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 > 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 ≤ 0.01 for all). Total HDL-C levels were unchanged, but the subfraction of large HDL-C was higher (p ≤ 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 ≤ 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.

MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

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.

Phenotypic and functional alterations of myeloid-derived suppressor cells during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system involving dysregulated encephalitogenic T cells. Myeloid-derived suppressor cells (MDSCs) have been recognized for their important function in regulating T-cell responses. Recent studies have indicated a role for MDSCs in autoimmune diseases, but their significance in MS is not clear. Here, we assessed the frequencies of CD14+ HLA-DRlow monocytic MDSCs (Mo-MDSCs) and CD33+ CD15+ CD11b+ HLA-DRlow granulocytic MDSCs (Gr-MDSCs) and investigated phenotypic and functional differences of Mo-MDSCs at different clinical stages of MS and in healthy subjects (HC). Increased frequencies of Mo-MDSCs (P < 0.05) and Gr-MDSCs (P < 0.05) were observed in relapsing-remitting MS patients during relapse (RRMS-relapse) compared to stable RRMS (RRMS-rem). Secondary progressive MS (SPMS) patients displayed a decreased frequency of Mo-MDSCs and Gr-MDSCs compared to HC (P < 0.05). Mo-MDSCs within RRMS patients expressed significantly higher cell surface protein levels of CD86 and CD163 compared to SPMS patients. Mo-MDSCs within SPMS exhibited decreased mRNA expression of interleukin-10 and heme oxygenase 1 compared to RRMS and HC. Analysis of T-cell regulatory function of Mo-MDSCs demonstrated T-cell suppressive capacity in RRMS and HCs, while Mo-MDSCs of SPMS promoted autologous T-cell proliferation, which aligned with a differential cytokine profile compared to RRMS and HCs. This study is the first to show phenotypic and functional shifts of MDSCs between clinical stages of MS, suggesting a role for MDSCs as a therapeutic target to prevent MS disease progression.

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 ±â€¯17.5 to 16.3 ±â€¯2.1 mg/dL, (p = 0.03), intermediate 38.3 ±â€¯0.6 to 5.0 ±â€¯3.5 mg/dL (p = 0.004) and small 5.0 ±â€¯2.6 to 0.2 ±â€¯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.

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 < .05), LDL cholesterol (LDL-C; -32.3 mg/dL, P < .0001), and apolipoprotein B (apoB; -62.9 µg/mL, P < .001). Bariatric surgery further reduced TGs (-36.7 mg/dL, P < .05), increased HDL cholesterol (+12 mg/dL, P < .0001), and reductions in LDL-C and apoB were sustained. Bariatric surgery reduced large, buoyant LDL (P < .0001), but had no effect on the small, dense LDL. The large HDL subfractions increased (P < .0001), but there was no effect on the smaller HDL subfractions. The ratio for SAA/PON1 was reduced after the lifestyle intervention (P < .01) and further reduced after bariatric surgery (P < .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.

Sensitization to omeprazole in the occupational setting.

BACKGROUND: Omeprazole is a proton pump inhibitor for the treatment of gastric acid-related disorders. In recent years, reports of dermatitis upon exposure to omeprazole during manufacture have been noted. OBJECTIVE: To present diagnostic findings in workers who reported suspected hypersensitivity reactions resulting from occupational exposure to omeprazole. METHODS: Ninety-six workers exposed to omeprazole during the manufacturing process underwent investigation by the AstraZeneca Occupational Health Centre (Södertälje, Sweden) for suspected allergy. All subjects underwent a lymphocyte transformation test (LTT) and a skin test within 6 months of the clinical reaction. Predictive tests on guinea-pigs were conducted to establish omeprazole's sensitizing potential. RESULTS: Thirty-one subjects with clinical symptoms had a positive LTT result. Twenty-eight subjects had positive patch test results; of these, 23 also had a positive LTT result (sensitivity of the LTT: 82%). Fifty-six subjects had negative patch test results; 46 of these had a negative LTT result (specificity: 82%). All subjects who underwent prick testing (n = 18) had negative results. Delayed contact hypersensitivity was observed in 18 of 20 test animals. CONCLUSIONS: These findings confirm the risk of sensitization to omeprazole from occupational exposure. They are of importance for the development of new formulations of omeprazole, or its enantiomers, in light of the potential for inducing skin allergy.

Tesaglitazar, a dual PPAR-α/γ agonist, hamster carcinogenicity, investigative animal and clinical studies.

Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.

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.

A systems biology approach to understanding elevated serum alanine transaminase levels in a clinical trial with ximelagatran.

Ximelagatran was developed for the prevention and treatment of thromboembolic conditions. However, in long-term clinical trials with ximelagatran, the liver injury marker, alanine aminotransferase (ALT) increased in some patients. Analysis of plasma samples from 134 patients was carried out using proteomic and metabolomic platforms, with the aim of finding predictive biomarkers to explain the ALT elevation. Analytes that were changed after ximelagatran treatment included 3-hydroxybutyrate, pyruvic acid, CSF1R, Gc-globulin, L-glutamine, protein S and alanine, etc. Two of these analytes (pyruvic acid and CSF1R) were studied further in human cell cultures in vitro with ximelagatran. A systems biology approach applied in this study proved to be successful in generating new hypotheses for an unknown mechanism of toxicity.

A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies.

A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems.Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5ml peripheral blood, 1.25x10(5) effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled.

NK-cell activity in immunotoxicity drug evaluation.

NK-cell activity as a tool for detection of immunotoxic effects of new human drugs has gained further attention when the recent European note for guidance CPMP/SWP/1042/99 was adopted. The inclusion of NK-cell activity plus distribution of lymphocyte subsets were suggested as an alternative to the primary antibody response to a T-cell dependent antigen. Either of the two test alternatives should be included as a routine parameter in at least one repeated dose-toxicity study, rats or mice being the species of choice. The standard procedure for measuring NK-cell activity is the 51Cr-release assay. However, a new flow-cytometric assay, adapted for rat peripheral blood, does not require dedicated groups of animals, offers the possibility of repeated testing, and shows at least as sensitive as the conventional 51Cr-release assay.