Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc gammaRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc gammaRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc gammaRI and Fc gammaRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc gammaRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc gammaRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.     

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P₂. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.     

Lipofection of Insulin-Producing RINm5F Cells: Methodological Improvements

Cationic lipid/DNA-complexes have been widely used as gene transfer vectors because they are less toxic and immunogenic than viral vectors. The aim of the present study was to improve and characterize lipofection of an insulin-producing cell line. We compared the transfection efficiency of seven commercially available lipid formulations (Lipotaxi, SuperFect, Fugene, TransFast, Dosper, GenePORTER and LipofectAMINE) by flow cytometry analysis of GFP-expression. In addition, we have determined the influences of centrifugation, serum and a nuclear localization signal peptide on the lipofection efficiency. We observed that two lipid formulations, GenePORTER and LipofectAMINE, were able to promote efficient gene transfer in RINm5F cells. However, GenePORTER exhibited the important advantage of being able to transfect cells in the presence of serum and with less cytotoxicity than LipofectAMINE. LipofectAMINE-induced RINm5F cell death could partially be counteracted by TPA, forskolin or fumonisin β₁. Finally, both centrifugation and a nuclear localization signal peptide increased transfection efficiency.     

Variables Involved in the Discordance between HbA1c and Fructosamine: The Glycation Gap Revisited

Aims

Glycation gap (GG) is defined as the difference between the measured level of HbA1c and the level that would be predicted from its regression on the fructosamine level. The aims of the study were: 1) To determine the reproducibility and consistency of GC; 2) To discover factors related to GG value. Given that metformin might increase glucose transport through the erythrocyte membrane, this treatment was also considered in the analyses of the results.

Methods

GG was calculated in two blood samples separated 30.6 (SD 7.3) weeks, obtained in 508 type 2 diabetic patients. The following variables were considered: HbA1c, fructosamine, glucose, creatinine, hematological parameters and treatment with metformin. Multivariate and logistic regression analyses were performed to explore the variables independently related to CG.

Results

GG was reproducible and consistent over time. Creatinine, mean corpuscular hemoglobin concentration (MCHC), and glycemia (inverse relationship); and HbA1c and treatment with metformin (direct relationship) were independently related to GG. Patients treated with metformin showed higher HbA1c values, despite similar fructosamine concentrations, than patients not treated with the drug.

Conclusions

GG is independently related to serum levels of creatinine, MCHC and treatment with metformin. The spurious effect of metformin on Hb glycation could have serious clinical implications and should be considered when interpreting the results of clinical trials.     

Combination of Bortezomib and Mitotic Inhibitors Down-Modulate Bcr-Abl and Efficiently Eliminates Tyrosine-Kinase Inhibitor Sensitive and Resistant Bcr-Abl-Positive Leukemic Cells

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.     

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched.Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner.Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.     

Adiposity and hand osteoarthritis: the Netherlands Epidemiology of Obesity study

Introduction

Obesity, usually characterized by the body mass index (BMI), is a risk factor for hand osteoarthritis (OA). We investigated whether adipose tissue and abdominal fat distribution are associated with hand OA.

Methods

The Netherlands Epidemiology of Obesity (NEO) study is a population-based cohort aged 45 to 65 years, including 5315 participants (53% women, median BMI 29.9 kg/m²). Fat percentage and fat mass (FM) (kg) were estimated using bioelectrical impedance analysis. The waist-to-hip ratio (WHR) was calculated. In 1721 participants, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) (cm²) were assessed using abdominal MR imaging. Hand OA was defined according to the ACR criteria.Odds ratios (OR) with 95% confidence intervals (CI) were calculated for the association of fat percentage, FM, WHR, VAT and SAT with hand OA using logistic regression analyses per standard deviation, stratified by sex and adjusted for age.

Results

Hand OA was present in 8% of men and 20% of women. Fat percentage was associated with hand OA in men (OR 1.34 (95% CI 1.11 to 1.61)) and women (OR 1.26 (1.05 to 1.51)), as was FM. WHR was associated with hand OA in men (OR 1.45 (1.13 to 1.85)), and to a lesser extent in women (OR 1.17 (1.00 to 1.36)). Subgroup analysis revealed that VAT was associated with hand OA in men (OR1.33 (1.01 to 1.75)). This association increased after additional adjustment for FM (OR 1.51 (1.13 to 2.03)).

Conclusions

Fat percentage, FM and WHR were associated with hand OA. VAT was associated with hand OA in men, suggesting involvement of visceral fat in hand OA.     

Inferring on the Intentions of Others by Hierarchical Bayesian Learning

Inferring on others'' (potentially time-varying) intentions is a fundamental problem during many social transactions. To investigate the underlying mechanisms, we applied computational modeling to behavioral data from an economic game in which 16 pairs of volunteers (randomly assigned to “player” or “adviser” roles) interacted. The player performed a probabilistic reinforcement learning task, receiving information about a binary lottery from a visual pie chart. The adviser, who received more predictive information, issued an additional recommendation. Critically, the game was structured such that the adviser''s incentives to provide helpful or misleading information varied in time. Using a meta-Bayesian modeling framework, we found that the players'' behavior was best explained by the deployment of hierarchical learning: they inferred upon the volatility of the advisers'' intentions in order to optimize their predictions about the validity of their advice. Beyond learning, volatility estimates also affected the trial-by-trial variability of decisions: participants were more likely to rely on their estimates of advice accuracy for making choices when they believed that the adviser''s intentions were presently stable. Finally, our model of the players'' inference predicted the players'' interpersonal reactivity index (IRI) scores, explicit ratings of the advisers'' helpfulness and the advisers'' self-reports on their chosen strategy. Overall, our results suggest that humans (i) employ hierarchical generative models to infer on the changing intentions of others, (ii) use volatility estimates to inform decision-making in social interactions, and (iii) integrate estimates of advice accuracy with non-social sources of information. The Bayesian framework presented here can quantify individual differences in these mechanisms from simple behavioral readouts and may prove useful in future clinical studies of maladaptive social cognition.