Role of IFN‐λs,IFN‐λs related genes and the DEPDC5 gene in Hepatitis B virus‐related liver disease

Recent studies have associated genetic variation near the interleukin 28B (IL28B/IFN‐λ3) gene with natural clearance of the hepatitis C virus (HCV) infection, and a common variant in the DEP domain containing 5 (DEPDC5) locus on chromosome 22 has been shown to affect susceptibility to hepatocellular carcinoma (HCC) in Japanese individuals with chronic HCV infection. This study was conducted to determine whether polymorphisms near or in interferon‐lambda (IFN‐λs) genes and their receptor genes such as interleukin 28 receptor, alpha (IL28RA) and interleukin 10 receptor, beta (IL10RB) as well as p21_activated kinases 4 (PAK4) and iron/zinc purple acid phosphatase‐like protein (PAPL), which are locate upstream of IFN‐λs, and lastly the DEPDC5 gene are associated with hepatitis B virus‐related liver disease in Han Chinese. The study subjects included 507 normal healthy controls, 350 individuals with natural clearance of HBV and 792 HBV‐infected patients. The patients were categorized into 157 inactive carriers (Case I), 216 active carriers (Case II), 111 cirrhotics (Case III) and 308 HCC patients (Case IV) subgroups. Seven single nucleotide polymorphisms (SNPs) were genotyped using the Matrix‐assisted Laser Desorption/Ionisation mass spectrometric (MALDI‐TOF MS) SNP genotyping assay. Rs423058 upstream of PAPL, rs2834167 in IL10RB and rs1012068 in DEPDC5 were associated with chronic HBV status, HBV natural clearance and the presence of HCC (P = 0.0004–0.024), respectively. PAPL, IL10RB and DEPDC5 polymorphisms have an impact on progression of HBV‐related liver disease. However, IFN‐λs genes as a tool to differentiate between different clinical courses of HBV infection were not useful in the Han Chinese population.     

Identification and comparison of insulin pharmacokinetics injected with a new 4‐mm needle vs 6‐ and 8‐mm needles accounting for endogenous insulin and C‐peptide secretion kinetics in non‐diabetic adult males

Aims/Introduction

Many patients with diabetes now use 5‐, 6‐ or 8‐mm needles for insulin injection. However, it is unclear whether needle length, particularly for shorter needles, affects the pharmacokinetic properties of insulin.

Materials and Methods

This was a three‐way, randomized, cross‐over, single‐center study involving 12 healthy Japanese adult males (age 27.4 ± 4.14 years; weight 64.2 ± 5.2 kg; body fat percentage 18.2 ± 1.5%). Participants received a subcutaneous (abdomen) dose of insulin lispro (1.5 U for participants weighing 55 to <65.0 kg; 2.0 U for participants weighing 65.0 to <80.0 kg) delivered using a 32‐G × 4 mm (32G × 4), 31‐G × 8 mm (31G × 8) or 32‐G × 6 mm (32G × 6) needle with a 3–7‐day washout between doses. Pharmacokinetic parameters of exogenous insulin were identified using non‐linear least squares, where the total insulin concentration was fit to the measured plasma insulin concentration using an overall combined model that accounted for C‐peptide/insulin secretion in addition to the injected dose.

Results

Maximum concentration and area under the curve for 0 to infinity min for insulin were bioequivalent for the 32G × 4 needle relative to the 32G × 6 and the 31G × 8 needles. The time to the maximum insulin concentration was bioequivalent for the 32G × 4 needle relative to the 32G × 6 needle, but not the 31G × 8 needle.

Conclusions

The use of 4‐mm needles is unlikely to change the pharmacokinetic properties of insulin when injected subcutaneously in adults. This trial was registered with UMIN‐CTR (no. UMIN000004469).     

Comparative analysis between RQ‐PCR and digital‐droplet‐PCR of immunoglobulin/T‐cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia

Real‐time quantitative polymerase chain reaction (RQ‐PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow‐up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ‐PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value.     

Evolution of glomerular filtration rate in HIV‐infected,HIV–HBV‐coinfected and HBV‐infected patients receiving tenofovir disoproxil fumarate

We aimed to compare the evolution of estimated glomerular filtration rate (eGFR) in HIV‐, HIV–HBV‐ and HBV‐infected patients treated with tenofovir disoproxil fumarate (TDF). Three groups of patients receiving TDF > 12 months were recruited: 194 HIV‐infected patients, 85 HIV–HBV‐coinfected patients and 50 HBV‐infected patients. eGFR was estimated using the Modification of the Diet in Renal Disease (MDRD) equation. Multivariate regression models were constructed to estimate factors associated with eGFR decrease from baseline. A total of 329 patients were studied. Median follow‐up was 2.7 years. Median eGFR decrease was ?4.9 (?16.6 to +7.2) mL/min/1.73 m². After multivariate stepwise regression analysis, age (P = 0.0002), non‐African origin (P < 0.0001), baseline eGFR (P < 0.0001) and TDF duration (P = 0.02) were associated with eGFR decrease in the whole population, while hypertension, diabetes and type of infection were not. Age (P < 0.0001), non‐African origin (P = 0.0004), baseline eGFR (P < 0.0001) and TDF duration (P = 0.007) remained associated with eGFR decline in HIV and HIV–HBV‐infected patients, while other variables including HIV risk factor, CDC stage, CD4 and HIV‐RNA levels were not. Age (P = 0.03), non‐African origin (P = 0.004), baseline eGFR (P < 0.0001) and baseline HBV–DNA > 2000 IU/mL (P = 0.04) were associated with eGFR decline in HBV and HIV–HBV‐infected patients, while other variables including HBV risk factor and fibrosis stage were not. Estimated glomerular filtration rate decline under TDF therapy appears mainly associated with older age, non‐African origin, higher baseline eGFR and longer TDF administration but not with the type of viral infection. Regular follow‐up of renal function, especially tubular function is recommended during TDF therapy.     

Relative expression analysis of IL‐5 and IL‐6 genes in tropical sheep breed Pelibuey infected with Haemonchus contortus

Haemonchus contortus is a parasitic nematode of Pelibuey sheep, a meat breed used in tropical regions. Due to anthelmintic problems, the identification of hosts resistant to H. contortus is another option of control. The aim of this study was to analyse the relative expression of IL‐5 and IL‐6 genes in Pelibuey sheep after H. contortus infection. Nineteen lambs infected with H. contortus and three more lambs without infection were studied. The haemonchosis was determined by the number of eggs per gram of faeces (epg) and by the estimation of the percentage of the packed cell volume (%pcv). Peripheral blood mononuclear cells (PBMCs) were obtained to extract RNA at 0, 1, 2, 7, 14, 21 and 28 days after infection to quantify the relative expression of IL‐5, IL‐6 and GAPDH by real‐time PCR. Five lambs were classified as low responders (lr) to haemonchosis with averages of 1519 ± 315·3 epg and 31·49 ± 5·13%pcv, and 14 lambs were identified as high responders (hr) with averages of 530 ± 132 epg and 34·88 ± 3·75%pcv. The expression ratio of IL‐5 was significantly different compared with control lambs at 2, 7 and 14 days post‐infection (PI), and IL‐6 was significantly different after 14 days. The highest level of relative expression for IL‐5 and IL‐6 genes was 9·9‐fold and 12‐fold after 2 and 14 days for hr hosts (P < 0·05) compared with control group, respectively. In conclusion, the Pelibuey breed in grazing areas exhibited different expression of IL‐5 and IL‐6 obtained from PBMCs against H. contortus, suggesting the importance of these cytokines in regulating the nematode infection.     

IL‐28B (IFN‐λ3) and IFN‐α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however , no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon ‐ stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.