A placebo-controlled proof-of-concept study of alirocumab on postprandial lipids and vascular elasticity in insulin-treated patients with type 2 diabetes mellitus.

AIM: Type 2 diabetes mellitus (T2DM) is associated with an increased risk of cardiovascular disease (CVD) linked to atherogenic dyslipidaemia and postprandial hyperlipidaemia. Alirocumab, a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor, improves CVD risk by reducing the concentration of low-density lipoprotein-cholesterol (LDL-C). However, effects of PCK9 inhibitors on other aspects of diabetic dyslipidaemia, particularly in the postprandial situation, are less clear. MATERIAL AND METHODS: Twelve male patients with T2DM on an intensive insulin regimen completed a 6-week randomized, double-blind, placebo-controlled, proof-of-concept study. Participants received three biweekly dosages of subcutaneous alirocumab (150 mg) or placebo. Before and after the intervention, fasting and postprandial triglyceride (TG) plasma levels, apolipoprotein (apo) B48, lipoprotein composition isolated by ultracentrifugation, vascular function and markers of inflammation were evaluated. RESULTS: Alirocumab treatment reduced fasting plasma TG levels (between group median change -24.7%; P = 0.018) and fasting apoB48 serum levels (-35.9%; P = 0.039) compared with placebo. Alirocumab reduced the plasma TG area under the curve (AUC) (-26.4%; P = 0.006) and apoB48 AUC (-55.7%; P = 0.046), as well as plasma TG incremental AUC (-21.4%; P = 0.04) and apoB48 incremental AUC (-26.8%; P = 0.02). In addition, alirocumab reduced fasting and postprandial TG levels in very low-density lipoprotein (VLDL) and LDL. Alirocumab improved fasting pulse wave velocity, but no changes in postprandial markers of inflammation were observed. CONCLUSIONS: In addition to the well-known LDL-C-reducing effects, 6 weeks of alirocumab treatment lowered both fasting and postprandial plasma TG levels by reducing the TG levels in VLDL and LDL and the concentration of intestinal remnants.

The alternative Thomas-plot: A new tool for effective anemia diagnostics.

INTRODUCTION: The Thomas-plot has proven to be a helpful tool to discriminate between different types of anemia. This plot combines the reticulocyte hemoglobin content (Ret-He) with the soluble transferrin receptor (sTfR)/log ferritin (fer) ratio. In this study, we designed an alternative Thomas-plot in which Ret-He is combined with the transferrin (Tf)/log ferritin ratio. We validated both Thomas-plots in a population of anemic patients and compared the performance to the current laboratory diagnostics of anemia. METHODS: A total of 536 anemic patients were included. The first 188 patients were used to generate ROC curves to define the optimal cut-off values for both Thomas-plots. With the following 348 patients included we studied the performance of the alternative and classical Thomas-plots compared to current anemia diagnostics. RESULTS: Cut-off values were defined (Ret-He: 31.2 pg, sTfR/log(fer): 0.91, and Tf/log(fer): 1.71). With both Thomas-plots the amount of e causa ignota (ECI) cases dropped from 39% to 27%. A more in depth analysis on the iron status of anemia of chronic disease (ACD) patients and a subdivision between latent and classical iron deficiencies could be made with the help of both plots. A shift from classical iron deficiency anemia (IDA) cases according to the classical Thomas-plot toward functional IDA according to the alternative Thomas-plot was observed. CONCLUSION: The alternative Thomas-plot is an effective tool that gives a more in depth view on the iron status of anemic patients. In addition, it is easier to implement due to the use of transferrin rather than the soluble transferrin receptor.

Effects of dapagliflozin on postprandial lipid metabolism in type 2 diabetes mellitus.

Objectives: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) modulate lipid metabolism and improve cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). The exact cardioprotective mechanism of SGLT2i is unclear. We evaluated the effects of SGLT2i on postprandial lipids, lipoprotein concentrations, glucose and fatty acids. Design: A placebo-controlled randomized, proof-of-concept study. Methods: Fourteen male patients with T2DM on intensive insulin regimen were randomly and double-blind allocated to 12 weeks dapagliflozin (10 mg) or placebo. Postprandial effects were assessed with an 8-h standardized oral fat loading test. Results: Mean glycated A1c did not change by dapagliflozin, but the mean daily insulin dose was significantly reduced. Although dapagliflozin did not affect fasting or postprandial levels of glucose and insulin, it increased the postprandial levels of glucagon. While fasting levels of free fatty acids and beta-hydroxybutyrate (bHBA) were unchanged, dapagliflozin significantly increased the postprandial bHBA response. This was seen in the context of increased postprandial glucagon levels by dapagliflozin, without influencing postprandial insulin or glucose levels. Dapagliflozin did not affect fasting or postprandial plasma cholesterol and triglycerides nor postprandial inflammatory markers. Fasting apolipoprotein B48 was decreased without affecting the postprandial response. Markers of inflammation and vascular function did not change. Conclusion: Treatment with dapagliflozin of patients with T2DM led to a reduction of fasting chylomicron remnants and increased postprandial ketone bodies compared to placebo suggesting enhanced hepatic fatty acid oxidation. The latter may have been caused by decreasing the insulin-glucagon ratio. The beneficial clinical effects seen in the trials using dapagliflozin most likely are not due to effects on postprandial inflammation nor postprandial lipemia.

Gene transfer of human TCR in primary murine T cells is improved by pseudo-typing with amphotropic and ecotropic envelopes.

BACKGROUND: T cell receptor (TCR) gene therapy represents an attractive anti-cancer treatment but requires further optimization of its efficacy and safety in clinically relevant models, such as those using a tumor antigen and TCR of human origin. Currently, however, there is no consensus as to what protocol is most optimal for retroviral human TCR gene transfer into primary murine T cells, most notably with respect to virus pseudo-type. METHODS: Primary murine T cells were transduced, expanded and subsequently tested for transgene expression, proliferation and antigen-specific function. To this end, murine leukemia virus (MLV) retroviruses were produced upon transfection of various packaging cells with genes encoding either green fluorescent protein (GFP) or TCRalphabeta specific for human melanoma antigen gp100(280-288) and the helper elements GAG/POL and ENV. Next to viral pseudotyping, the following parameters were studied: T cell densities; T cell activation; the amounts of IL-2 and the source of serum used to supplement medium. RESULTS: The pseudo-type of virus produced by packaging cells critically determines T cell transduction efficiencies. In fact, MLV-A and MLV-E pseudo-typed viruses derived from a co-culture of Phoenix-A and 293T cells resulted in T cell transduction efficiencies that were two-fold higher than those based on retroviruses expressing either VSV-G, GALV, MLV-A or MLV-E envelopes. In addition, T cell densities during transduction were inversely related to transduction efficiencies. Further optimization resulted in transduction efficiencies of over 90% for GFP, and 68% for both a murine and a human (i.e. murinized) TCR. Importantly, TCR-transduced T cells proliferate (i.e. showing a log increase in cell number in a few days) and show antigen-specific function. CONCLUSIONS: We set up a quick and versatile method to genetically modify primary murine T cells based on transient production of TCR-positive retroviruses, and show that retroviral gene transfer of a human TCR into primary murine T cells is critically improved by viral pseudo-typing with both MLV-A and MLV-E envelopes.