Plasma Cytokeratin-18 Fragment Level Reflects the Metabolic Phenotype in Obesity.

There is growing interest in the non-invasive identification and monitoring of the outcome of liver damage in obese patients. Plasma cytokeratin-18 (CK-18) fragment levels correlate with the magnitude of hepatocyte apoptosis and have recently been proposed to independently predict the presence of non-alcoholic steatohepatitis (NASH). The aim of the study was to analyze the associations of CK-18 with obesity and related complications: insulin resistance, impaired lipid metabolism and the secretion of hepatokines, adipokines and pro-inflammatory cytokines. The study involved 151 overweight and obese patients (BMI 25-40), without diabetes, dyslipidemia or apparent liver disease. Liver function was assessed based on alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and the fatty liver index (FLI). CK-18 M30 plasma levels, FGF-21, FGF-19 and cytokines were determined by ELISA. CK-18 values >150 U/l were accompanied by high ALT, GGT and FLI, insulin resistance, postprandial hypertriglyceridemia, elevated FGF-21 and MCP-1 and decreased adiponectin. ALT activity was the strongest independent factor influencing high CK-18 plasma levels, even after an adjustment for age, sex and BMI [ß coefficient (95%CI): 0.40 (0.19-0.61)]. In conclusion, the applied CK-18 cut-off point at 150 U/l allows to distinguish between two metabolic phenotypes in obesity.

Glucose-Dependent Insulinotropic Polypeptide Plasma Level Influences the Effect of n-3 PUFA Supplementation.

Elevated glucose-dependent insulinotropic peptide (GIP) levels in obesity may predict the metabolic benefits of n-3 PUFA supplementation. This placebo-controlled trial aimed to analyze fasting and postprandial GIP response to 3-month n-3 PUFA supplementation (1.8 g/d; DHA:EPA, 5:1) along with caloric restriction (1200-1500 kcal/d) in obese subjects. Compliance was confirmed by the incorporation of DHA and EPA into red blood cells (RBCs). Blood analyses of glucose, insulin, non-esterified fatty acids (NEFAs), GIP and triglycerides were performed at fasting, and during an oral glucose tolerance test and a high fat mixed-meal tolerance test. Fatty acid composition of RBC was assessed by gas chromatography and total plasma fatty acid content and composition was measured by gas-liquid chromatography. The DHA and EPA content in RBCs significantly increased due to n-3 PUFA supplementation vs. placebo (77% vs. -3%, respectively). N-3 PUFA supplementation improved glucose tolerance and decreased circulating NEFA levels (0.750 vs. 0.615 mmol/L), as well as decreasing plasma saturated (1390 vs. 1001 µg/mL) and monounsaturated (1135 vs. 790 µg/mL) fatty acids in patients with relatively high GIP levels. The effects of n-3 PUFAs were associated with the normalization of fasting (47 vs. 36 pg/mL) and postprandial GIP levels. Obese patients with elevated endogenous GIP could be a target group for n-3 PUFA supplementation in order to achieve effects that obese patients without GIP disturbances can achieve with only caloric restriction.

The Effect of Caloric Restriction with and without n-3 PUFA Supplementation on Bone Turnover Markers in Blood of Subjects with Abdominal Obesity: A Randomized Placebo-Controlled Trial.

Weight loss contributes to an increased risk of hip fracture, especially in postmenopausal women. Omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation could diminish the adverse effect of weight loss on bone health. The aim of this randomized, placebo-controlled, double-blind parallel trial was to investigate the effect of caloric restriction and n-3 PUFA supplement intake on osteogenic markers (carboxylated osteocalcin (Gla-OC); procollagen I N-terminal propeptide (PINP)), as well as a bone resorption marker (C-terminal telopeptide of type I collagen (CTX-I)) in a serum of 64 middle aged individuals (BMI 25-40 kg/m2) with abdominal obesity. Bone remodeling, metabolic and inflammatory parameters and adipokines were determined before and after 3 months of an isocaloric diet (2300-2400 kcal/day) or a low-calorie diet (1200 kcal/day for women and 1500 kcal/day for men) along with n-3 PUFA (1.8 g/day) or placebo capsules. CTX-I and adiponectin concentrations were increased following 7% weight loss independently of supplement use. Changes in CTX-I were positively associated with changes in adiponectin level (rho = 0.25, p = 0.043). Thus, an increase in serum adiponectin caused by body weight loss could adversely affect bone health. N-3 PUFAs were without effect.

Expression of VEGFA-mRNA in classical and MSX2-mRNA in non-classical monocytes in patients with spondyloarthritis is associated with peripheral arthritis.

Spondyloarthritis (SpA) is characterized by chronic inflammation and structural damage involving spine and peripheral joints. Monocytes, as part of innate immune system, following migration into affected tissue, may play a role in the pathogenesis of SpA. Here, potential associations between osteogenesis-linked gene expression profile in particular monocyte subpopulations and clinical signs of SpA were investigated. The 20 patients with axial and 16 with peripheral SpA were enrolled in the study. Monocyte subpopulations (classical-CD14++CD16-, intermediate-CD14++CD16+ and non-classical-CD14+CD16++) were isolated from blood using flow cytometry and gene expression analysis was performed using real-time PCR method and TaqMan Array, Human Osteogenesis, Fast 96-well plates. Next, the characteristic clinical features shared by axial and peripheral SpA were analyzed in the context of the expression of selected genes in the three subpopulations of monocytes. We demonstrated that expression of VEGFA in classical and MSX2 in non-classical monocytes were associated with the number of swollen and painful peripheral joints of SpA patients. We conclude that monocytes may contribute to the development of peripheral arthritis in SpA patients. This might be possible through subpopulation specific effects, linking number of inflamed joints with expression of VEGFA in classical monocytes and MSX2 in non-classical monocytes.

DNA methylation microarrays identify epigenetically regulated lipid related genes in obese patients with hypercholesterolemia.

BACKGROUND: Epigenetics can contribute to lipid disorders in obesity. The DNA methylation pattern can be the cause or consequence of high blood lipids. The aim of the study was to investigate the DNA methylation profile in peripheral leukocytes associated with elevated LDL-cholesterol level in overweight and obese individuals. METHODS: To identify the differentially methylated genes, genome-wide DNA methylation microarray analysis was performed in leukocytes of obese individuals with high LDL-cholesterol (LDL-CH, ≥ 3.4 mmol/L) versus control obese individuals with LDL-CH, < 3.4 mmol/L. Biochemical tests such as serum glucose, total cholesterol, HDL cholesterol, triglycerides, insulin, leptin, adiponectin, FGF19, FGF21, GIP and total plasma fatty acids content have been determined. Oral glucose and lipid tolerance tests were also performed. Human DNA Methylation Microarray (from Agilent Technologies) containing 27,627 probes for CpG islands was used for screening of DNA methylation status in 10 selected samples. Unpaired t-test and Mann-Whitney U-test were used for biochemical and anthropometric parameters statistics. For microarrays analysis, fold of change was calculated comparing hypercholesterolemic vs control group. The q-value threshold was calculated using moderated Student's t-test followed by Benjamini-Hochberg multiple test correction FDR. RESULTS: In this preliminary study we identified 190 lipid related CpG loci differentially methylated in hypercholesterolemic versus control individuals. Analysis of DNA methylation profiles revealed several loci engaged in plasma lipoprotein formation and metabolism, cholesterol efflux and reverse transport, triglycerides degradation and fatty acids transport and ß-oxidation. Hypermethylation of CpG loci located in promoters of genes regulating cholesterol metabolism: PCSK9, LRP1, ABCG1, ANGPTL4, SREBF1 and NR1H2 in hypercholesterolemic patients has been found. Novel epigenetically regulated CpG sites include ABCG4, ANGPTL4, AP2A2, AP2M1, AP2S1, CLTC, FGF19, FGF1R, HDLBP, LIPA, LMF1, LRP5, LSR, NR1H2 and ZDHHC8 genes. CONCLUSIONS: Our results indicate that obese individuals with hypercholesterolemia present specific DNA methylation profile in genes related to lipids transport and metabolism. Detailed knowledge of epigenetic regulation of genes, important for lipid disorders in obesity, underlies the possibility to influence target genes by changing diet and lifestyle, as DNA methylation is reversible and depends on environmental factors. These findings give rise for further studies on factors that targets methylation of revealed genes.

Resonance Raman Optical Activity Shows Unusual Structural Sensitivity for Systems in Resonance with Multiple Excited States: Vitamin B12 Case.

In this work, cobalamins with different upper axial substituents and a cobalamin derivative with a ring modification were studied using chiroptical spectroscopies, in particular resonance Raman optical activity (RROA), to shed light on the influence of structural modifications on RROA spectra in these strongly chiral systems in resonance with multiple excited states at 532 nm excitation. We have demonstrated that for these unique systems RROA possesses augmented structural specificity, surpassing resonance Raman spectroscopy and enabling at the same time measurement of cobalamins at fairy low concentrations of ∼10-5 mol dm-3. The enhanced structural specificity of RROA is a result of bisignate spectra due to resonance via more than one electronic state. The observation of increased structural capability of RROA for cobalamins opens a new perspective for studying chiral properties of other biological systems incorporating d-metal ions.

Enhanced GIP Secretion in Obesity Is Associated with Biochemical Alteration and miRNA Contribution to the Development of Liver Steatosis.

Nutrient excess enhances glucose-dependent insulinotropic polypeptide (GIP) secretion, which may in turn contribute to the development of liver steatosis. We hypothesized that elevated GIP levels in obesity may affect markers of liver injury through microRNAs. The study involved 128 subjects (body mass index (BMI) 25-40). Fasting and postprandial GIP, glucose, insulin, and lipids, as well as fasting alanine aminotransferase (ALT), γ-glutamyltransferase (GGT), cytokeratin-18, fibroblast growth factor (FGF)-19, and FGF-21 were determined. TaqMan low density array was used for quantitative analysis of blood microRNAs. Fasting GIP was associated with ALT [ß = 0.16 (confidence interval (CI): 0.01-0.32)], triglycerides [ß = 0.21 (95% CI: 0.06-0.36], and FGF-21 [ß = 0.20 (95%CI: 0.03-0.37)]; and postprandial GIP with GGT [ß = 0.17 (95%CI: 0.03-0.32)]. The odds ratio for elevated fatty liver index (>73%) was 2.42 (95%CI: 1.02-5.72) for high GIP versus low GIP patients. The miRNAs profile related to a high GIP plasma level included upregulated miR-136-5p, miR-320a, miR-483-5p, miR-520d-5p, miR-520b, miR-30e-3p, and miR-571. Analysis of the interactions of these microRNAs with gene expression pathways suggests their potential contribution to the regulation of the activity of genes associated with insulin resistance, fatty acids metabolism, and adipocytokines signaling. Exaggerated fasting and postprandial secretion of GIP in obesity are associated with elevated liver damage markers as well as FGF-21 plasma levels. Differentially expressed microRNAs suggest additional, epigenetic factors contributing to the gut-liver cross-talk.

Bradykinin and oxidative stress in patients with hereditary angioedema due to C1 inhibitor deficiency.

INTRODUCTION: Hereditary angioedema (HAE) is a rare autosomal dominant disease caused by genetic dysfunction of C1 inhibitor (C1-INH) due to mutations in the SERPING1 gene. The disorder is mediated mainly by bradykinin. The clinical course of the disease is varied and not related to genetic changes. OBJECTIVES: We aimed to evaluate redox homeostasis of peripheral blood mononuclear cells (PBMCs) in patients with HAE due to C1-INH deficiency (C1 INH HAE) by measuring the levels of reactive oxygen species (ROS) of PBMCs as well as plasma advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs). We also aimed to assess the effect of bradykinin on ROS levels. PATIENTS AND METHODS: We enrolled 30 adults with C1-INH-HAE and 15 healthy individuals. The levels of ROS were measured by flow cytometry, while the plasma levels of AGEs and AOPPs were determined spectrophotometrically by enzyme­ linked immunosorbent assays. RESULTS: Basal and hydrogen peroxide (H2O2)-induced ROS levels were higher in patients with HAE when compared with controls (P = 0.002 and P = 0.001, respectively), indicating abnormalities in redox homeostasis. Plasma AOPP and AGE levels were similar in both groups. Bradykinin reduced basal and H2O2-induced ROS generation in PBMCs only in patients with HAE (P = 0.03). CONCLUSIONS: The higher basal and H2O2-induced ROS levels in patients with C1 INH HAE indicate redox imbalance. However, by reducing basal and H2O2-induced ROS levels, bradykinin shows antioxidant action in this disorder.

Effect of insulin resistance on whole blood mRNA and microRNA expression affecting bone turnover.

OBJECTIVE: To evaluate the effect of insulin resistance in obesity on the expression in whole blood of mRNA and miRNA affecting bone homeostasis as well as to estimate the influence of oral glucose load (OGTT) on serum osteocalcin concentration in obese individuals with and without insulin resistance. DESIGN: Cross-sectional study. METHODS: Carboxylated (cOC), undercarboxylated (ucOC) and total osteocalcin were measured by ELISA in the serum of obese subjects with insulin resistance (n = 41) and obese subjects without insulin resistance (n = 41) (control group) during OGTT. Analysis of gene expression (microarray) and miRNAs (real-time PCR) was performed in venous blood (representating samples) collected before OGTT from obese with insulin resistance and controls. RESULTS: Obese subjects with insulin resistance (higher HOMA-IR and lower oral glucose insulin sensitivity index) presented significantly increased expression of WNT signalling inhibitors (DKK1, DKK2, SOST, SFRP1) and downregulation of the key factor in WNT signalling - ß catenin participating in osteoblasts differentiation. Expression of miRNA involved in osteoblastogenesis was also inhibited (miR-29b, miR-181a, miR-210, miR-324-3p). During OGTT, contrary to the control group, subjects with insulin resistance presented suppression of cOC and total OC decrease after 1 and 2 h of oral glucose load. CONCLUSIONS: Obese subjects with insulin resistance may have defects in osteoblastogenesis that was demonstrated via key signalling molecules mRNA downregulation, and increased expression of WNT antagonists as well as inhibition of expression of miRNA participating in the regulation of osteoblast differentiation. Disturbed osteoblastogenesis in insulin-resistant subjects results in the suppression of blood carboxylated and total osteocalcin decrease during OGTT.

Sera of patients with axial spondyloarthritis (axSpA) enhance osteoclastogenic potential of monocytes isolated from healthy individuals.

BACKGROUND: Axial spondyloarthritis (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated, with sDMARDs and/or bDMARDs, axSpA patients and to test whether these sera influence differentiation of healthy monocytes towards osteoclast lineage. METHODS: Bone remodeling molecules (RANKL, M-CSF, OPG, IL-6, OSM, IL-17A, TGFß, and TNFα) were evaluated in 27 patients with axSpA and 23 age and sex-matched controls. Disease activity (BASDAI, ASDAS) and inflammatory markers (ESR, CRP) were assessed. Monocytes obtained from healthy individuals were cultured in vitro in presence of sera from 11 randomly chosen axSpA patients and 10 controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K and RANK at mRNA level) and with osteoclast-specific staining. RESULTS: axSpA patients' sera levels of soluble RANKL were significantly lower and M-CSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls, whereas of OPG and TGFß were comparable in both groups. Numbers of generated in vitro osteoclasts and cathepsin K mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence and presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Furthermore, sera from axSpA patients induced substantially higher levels of RANK mRNA, independently of M-CSF and RANKL stimulation. CONCLUSION: We show that, paradoxically, serum levels of soluble RANKL observed in axSpA are in fact significantly lower in comparison to healthy blood donors. Our results indicate that sera of axSpA patients - in contrary to healthy subjects - contain circulating, soluble factors (presumably IL-6, OSM, IL-17A, TNFα and others) able to stimulate healthy monocytes responsiveness to even relative low RANKL serum levels, by inducing high RANK mRNA expression and - as a net effect - boosting their osteoclastogenic potential. We suggest also that locally produced RANKL in axSpA may induce overactive osteoclasts from their precursors.

High Fat Mixed Meal Tolerance Test Leads to Suppression of Osteocalcin Decrease in Obese Insulin Resistant Subjects Compared to Healthy Adults.

Nutrients influence bone turnover. Carboxylated osteocalcin (Gla-OC) participates in bone formation whereas its undercarboxylated form (Glu-OC) acts as a hormone in glucose metabolism. The aim of the study was to determine the responses of Gla-OC, Glu-OC, and total-OC (calculated as the sum of Gla-OC and Glu-OC) to a high fat mixed meal tolerance test (HFMTT) in non-obese (body mass index (BMI) < 30 kg/m², n = 24) and obese subjects (30 < BMI < 40 kg/m², n = 70) (both sexes, aged 25⁻65 years). Serum Gla-OC and Glu-OC were measured at baseline as well as at 2 and 6 h during a HFMTT by enzyme-linked immunosorbent assay (ELISA). Baseline Gla-OC, Glu-OC, and total-OC levels were lower in obese individuals compared to non-obese participants (p = 0.037, p = 0.016 and p = 0.005, respectively). The decrease in Gla-OC and total-OC, but not in Glu-OC, concentrations during the HFMTT was suppressed in obese, but not in non-obese controls (p < 0.05, p < 0.01, p = 0.08, respectively). Subjects with the highest homeostatic model assessment for insulin resistance (HOMA-IR) index values had a less pronounced decrease in total-OC compared to patients with values of HOMA-IR index in the 1st quartile (p < 0.05). Net incremental area under Gla-OC inversely correlated with adiponectin (rho = -0.35, p = 0.001). Increase in insulin sensitivity and adiponectin level in obese subjects could beneficially influence postprandial bone turnover expressed by osteocalcin concentration.

Neurodegenerative changes detected by neuroimaging in a patient with contiguous X-chromosome deletion syndrome encompassing BTK and TIMM8A genes.

INTRODUCTION: In this study we describe a patient with gross deletion containing the BTK and TIMM8A genes. Mutations in these genes are responsible for X-linked agammaglobulinemia and Mohr-Tranebjaerg syndrome, respectively. X linked agammaglobulinemia is a rare primary immunodeficiency characterized by low levels of B lymphocytes and recurrent microbial infections, whereas, Mohr-Tranebjaerg syndrome is a progressive neurodegenerative disorder with early onset of sensorineural deafness. MATERIAL AND METHODS: For neuroimaging, the magnetic resonance imaging and magnetic resonance spectroscopy of the brain were performed. Microarray analysis was performed to establish the extent of deletion. RESULTS: The first clinical symptoms observed in our patient at the age of 6 months were connected with primary humoral immunodeficiency, whereas clinical signs of MTS emerged in the third year of live. Interestingly, the loss of speech ability was not accompanied by hearing failure. Neuroimaging of the brain suggested leukodystrophy. Molecular tests revealed contiguous X-chromosome deletion syndrome encompassing BTK (from exons 6 through 19) and TIMM8A genes. The loss of the patient's DNA fragment was accurately localized from 100 601 727 to 100 617 576 bp on chromosome's loci Xq22.1. CONCLUSIONS: We diagnosed XLA-MTS in the first Polish patient on the basis of particular molecular methods. We detected neurodegenerative changes in MRI and MR spectroscopy in this patient. Our results provide further insight into this rare syndrome.

The level of myeloid-derived suppressor cells positively correlates with regulatory T cells in the blood of children with transient hypogammaglobulinaemia of infancy.

INTRODUCTION: Transient hypogammaglobulinaemia of infancy (THI) is a primary immunodeficiency characterised by low levels of immunoglobulin G (often with concomitant decrease of IgA and sometimes also of IgM) with still unknown exact reason. A delayed normalisation of the immunoglobulin level in THI may be associated with a transiently elevated number of regulatory T cells (Treg). Although in cancer and chronic inflammation it was shown that the level of Treg cells can be increased by myeloid-derived suppressor cells (MDSCs), until now no studies have been performed in the context of the role of MDSCs in THI and their correlation with Treg cells. Consequently, we aimed to determine the occurrence of MDSCs in the peripheral blood of children with THI and correlate their level with the level of Treg cells. MATERIAL AND METHODS: Flow cytometry analyses of Mo-MDSCs and Gr-MDSCs, characterised as HLA-DR-CD11b+CD15-CD14+ and HLA-DR-CD11b+CD15+CD14-, respectively, and Treg (CD4+CD25+Foxp3+) cells were performed. RESULTS: The proportion of Mo-MDSCs and Gr-MDSCs was significantly higher in the group of THI patients with elevated level of Treg cells (from the 95% confidence interval level of healthy controls). The cells with Mo-MDSC and Gr-MDSC characteristics positively correlated with the level of Treg cells. Moreover, children with a higher proportion of circulating Treg cells, and thereby higher level of MDSCs, showed delayed normalisation of IgG level and recovery. CONCLUSIONS: These findings show for the first time that MDSCs may be involved in the pathomechanism of THI, probably acting through the induction of Treg cells.

Glucagon-like peptide-1 receptor agonist stimulates mitochondrial bioenergetics in human adipocytes.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) are relatively new pharmacological agents used to normalize glucose level in type 2 diabetes. Recently, GLP-1RAs have been approved for the treatment of obesity to reduce body weight in non-diabetic patients. The extra-pancre-atic effects of GLP-1RAs, as well as their molecular mechanism of action, are still poorly understood. Thus this study was aimed to verify the hypothesis that the mechanism of action of the GLP-1RAs involves mitochondria and that GLP-1RAs administration can improve mitochondrial functions. For this purpose, preadipocytes CHUBS7 were differentiated to mature adipocytes and then stimulated with GLP-1RA, exendin-4 at 100 nM for 24 h. Oxygen consumption rates, mitochondrial membrane potential, intracellular ATP (adenosine triphosphate) level, SIRT1 and SIRT3 gene expression and the histone deacetylases' activity were measured. Exendin-4 was found to uncouple mitochondrial electron transport from ATP synthesis, slightly decreasing mitochondrial membrane potential in mature adipocytes. Routine respiration and uncoupled oxy- gen consumption rates were higher in exendin-4 treated adipocytes than in the non-treated cells. The ATP level remained unchanged. Exendin-4 enhanced SIRT1 and SIRT3 genes expression. Histone deacetylases' activity in the nuclear fraction was not affected by exendin-4, although the activity of class III histone deacetylases was increased. All of the effects on mitochondrial bioenergetics induced by exendin-4 were abolished by addition of glucagon-like peptide 1 receptor antagonist. In conclusion, exendin-4 activates the sirtuin pathway and increases energy expenditure in human adipocytes. Our results suggest another mechanism that may be responsible for body weight reduction observed in patients using GLP-1RAs.

Reprint of: Alterations of TRIM21-mRNA expression during monocyte maturation.

Tripartite motif-containing protein 21 (TRIM21) play a dual role in the cytoplasm of the cells where it facilitates destruction of some antibody-coated viruses and some bacteria, and initiates synthesis of proinflammatory cytokines. Macrophages and CD16+ monocyte subset can particularly participate in a proinflammatory response caused by viral infection, however, the molecular mechanisms underlying these processes are not fully understood. The aim of this study was to determine the level of TRIM21-mRNA expression in monocyte subsets including: classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes, as well as during in vitro differentiation of the isolated monocytes towards dendritic cells or macrophages. Our results revealed that the level of TRIM21 mRNA expression was significantly lower in CD16- monocytes, when compared to CD16+ cells and the whole monocyte population, yet no significant differences were observed when CD16+ population was divided into intermediate and non-classical subsets. More pronounced differences were observed in the case of monocyte-derived macrophages (MDM) and dendritic cells (DCs). TRIM21-mRNA expression level was app. 6-fold higher in DCs, and app. 16-fold higher in MDM (p<0,01), when compared to freshly isolated monocytes. Our results may suggest the new mechanism of increased proinflammatory cytokine production by CD16+ (intermediate and non-classical) monocytes and macrophages, at least in patients with acute or chronic infections, caused by enveloped viruses. We suggest that TRIM21 may be one of the factors associated with the "switching on" the proinflammatory programme in CD16+ monocytes or monocyte-derived macrophages.

Comparison of 6B11 mAb and α-GalCer-loaded CD1d dextramers for detection of iNKT cells by flow cytometry.

Invariant natural killer T (iNKT) cells are a small population of thymus-derived T cells that are restricted by non-classical MHC class I molecule CD1d and express an evolutionary conserved TCR with an invariant α-chain. The frequency of iNKT cells in peripheral blood is very low, thus, accurate methods to identify and enumerate iNKT cells are needed. The aim of the study was to compare 6B11 mAb or α-GalCer-loaded CD1d dextramers usage in iNKT cell detection. The frequency of CD3+CD56+ lymphocytes is much higher, with statistical significance (p<0,001), than real iNKT cells detected by 6B11 mAb or α-GalCer-loaded CD1d dextramers. The frequency of iNKT cells, recognized by 6B11 mAb or α-GalCer-loaded CD1d dextramers, was in a similar range. Nonetheless, when we compared whether 6B11+ and α-GalCer-loaded CD1d dextramers+ are the same populations, it turned out that by this approach we were able to identify three distinct subsets of iNKT cells: i) 6B11+/α-GalCer-loaded dextramer- cells, ii) 6B11+/α-GalCer-loaded dextramer+ cells, and iii) 6B11-/α-GalCer-loaded dextramer+. Thus, although 6B11 mAb and α-GalCer-loaded dextramers may identify not exactly the same cells, application of these methods seems to give similar results of iNKT cell frequency in peripheral blood. It seems that both approaches for iNKT detection can be used for precise identification of these cells. Moreover, our results indicate that CD3+CD56+ lymphocytes are a heterogeneous population of T cells, expressing activation markers of both NK and T lymphocytes, yet with not well characterized properties.

Alterations of TRIM21-mRNA expression during monocyte maturation.

Tripartite motif-containing protein 21 (TRIM21) play a dual role in the cytoplasm of the cells where it facilitates destruction of some antibody-coated viruses and some bacteria, and initiates synthesis of proinflammatory cytokines. Macrophages and CD16+ monocyte subset can particularly participate in a proinflammatory response caused by viral infection, however, the molecular mechanisms underlying these processes are not fully understood. The aim of this study was to determine the level of TRIM21-mRNA expression in monocyte subsets including: classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes, as well as during in vitro differentiation of the isolated monocytes towards dendritic cells or macrophages. Our results revealed that the level of TRIM21 mRNA expression was significantly lower in CD16- monocytes, when compared to CD16+ cells and the whole monocyte population, yet no significant differences were observed when CD16+ population was divided into intermediate and non-classical subsets. More pronounced differences were observed in the case of monocyte-derived macrophages (MDM) and dendritic cells (DCs). TRIM21-mRNA expression level was app. 6-fold higher in DCs, and app. 16-fold higher in MDM (p<0,01), when compared to freshly isolated monocytes. Our results may suggest the new mechanism of increased proinflammatory cytokine production by CD16+ (intermediate and non-classical) monocytes and macrophages, at least in patients with acute or chronic infections, caused by enveloped viruses. We suggest that TRIM21 may be one of the factors associated with the "switching on" the proinflammatory programme in CD16+ monocytes or monocyte-derived macrophages.

Carboxylated and undercarboxylated osteocalcin in metabolic complications of human obesity and prediabetes.

BACKGROUND: Carboxylated osteocalcin (Gla-OC) participates in bone remodeling, whereas the undercarboxylated form (Glu-OC) takes part in energy metabolism. This study was undertaken to compare the blood levels of Glu-OC and Gla-OC in nonobese, healthy obese, and prediabetic volunteers and correlate it with the metabolic markers of insulin resistance and early markers of inflammation. METHODS: Nonobese (body mass index [BMI] <30 kg/m2 ; n = 34) and obese subjects (30

Dicarbonyl stress in clinical obesity.

The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure.

An In Vitro Study of the Neurotoxic Effects of N-Benzylpiperazine: A Designer Drug of Abuse.

Recently, the number of new psychoactive substances has significantly increased. Despite the systematic introduction of prohibition in trade of medicinal products which mimic the effects of illegal drugs, the problem concerning this group of drugs is still important although knowledge about the mechanism of action of those types of substances is scarce. This study aimed to follow the neurotoxic effect of N-benzylpiperazine (BZP), the central nervous system psychostimulant, using the human cancer LN-18 cell model. The statistically significant elevation of LDH levels, increased mitochondrial membrane potential, decreased ATP and increased ROS production, increased levels of DNA damage marker (8-OHdG) and activation of caspases: -3 and -9 confirmed by Real-Time PCR imply the activation of mitochondrial proapoptotic pathways induced by BZP after 24 h incubation. This study is a novel, preliminary attempt to explain the toxicity of one of the most popular designer drug of abuse at the cellular level.