Microvesicles Correlated with Components of Metabolic Syndrome in Men with Type 2 Diabetes Mellitus and Lowered Testosterone Levels But Were Unaltered by Testosterone Therapy

Aims. To investigate how circulating microvesicle phenotypes correlate with insulin sensitivity, body composition, plasma lipids, and hepatic fat accumulation. We hypothesized that changes elicited by testosterone replacement therapy are reflected in levels of microvesicles. Methods. Thirty-nine type 2 diabetic males with lowered testosterone levels were assigned to either testosterone replacement therapy or placebo and evaluated at baseline and after 24 weeks. Microvesicles were analysed by flow cytometry and defined as lactadherin-binding particles within the 0.1–1.0 μm gate. Microvesicles of platelet, monocyte, and endothelial cell origin were identified by cell-specific markers and their expression of CD36 was investigated. Results. Triglycerides correlated positively with all investigated microvesicle phenotypes in this study (p<0.05), and indicators of hepatic fat accumulation, alanine aminotransferase, and gamma glutamyltransferase correlated with platelet and endothelial microvesicles and CD36-expressing microvesicles from platelets and monocytes (p<0.05). BMI, waist circumference, and fat percentage correlated with CD36-expressing monocyte microvesicles (p<0.05), while insulin sensitivity did not correlate with any microvesicle phenotypes. Microvesicle levels were unaffected by testosterone therapy. Conclusions. Metabolic syndrome components and hepatic fat accumulation correlated with microvesicle phenotypes, supporting the involvement of especially CD36 on monocytes in metabolic syndrome pathogenesis. Although testosterone therapy improved body composition measures, microvesicle phenotype levels were unaffected. This trial was registered at ClinicalTrials.gov (NCT01560546)

Probabilistic approach for assessing cancer risk due to benzo[a]pyrene in barbecued meat: Informing advice for population groups

<div><p>Background</p><p>Consumption of meat prepared by barbecuing is associated with risk of cancer due to formation of carcinogenic compounds including benzo[<i>a</i>]pyrene (BaP). Assessment of a population’s risk of disease and people’s individual probability of disease given specific consumer attributes may direct food safety strategies to where impact on public health is largest. The aim of this study was to propose a model that estimates the risk of cancer caused by exposure to BaP from barbecued meat in Denmark, and to estimate the probability of developing cancer in subgroups of the population given different barbecuing frequencies.</p><p>Methods</p><p>We developed probabilistic models applying two dimensional Monte Carlo simulation to take into account the variation in exposure given age and sex and in the individuals’ sensitivity to develop cancer after exposure to BaP, and the uncertainty in the dose response model. We used the Danish dietary consumption survey, monitoring data of chemical concentrations, data on consumer behavior of frequency of barbecuing, and animal dose response data.</p><p>Findings</p><p>We estimated an average extra lifetime risk of cancer due to BaP from barbecued meat of 6.8 × 10⁻⁵ (95% uncertainty interval 2.6 × 10⁻⁷ − 7.0 × 10⁻⁴) in the Danish population. This corresponds to approximately one to 4,074 extra cancer cases over a lifetime, reflecting wide uncertainty. The impact per barbecuing event on the risk of cancer for men and women of low body weight was higher compared to higher bodyweight. However, the difference due to sex and bodyweight between subgroups are dwarfed by the uncertainty.</p><p>Interpretation</p><p>This study proposes a model that can be applied to other substances and routes of exposure, and allows for deriving the change in risk following a specific change in behaviour. The presented methodology can serve as a valuable tool for risk management, allowing for the formulation of behaviour advice targeted to specific sub-groups in the population.</p></div

In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry

Microvesicles (MVs) are submicron vesicles with sizes of 0.1–1.0 μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5 μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5 μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as <1 μm and positive for lactadherin binding and cell-specific markers. Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41(+) and CD41(+)CD36(+) MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of standardized protocols for MV quantification by flow cytometry

The effect of oxLDL on microvesicle release from platelets, measured by a sensitive flow cytometry method.

Microvesicles (MVs) are submicron vesicles with sizes of 0.1-1.0-µm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxLDL, a platelet ligand, induce platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5-µm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5-µm limit. MV size-distribution was analysed in plasma from 11 healthy volunteers (4 females, 7 males). MVs were identified as < 1-μm and positive for lactadherin binding and cell specific markers. Platelet rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by 6-8 fold in PRP, when left at room temperature, and the presence of cell specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large inter-individual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that pro-coagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great inter-individual variability in size-distribution of platelet derived MVs and thereby stresses the importance for generation of standardized protocols for MV quantification by flow cytometry