Elevated sodium leads to the increased expression of HSP60 and induces apoptosis in HUVECs

<div><p>Atherosclerosis is the leading cause of death in the world. We have previously shown that expression of heat shock protein 60 (HSP60) on the surface of endothelial cells is the main cause of initiating the disease as it acts as a T cell auto-antigen and can be triggered by classical atherosclerosis risk factors, such as infection (e.g. <i>Chlamydia pneumoniae</i>), chemical stress (smoking, oxygen radicals, drugs), physical insult (heat, shear blood flow) and inflammation (inflammatory cytokines, lipopolysaccharide, oxidized low density lipoprotein, advanced glycation end products). In the present study, we show that increasing levels of sodium chloride can also induce an increase in intracellular and surface expression of HSP60 protein in human umbilical vein endothelial cells. In addition, we found that elevated sodium induces apoptosis.</p></div

Quantification of total cells and apoptotic cells in HUVECs treated with increasing sodium concentration.

<p>(A) Representative images of HUVECs treated with 137 mM and 188 mM sodium, fixed with 2% PFA + MetOH and stained with Hoechst and anti-β-actin A488, and their subsequent enumeration using FIJI, as explained in Material and Methods. The number of cells per image is displayed in red under particle analysis. Scale bar = 100 μm. (B) Scatter plot of enumeration from acquired images of HUVECs treated with increasing sodium concentrations. Each dot represents number of cells per image. Mean ± SEM (C) Linear regression of values shown in (B). Mean ± SD (D) Representative flow cytometric gating strategies for enumeration of cells and Annexin V⁺ cells (apoptotic cells). Linear regression of FACS results for number of cells and percentage of apoptotic cells. Mean ± SD (E) Summary of flow cytometric enumeration of live and apoptotic cells (Annexin V⁺). The percentage of apoptotic cells within the total number of cells is shown on top of the bars. Data are from one experiment (n = 3). ***<i>p</i><0.001. CTCF = corrected total cell fluorescence. mM = millimolar. B&W = black and white. No. = number.</p

Surface staining of HSP60 on HUVECs treated with increasing sodium concentrations.

<p>(A) Representative images of nuclei (Hoechst), β-actin (A488) and HSP60 (A568) on HUVECs fixed with 2% PFA + MetOH or with 1% PFA. Scale bar = 50 μm. (B) MFI of surface HSP60 expression out of CD31⁺ endothelial cells. MFI of three donors ± SEM is shown. One-way ANOVA analysis was performed, p-value = 0.3916. (C) Representative images of surface staining of HSP60 under each experimental condition. Scale bar = 50 μm. (D) Quantification of surface HSP60 fluorescence staining intensity as explained in the Material and Methods. Mean CTCF values ± SEM combined from two independent experiments. Each dot represents the CTCF readout from one donor (n = 6). (E) Linear regression of values presented in (D). Dotted lines show the 95% confidence interval. Mean ± SD. CTCF = corrected total cell fluorescence. mM = millimolar. MFI = median fluorescence intensity.</p

Intracellular HSP60 expression in HUVECs treated with increasing sodium concentrations.

<p>(A) Representative images of nuclei (Hoechst) and HSP60 (A568) staining under each experimental condition, on HUVECs fixed with 2% PFA + MetOH. Scale bar = 20 μm. (B) Quantification of HSP60 fluorescence staining intensity as explained in Material and Methods. Mean CTCF values ± SEM combined from two independent experiments. Each dot represents the CTCF readout from one donor (n = 6). (C) Linear regression of values presented in (B). Dotted lines show 95% confidence intervals. Mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01. CTCF = corrected total cell fluorescence. mM = millimolar.</p

Correlation between HSP60 expression and apoptosis in HUVECS.

<p>Pearson correlation analysis. shows a significant correlation of intracellular and surface expression of HSP60 with the number of apoptotic cells, as determined by flow cytometry. Each dot represents the mean CTCF and corresponding percentage of apoptosis (Annexin V⁺) of 6 samples. CTCF = corrected total cell fluorescence.</p

Analisis Pohon Berstruktur Menggunakan Metode CHAID pada Data Respons Ordinal

Eksplorasi data merupakan suatu hal yang penting dilakukan sebelum menganalisa data dengan metode lain. Salah satu metode eksplorasi untuk data respons kategorik adalah metode CHAID (Chi-square Automatic Interaction Detection). Tulisan ini berisi tentang kajian teori metode CHAID dan penerapannya yaitu menganalisis faktor-faktor yang berpengaruh pada peningkatan omset USAha anggota koperasi simpan pinjam (KSP) sebagai respons ordinal. Hasil kajian menunjukkan bahwa metode CHAID tidak hanya dapat digunakan sebagai metode eksplorasi tetapi juga mampu menggunakan struktur hubungan antara variabel respons kategorik dengan serangkaian variabel penjelas serta interaksi antar variabel penjelas. Struktur hubungan ini digambarkan dengan suatu pohon berstruktur

Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin

<div><p>Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and (c) demonstrated the immunomodulatory effect of ATG under these experimental circumstances by prolongation of heart allograft survival.</p></div

Immunophenotypic characterization of human T cells after <i>in vitro</i> exposure to different silicone breast implant surfaces - Fig 3

<p><b>IL-1β (A) IL-6 (B), and TNF-α(C) levels in the supernatants collected from PBMC cultured on 8 surfaces as indicated.</b> Results are expressed as mean ± SEM (n = 5). Each data point represents an individual donor. Friedman ANOVA test was used to compare the means (Dunn’s multiple comparison test; *p<0.05, ***p<0.001).</p

Immunophenotype of CD8+T cells cultured on silicone surfaces.

<p>A) Representative FACS plots showing gating strategy of 4 different CD8+ T cells. A viability dye was used to discriminate live and dead cells. After gating on CD3+ T cells, naïve /memory cells were discriminated on the basis of expression of CD45RA and CD197 markers, respectively. B) PBMC from seven donors were cultured on implants with different surfaces for 4 days. Cells were gated on CD8+ cells: naïve (N, CD45RA⁺CD197⁺), central memory (CM, CD45RA^-CD197⁺), effector memory (EM, CD45RA^-CD197^-) and terminally differentiated central memory cells (TEMRA, CD45RA⁺CD197^-) were determined and shown as % of CD8+. Three independent experiments (2–3 donors/experiment) were performed. Each data point represents an individual donor. Results are expressed as mean ± SEM. Friedman's ANOVA test was used to compare the means (Dunn’s multiple comparison test; * p<0.05,** p<0.01).</p

Murine cervical heart allotransplantation.

<p>BALB/c huCD3Ɛ were injected i.v with ATG or control rabbit Ig. Cervical heart transplantation was performed and graft function was evaluated by daily regular heart palpation. (A) Cardiac graft function and (B) graft survival (days). Graft function was expressed as the beating score, 0 no organ function, 1: fibrillation, only visible through magnification, 2: poor or partial organ function, 3: impairment in frequency or intensity of heart beating, 4: physiological organ function). Four to five mice per group, two independent experiments were performed. Data are shown as means ± SEM. Mann-Whitney statistical test was used, *p<0.05, ** p<0.001.</p