Meso Scale Discovery and Luminex Comparative Analysis of Calbindin D28K

The sensitivity of different renal regions to xenobiotics requires the development of a multiplex immunoassay for the simultaneous analysis of kidney biomarkers. Calbindin D28K is a distal tubule-specific protein that can be detected in urine under pathological conditions. In this study, a pair of anti-calbindin D28K antibodies was used in an immunoassay for the detection of calbindin D28K expression in rat and human kidney and urine. Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms. Analysis on both platforms detected calbindin D28K concentrations between 100 ng/mL and 100 pg/mL. Luminex detected 10-fold the amount of calbindin D28K in samples analyzed as compared to MSD, whereas calbindin D28K level in rat and human urine was below detection limit in both platforms. The application of the immunoassays described herein may be useful in toxicological and pathological studies of distal tubular damage in rats and human

Управление деятельностью транспортной службы предприятия (на примере КЖРЭУП «Центральное»)

BACKGROUND: Drug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood. METHODS: To study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView. RESULTS: We demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity. CONCLUSIONS: Our bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI

Meso scale discovery and luminex comparative analysis of calbindin D28K

The sensitivity of different renal regions to xenobiotics requires the development of a multiplex immunoassay for the simultaneous analysis of kidney biomarkers. Calbindin D28K is a distal tubule-specific protein that can be detected in urine under pathological conditions. In this study, a pair of anti-calbindin D28K antibodies was used in an immunoassay for the detection of calbindin D28K expression in rat and human kidney and urine. Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms. Analysis on both platforms detected calbindin D28K concentrations between 100 ng/mL and 100 pg/mL. Luminex detected 10-fold the amount of calbindin D28K in samples analyzed as compared to MSD, whereas calbindin D28K level in rat and human urine was below detection limit in both platforms. The application of the immunoassays described herein may be useful in toxicological and pathological studies of distal tubular damage in rats and human

HLA-DR7 and HLA-DQ2 : Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immunemediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202, -DQA1*0201) were created. These two lines were crossed with a human (h) CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a preclinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.Funding Agencies|AstraZeneca; AstraZeneca R&amp;D as part of safety problem-solving activities initiated for ximelagatran (Exanta(R))</p

Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles - an interdisciplinary approach

Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle-protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both danger signals, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations.Funding Agencies|Region Ostergotland ALF [LIO-606891]; AFA insurances [150246]; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [2012.0083 CTS, 18:399]; Center in Nano Science and Nano technology at LiTH (CeNano) at Linkoping University</p

Lipoprotein apheresis affects lipoprotein particle subclasses more efficiently compared to the PCSK9 inhibitor evolocumab, a pilot study.

Lipoprotein apheresis and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are last therapeutic resorts in patients with familial hypercholesterolemia (FH). We explored changes in lipoprotein subclasses and high-density lipoprotein (HDL) function when changing treatment from lipoprotein apheresis to PCSK9 inhibition. We measured the levels of low-density lipoprotein (LDL) and HDL particle subclasses, serum amyloid A1 (SAA1), paraoxonase-1 (PON1) activity and cholesterol efflux capacity (CEC) in three heterozygous FH patients. Concentrations of all LDL particle subclasses were reduced during apheresis (large 68.0 ± 17.5 to 16.3 ± 2.1 mg/dL, (p = 0.03), intermediate 38.3 ± 0.6 to 5.0 ± 3.5 mg/dL (p = 0.004) and small 5.0 ± 2.6 to 0.2 ± 0.1 mg/dL (p = 0.08)). There were non-significant reductions in the LDL subclasses during evolocumab treatment. There were non-significant reductions in subclasses of HDL particles during apheresis, and no changes during evolocumab treatment. CEC was unchanged throughout the study, while the SAA1/PON1 ratio was unchanged during apheresis but decreased during evolocumab treatment. In conclusion, there were significant reductions in large and intermediate size LDL particles during apheresis, and a non-significant reduction in small LDL particles. There were only non-significant reductions in the LDL subclasses during evolocumab treatment.Funding agencies: Amgen, Norway</p

Effect of N-3 Polyunsaturated Fatty Acids on Lipid Composition in Familial Hypercholesterolemia : A Randomized Crossover Trial

Individuals with familial hypercholesterolemia (FH) have an increased risk of cardiovascular disease. Treatment is mainly low-density lipoprotein cholesterol (LDL-C) reduction. How omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplements affect lipoproteins in FH subjects is unknown. We hypothesized that a high-dose n-3 PUFA supplement would reduce atherogenic lipoproteins and influence the high-density lipoprotein cholesterol (HDL-C) function. We performed a randomized, double-blinded crossover study with 34 genetically verified FH individuals (18-75 years, clinically stable, statin treatment &amp;gt; 12 months). Treatment was 4 g n-3 PUFAs (1840 mg eicosapentaenoic acid and 1520 mg docosahexaenoic acid daily) or four capsules of olive oil for three months in a crossover design with a washout period of three months. The defined outcomes were changes in triglycerides, lipoproteins, lipoprotein subfractions, apolipoproteins, and HDL-C function. After treatment with n-3 PUFAs, total cholesterol, LDL-C, and triglycerides were reduced compared to placebo (p &amp;lt;= 0.01 for all). Total HDL-C levels were unchanged, but the subfraction of large HDL-C was higher (p &amp;lt;= 0.0001) after n-3 PUFAs than after placebo, and intermediate HDL-C and small HDL-C were reduced after n-3 PUFAs compared to placebo (p = 0.02 and p &amp;lt;= 0.001, respectively). No changes were found in apolipoproteins and HDL-C function. N-3 PUFAs supplements reduced atherogenic lipoproteins in FH subjects, leaving HDL-C function unaffected.Funding Agencies|Northern Norway Regional Health Authority (Helse Nord RHF) [SFP1311-16]; UiT The Arctic University of Norway</p

Bariatric surgery improves lipoprotein profile in morbidly obese patients by reducing LDL cholesterol, apoB, and SAA/PON1 ratio, increasing HDL cholesterol, but has no effect on cholesterol efflux capacity

BACKGROUND: Bariatric surgery has been shown to reduce cardiovascular events and cause specific mortality for coronary artery disease in obese patients. Lipoprotein biomarkers relating to low-density lipoprotein (LDL), high-density lipoprotein (HDL), their subfractions, and macrophage cholesterol efflux have all been hypothesized to be of value in cardiovascular risk assessment. OBJECTIVES: The objective of this study was to examine the effect of a lifestyle intervention followed by bariatric surgery on the lipid profile of morbidly obese patients. METHODS: Thirty-four morbidly obese patients were evaluated before and after lifestyle changes and then 1 year after bariatric surgery. They were compared with 17 lean subjects. Several lipoprotein metrics, serum amyloid A (SAA), serum paraoxonase-1 (PON1), and macrophage cholesterol efflux capacity (CEC) were assessed. RESULTS: Average weight loss after the lifestyle intervention was 10.5% and 1 year after bariatric surgery was 33.9%. The lifestyle intervention significantly decreased triglycerides (TGs; 28.7 mg/dL, P amp;lt; .05), LDL cholesterol (LDL-C; 32.3 mg/dL, P amp;lt; .0001), and apolipoprotein B (apoB; 62.9 mu g/mL, P amp;lt; .001). Bariatric surgery further reduced TGs (-36.7 mg/dL, P amp;lt; .05), increased HDL cholesterol (+12 mg/dL, P amp;lt; .0001), and reductions in LDL-C and apoB were sustained. Bariatric surgery reduced large, buoyant LDL (P amp;lt; .0001), but had no effect on the small, dense LDL.The large HDL subfractions increased (P amp;lt; .0001), but there was no effect on the smaller HDL sub fractions. The ratio for SAA/PON1 was reduced after the lifestyle intervention (P amp;lt; .01) and further reduced after bariatric surgery (P amp;lt; .0001). Neither the lifestyle intervention nor bariatric surgery had any effect on CEC. CONCLUSIONS: Lifestyle intervention followed by bariatric surgery in 34 morbidly obese patients showed favorable effects on TGs, LDL-C, and apoB. HDL cholesterol and apoA1 was increased, apoB/apoA1 ratio as well as SAA/PON1 ratio reduced, but bariatric surgery did not influence CEC. (C) 2017 National Lipid Association. All rights reserved

Surface markers.

<p>Surface markers.</p