs across Europe in order to find out whether there is unequal access to optimised therapy. Material and Methods We used IMS launch and sales data (April 2000 to December 2005) for peginterferons and ribavirin for 21 countries of the WHO European region. Market uptake was investigated by comparing the development of country-specific sales rates. For market access analysis, we converted sales figures into numbers of treated patients and related those to country-specific hepatitis C prevalence. To convert sales figures into patient figures, the amount of active pharmaceutical ingredients (API) sold was divided by average total patient doses (ATPD), derived by a probability tree-based calculation algorithm accounting for genotype distribution, early stopping rules, body weight, unscheduled treatment stops and dose reductions Ntotal=APIPegIFNalpha-2a/ATPDPegIFNalpha-2a+APIPegIFN&alpha-2b/ATPDPegIFNalpha-2b For more concise result presentation the 21 included countries were aggregated into four categories: 1. EU founding members (1957): Belgium, France, Germany, Italy and Netherlands; 2. Countries joining EU before 2000: Austria (1995), Denmark (1973), Finland (1995), Greece (1981), Republic of Ireland (1973), Spain (1986), Sweden and UK (1973) 3. Countries joining EU after 2000: Czech Republic (2004), Hungary (2004), Poland (2004) and Romania (2007); 4. EU non-member states: Norway, Russia, Switzerland and Turkey. Results Market launch and market uptake of the investigated drugs differed considerably across countries. The earliest, most rapid and highest increases in sales rates were observed in the EU founding member states, followed by countries that joined the EU before 2000, countries that joined the EU after 2000, and EU non-member states. Most new EU member states showed a noticeable increase in sales after joining the EU. Market access analysis yielded that until end of 2005, about 308 000 patients were treated with peginterferon in the 21 countries. Treatment rates differed across Europe. The number of patients ever treated with peginterferon per 100 prevalent cases ranged from 16 in France to less than one in Romania, Poland, Greece and Russia. Discussion Peginterferon market uptake and prevalence adjusted treatment rates were found to vary considerably across 21 countries in the WHO European region suggesting unequal access to optimised therapy. Poor market access was especially common in low-resource countries. Besides budget restrictions, national surveillance and prevention policy should be considered as explanations for market access variation. Although our results allowed for the ranking of countries in order of market access, no final conclusions on over- or undertreatment can be drawn, because the number of patients who really require antiviral treatment is unknown. Further research based on pan-European decision models is recommended to determine the fraction of not yet successfully treated but treatable patients among those ever diagnosed with HCV. ..

Wie wirksam sind neue Therapien für Hepatitis C? : Mathematische Modellierung charakterisiert den individuellen Behandlungserfolg

Die derzeitige Therapie der chronischen Hepatitis C ist komplex und mit zahlreichen Nebenwirkungen assoziiert. Um den Therapieerfolg frühzeitig vorhersagen zu können und die Behandlung individuell zu optimieren, greifen Forscher des Frankfurter Leberzentrums auf mathematische Modellierung zurück. ..

Prevalence of NS5A resistance associated substitutions in patients with hepatitis C virus genotypes 1a and 3: Impact on current therapeutic strategies

[Abstract] The presence of resistance‐associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). HCV resistance at NS5A region is mainly focused on patients with hepatitis C virus (HCV) genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (ie cirrhosis, prior treatment‐exposure, or HCV‐RNA >800 000 IU/mL). Herein, we evaluated in a cohort of HCV G1a and G3 infected patients the prevalence of RASs at domain I NS5A using population‐based sequencing and the impact of RASs on the optimization of current therapeutic strategies. The RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D, and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients naïve to NS5A inhibitors were included (166 G1a, 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored, at least, one factor of poor response (78.9% for G1a, and 75.8% for G3). Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. In conclusion, testing NS5A RASs in specific HCV‐infected populations (ie G1a & G3, cirrhosis, prior treatment experienced, HCV‐RNA >800 000 IU/mL) might be useful to optimize current NS5A‐based therapies avoiding ribavirin‐related toxicities, and shortening treatment duration in the majority of patients.Instituto de Salud Carlos III; CPII14/00014Instituto de Salud Carlos III; PI10/02166Instituto de Salud Carlos III; FI14/00557Instituto de Salud Carlos III; PI13/02266Instituto de Salud Carlos III; CM15/00233Instituto de Salud Carlos III; PI15/00713Instituto de Salud Carlos III; PI16/02159Xunta de Galicia; IN606A-2016/023Comisión Interministerial de Ciencia y Tecnología (España); RD12/0017/00

Interobserver agreement of Thyroid Imaging Reporting and Data System (TIRADS) and strain elastography for the assessment of thyroid nodules

Background: Thyroid Imaging Reporting and Data System (TIRADS) was developed to improve patient management and cost-effectiveness by avoiding unnecessary fine needle aspiration biopsy (FNAB) in patients with thyroid nodules. However, its clinical use is still very limited. Strain elastography (SE) enables the determination of tissue elasticity and has shown promising results for the differentiation of thyroid nodules. Methods: The aim of the present study was to evaluate the interobserver agreement (IA) of TIRADS developed by Horvath et al. and SE. Three blinded observers independently scored stored images of TIRADS and SE in 114 thyroid nodules (114 patients). Cytology and/or histology was available for all benign (n = 99) and histology for all malignant nodules (n = 15). Results: The IA between the 3 observers was only fair for TIRADS categories 2–5 (Coheńs kappa = 0.27,p = 0.000001) and TIRADS categories 2/3 versus 4/5 (ck = 0.25,p = 0.0020). The IA was substantial for SE scores 1–4 (ck = 0.66,p<0.000001) and very good for SE scores 1/2 versus 3/4 (ck = 0.81,p<0.000001). 92–100% of patients with TIRADS-2 had benign lesions, while 28–42% with TIRADS-5 had malignant cytology/histology. The negative-predictive-value (NPV) was 92–100% for TIRADS using TIRADS-categories 4&5 and 96–98% for SE using score ES-3&4 for the diagnosis of malignancy, respectively. However, only 11–42% of nodules were in TIRADS-categories 2&3, as compared to 58–60% with ES-1&2. Conclusions: IA of TIRADS developed by Horvath et al. is only fair. TIRADS and SE have high NPV for excluding malignancy in the diagnostic work-up of thyroid nodules

Issues in selecting HCV‐infected candidates for anti‐viral treatment

Watch the interview with the authors Answer questions and earn CMEPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91215/1/18_ftp.pd

SEMS vs cSEMS in duodenal and small bowel obstruction : high risk of migration in the covered stent group

Aim: To compare clinical success and complications of uncovered self-expanding metal stents (SEMS) vs covered SEMS (cSEMS) in obstruction of the small bowel. Methods: Technical success, complications and outcome of endoscopic SEMS or cSEMS placement in tumor related obstruction of the duodenum or jejunum were retrospectively assessed. The primary end points were rates of stent migration and overgrowth. Secondary end points were the effect of concomitant biliary drainage on migration rate and overall survival. The data was analyzed according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. Results: Thirty-two SEMS were implanted in 20 patients. In all patients, endoscopic stent implantation was successful. Stent migration was observed in 9 of 16 cSEMS (56%) in comparison to 0/16 SEMS (0%) implantations (P = 0.002). Stent overgrowth did not significantly differ between the two stent types (SEMS: 3/16, 19%; cSEMS: 2/16, 13%). One cSEMS dislodged and had to be recovered from the jejunum by way of laparotomy. Time until migration between SEMS and cSEMS in patients with and without concomitant biliary stents did not significantly differ (HR = 1.530, 95%CI 0.731-6.306; P = 0.556). The mean follow-up was 57 ± 71 d (range: 1-275 d). Conclusion: SEMS and cSEMS placement is safe in small bowel tumor obstruction. However, cSEMS is accompanied with a high rate of migration in comparison to uncovered SEMS

Interferon-free antiviral combination therapies without nucleosidic polymerase inhibitors

SummaryThe establishment of robust HCV cell culture systems and characterization of the viral life cycle provided the molecular basis for highly innovative, successful years in HCV drug development. With the identification of direct-acting antiviral agents (DAAs), such as NS3/4A protease inhibitors, NS5A replication complex inhibitors, nucleotide and non-nucleoside polymerase inhibitors, as well as host cell targeting agents, novel therapeutic strategies were established and competitively entered clinical testing. The first-in-class NS3/4A protease inhibitors telaprevir and boceprevir, approved in 2011, were recently outpaced by the pan-genotypic nucleotide polymerase inhibitor sofosbuvir that in combination with pegylated interferon and ribavirin, further shortens therapy durations and also offers the first interferon-free HCV treatment option. In the challenging race towards the goal of interferon-free HCV therapies, however, several oral DAA regimens without nucleotide polymerase inhibitors that combine a NS3/4A protease inhibitor, a NS5A inhibitor and/or a non-nucleoside polymerase inhibitor yielded competitive results. Second generation NS3/4A protease and NS5A inhibitors promise an improved genotypic coverage and a high resistance barrier. Results of novel DAA combination therapies without the backbone of a nucleotide polymerase inhibitor, as well as treatment strategies involving host targeting agents are reviewed herein

Retreatment with interferon-alpha and ribavirin in primary interferon-alpha non-responders with chronic hepatitis C

Background/Aims: Combination therapy with interferon-alpha (IFN-alpha) plus ribavirin is more efficacious than IFN-alpha monotherapy in previously untreated patients with chronic hepatitis C and patients with IFN-alpha relapse. Only limited data are available in IFN-alpha non-responders. In a multicenter trial we therefore evaluated the efficacy of combination therapy in IFN-alpha-resistant chronic hepatitis C. Methods: Eighty-two patients (mean age 46.8 years, 54 males, 28 females) with chronic hepatitis C were treated with IFN-alpha-2a (3 x 6 MIU/week) and ribavirin (14 mg/kg daily) for 12 weeks. Thereafter, treatment was continued only in virological responders (undetectable serum HCV RNA at week 12) with an IFN-alpha dose of 3 x 3 MIU/week and without ribavirin for a further 9 months. The primary study endpoint was an undetectable HCV RNA by RT-PCR at the end of the 24-week follow-up period. Results: After 12 weeks of combination therapy, an initial virological response was observed in 29 of 82 (35.4%) patients. Due to a high breakthrough rate after IFN-a dose reduction and ribavirin discontinuation, an end-of-treatment response was only achieved in 12 of 82 (14.6%) patients. After the follow-up period, a sustained virological response was observed in 8 of 82 (9.8%) patients. Infection with HCV genotype 3 was the only pretreatment parameter, which could predict a sustained response (HCV-1, 5%; HCV-3, 57.1%; p < 0.001). Conclusions: Despite a high initial response rate of 35.4%, sustained viral clearance was achieved only in 9.8% of the retreated primary IFN-alpha non-responders. Higher IFN-alpha induction and maintenance dose, as well as prolonged ribavirin treatment may possibly increase the virological response rates in non-responders, particularly in those infected by HCV-1