Epicardial, paracardial, and perivascular fat quantity, gene expressions, and serum cytokines in patients with coronary artery disease and diabetes

INTRODUCTION Obesity and diabetes mellitus (DM) are common disorders that increase cardiovascular risk and lead to coronary artery disease (CAD). OBJECTIVES The aim of our study was to assess the link between epicardial fat (EF) volume and paracardial fat (PF) volume, relative expressions of several genes in epicardial, paracardial, and perivascular fat and corresponding serum cytokines in patients with CAD in relation to DM. PATIENTS AND METHODS A total of 66 consecutive patients (33 with DM) with multivessel CAD were included. We obtained cardiac magnetic resonance, serum cytokines levels, and their relative mRNA expressions in EF, PF, and perivascular fat samples of the following: adrenomedullin (ADM), fibroblast growth factor 21 (FGF21), transforming growth factor β (TGFβ), phospholipid transfer protein (PLTP), receptor for advanced glycation endproducts (RAGE), thrombospondin 1 (THSB1), and uncoupling protein 1 (UCP1). RESULTS There were no differences in the anthropometric parameters or fat depots, except for higher epicardial fat volume in patients with DM (mean [SD], 105.6 [38.5] ml vs 84 [29.2] ml; P = 0.02). Patients with DM exhibited a significantly increased RAGE expression in EF (median [Q1–Q3], 0.17 [0.06–1.48] AU vs 0.08 [0.02–0.24] AU, P = 0.03). Diabetes was also associated with increased expression of ADM in EF and PF and decreased expression of FGF21 compared with patients without DM. CONCLUSIONS Patients with multivessel CAD and DM revealed increased volume and more dysfunctional profile of gene expressions in EF and significantly decreased expression of cardioprotective FGF21

Efficacy and complications of open and minimally invasive surgery in acute Achilles tendon rupture: a prospective randomised clinical study—preliminary report

PURPOSE: Surgical treatment of an acute Achilles tendon rupture can effectively reduce the risk of re-rupture, but it increases the probability of surgical complications. We postulated that a minimally invasive surgical treatment might reduce the number of complications related to open surgery and improve the functional results. METHOD: We enrolled 47 patients with acute Achilles tendon ruptures in a prospective, randomised trial to compare clinical results and complications between a minimally invasive procedure with the Achillon® device and traditional open surgery with Krackow-type sutures. The average patient age was 46 years. The follow up time was 24 months. RESULTS: No Achilles tendon re-rupture or nerve injury occurred in treated patients. There were two cases of wound infections in the open surgery group, and one superficial wound infection occurred in the minimally invasive group. The groups were not significantly different in the amount of pain, range of ankle movements, the single heel-rise test, calf circumference, or time to return to work and sports. CONCLUSION: After a two year follow-up period, we found no significant differences in clinical outcomes between groups treated with traditional open surgery or minimally invasive surgery

Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

<div><p>The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.</p></div

Effects of SR-A or CD36 deficiency on antigen uptake, expression of endocytic receptors and antigen presentation to CD4⁺ OT-II splenocytes.

<p>(<b>A</b>, <b>D</b>) Uptake of indicated, fluorescently labelled proteins, present at 5 μg/ml by BM-DC (A) and PEM (D) was assessed by flow cytometry. (<b>B</b>, <b>E</b>) Specific binding of Ab to receptors (geometric mean fluorescence intensity) on BM-DC (B) and PEM (E) which was obtained by subtracting binding of control Ab from the total binding of receptor-specific Ab. (<b>C</b>, <b>F</b>) IL-2 production in 1- or 2-days co-cultures of BM-DC (C) or PEM (F) with CD4⁺ OT-II lymphocytes. Directly before the co-incubation with lymphocytes, APC were pulsed for 3.5 h with 20 μg/ml OVA or 7 μg/ml OVA-Cl. The data shown on graphs A-G are means +SEM from 6–8 independent experiments. (<b>H</b>) Titers of OVA- or HSA-specific IgM in sera of mice immunized 8 days earlier with 20 μg OVA-Cl or HSA-Cl. Points represent titer values in individual mice and horizontal lines geometric means. The data were analysed with the regular (H) or repeated measures (A-G) ANOVA and the Dunnett’s post-test was used to make comparisons with the control groups (WT). *, p < 0.05.</p

LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.

<p>(<b>A</b>) Binding of rLOX-1 to plate-adsorbed proteins. (<b>B</b>, <b>C</b>) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (<b>D</b>) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (<b>E</b>) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (<b>F</b>) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.</p

The proposed mechanism of the immunoenhancing effect caused by HOCl-mediated oxidation of protein antigens.

<p>Administration of adjuvants, infection or sterile injury trigger acute inflammation, characterized by recruitment and activation of neutrophils. Activated neutrophils produce HOCl which causes non-selective oxidation of both self and, if present, pathogen-derived proteins, subsequently endocytosed by DC through MR and SR-A, processed and presented as complexes with MHC-II on their surface. TCR-mediated cognate interactions of Th lymphocytes with peptide-MHC-II complexes on DC induce IL-2 production in Th lymphocytes (not shown) and up-regulate expression of CD40 on DC and of CD40L on lymphocytes. Upon ligation with CD40L, CD40 induces expression of CD86, a ligand for CD28 on Th lymphocytes. In the absence of PRR ligands, presentation of low density of peptide-MHC-II complexes on DC stimulates differentiation of naive Th lymphocytes towards Th2 cells. The Th2 polarization is reinforced by intracellular signalling triggered upon binding of HOCl-oxidised proteins to SR-A or MR, leading to the suppression of IL-12 and enhancement of IL-10 production by DC.</p

OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.

<p>(<b>A</b>) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 10⁵ BM-DC were co-cultured for 2 or 3 days with 1.5 × 10⁵ CD4⁺ OT-II lymphocytes. One μCi of ³H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (<b>B</b>) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (<b>C</b>) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 10⁵/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (<b>D</b>) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.</p

LPS and SR-A ligands regulate uptake, acidification and proteolysis of endocytosed antigens.

<p>(<b>A</b>) Effects of 1-day pre-treatment with 100 ng/ml LPS on the total uptake of AF-OVA-Cl, internalisation of pHr-OVA-Cl and degradation of DQ-OVA by BM-DC and PEM. (<b>B</b>, <b>C</b>) Effects on indicated ligands on the acidification of pHr-OVA-Cl-containing endosomes (B) and proteolytic digestion of DQ-OVA (C) in BM-DC and PEM. The results shown are averages +SEM from 3 independent experiments, each performed in 4 replicates. The data were analysed by the Student’s t-test (A) or by ANOVA, with the Dunnett’s post-test applied to compare the control (“medium”) with other groups (B, C). *, p < 0.05.</p