Inhibition of calpains in both host and melanoma cells (global inhibition).

<p>C57BL/6 control (WT mice) and transgenic mice (CalpTG mice) were injected with one million melanoma B16-F10 cells transfected with control plasmid (WT/Ctrl-Mel) and calpastatin plasmid (TG/Calpast-Mel) respectively. Mice were sacrificed at day 16 for tissue analysis. <b>A</b>. Tumor growth was measured from day 9 to day 16. Calpastatin overexpression in both hosts and melanoma decreased significantly tumor growth. N = 10/group, * p<0.05. <b>B</b>. Tumor weight at day 16. Calpastatin overexpression in both hosts and melanoma decreased non-significantly tumor weight. N = 10/group, p = NS. <b>C</b>. Angiogenesis assessed by vessel count at 200×magnification after CD-31 staining. TG/Calpast-Mel mice had a trend to have less neo-angiogenesis than WT mice. N = 10/group, p = NS. <b>D,E,F,G.</b> CD3, CD4, CD8 and NK cell number/HPF (200×magnification). Immune cell infiltrate was significantly lower in TG/calpast Mel mice than in WT/Ctrl mice. N = 10/group, * p<0.05. <b>H.</b> Proportion of metastatic regional lymph nodes at day 16. TG/Calpast-Mel mice had a trend to have more metastatic lymph nodes than WT mice (9/10 vs 4/10 respectively, p = NS).</p

Survival studies.

<p><b>A</b>. Specific limitation of calpain activity in melanoma cells only by calpastatin overexpression in vivo. C57BL/6 WT mice were injected with one million melanoma B16-F10 cells either transgenic for calpastatin (Calpast-Mel) or transfected with a control plasmid (Ctrl-Mel), n = 10/group. Limitation of calpain activity in melanoma cells only did not modify survival at day 30. <b>B</b>. Specific limitation of calpain activity in mice transgenic for calpastatin. C57BL/6 control (WT mice) or transgenic mice (CalpTG mice) were injected with one million control melanoma B16F10 cells, n = 10/group. Limitation of calpain activity in host did not modify survival at day 30.</p

Inhibition of calpains in melanoma cells only: an in vivo approach.

<p>C57BL/6 WT mice were injected with one million melanoma B16-F10 cells either transgenic for calpastatin (Calpast-Mel) or transfected with a control plasmid (Ctrl-Mel) and sacrificed at day 16 for tissues analysis. <b>A</b>. Tumor growth was measured from day 9 to day 16. Calpastatin overexpression in melanoma decreased significantly tumor growth. N = 10/group, * p< 0.05. <b>B</b>. Tumor weight at day 16. Calpastatin overexpression reduced significantly melanoma weight. N = 10/group, * p<0.05. <b>C</b>. Proportion of metastatic regional (axillary) lymph nodes at day 16. Mice injected with melanoma cells with reduced calpain activity (Calpast-Mel) had significantly more metastatic lymph nodes than controls (Ctrl-Mel) (10/10 vs 5/10 respectively, * p<0.05). <b>D</b>. Angiogenesis assessed by vessel count at 200×magnification after CD-31 (PECAM) staining. Neo-angiogenesis was similar in transgenic and control melanomas. N = 10/group, p = NS. <b>E,F,G,H</b>: CD3, CD4, CD8 and NK cell number/HPF (200×magnification). Immune cell infiltrate was similar in transgenic and control tumors. N = 10/group, p = NS.</p

From arterial stiffness to kidney graft microvasculature: Mortality and graft survival within a cohort of 220 kidney transplant recipients

<div><p>Background</p><p>Aortic stiffness assessed by carotid-femoral pulse wave velocity (CF-PWV) is a predictor of mortality in several populations. However, little is known in kidney transplant recipients. Our objectives were to evaluate the ability of CF-PWV measured 3 months following transplantation to predict mortality, graft loss and its potential links to measured Glomerular Filtration Rate (mGFR) or kidney graft microvasculature parameters.</p><p>Methods</p><p>The study is based on a monocentric retrospective cohort including 220 adult kidney graft recipients evaluated three months after transplantation. CF-PWV measures, clinical, laboratory and histological data performed at 3 (M3) and 12 months (M12) following transplantation were retrospectively collected. The two primary endpoints were all-cause mortality and occurrence of end stage renal disease (ESRD) defined by initiation of dialysis.</p><p>Results</p><p>After a median follow up of 5.5 years [1.9; 8.8], death and graft loss occurred in 10 and 12 patients respectively. M3 CF-PWV was an independent mortality risk factor (HR = 1.29 [1.03; 1.61]; p = 0.03), despite no aortic stiffness variation during the first year of transplantation. Of notice, M3 CF-PWV was not associated with M12 mGFR or ESRD outcome. Graft microcirculation assessed by Banff vascular fibrous intimal thickening score (cv) worsened between M3 and M12 (p = 0.01), but no link was found with CF-PWV, mGFR or ESRD outcome. Surprisingly, acute rejections at M3 were associated after adjustment with mortality (p = 0.03) but not ESRD.</p><p>Conclusion</p><p>Aortic stiffness measured 3 months after kidney transplantation is a strong predictor of mortality with no obvious influence on kidney graft microvasculature or graft loss.</p></div

Inhibition of calpains in host mice only.

<p>C57BL/6 control (WT mice) or transgenic mice (CalpTG mice) were injected with one million melanoma B16F10 cells and sacrificed at day 16 for tissue analysis.<b>A</b>. Tumor growth was measured from day 11 to day 16. Calpastatin overexpression in host did not modify tumor growth. N = 10/group. <b>B</b>. Tumor weight at day 16. Calpastatin overexpression in hosts did not modify tumor weight. N = 10/group, p = NS. <b>C</b>. Proportion of metastatic regional lymph nodes at day 16. CalpTG mice had a trend to have more metastatic lymph nodes than WT mice (9/10 vs 4/10 respectively, N = 10/group, p = NS). <b>D,E,F</b>. Angiogenesis as assessed by vessel count at 200×magnification after CD-31 staining. Neo-angiogenesis was significantly decreased in CalpTG mice when compared to WT mice. N = 10/group, * p<0.05. <b>G,H,I,J</b>: CD3, CD4, CD8 and NK cell number/HPF (200×magnification). Immune cell infiltrate was significantly lower in CalpTG mice than in WT mice. N = 10/group, * p<0.05.</p

Crude hazard ratios (95% IC) of death and ESRD.

<p>Crude hazard ratios (95% IC) of death and ESRD.</p

Comparison of clinical and demographical characteristics at baseline according to M3 CF-PWV threshold of 10.6 m/s.

<p>Comparison of clinical and demographical characteristics at baseline according to M3 CF-PWV threshold of 10.6 m/s.</p

Survival study and kidney injury in 129S2/SvPasCrl and C57BL/6J mice.

<p><b>A</b>, Survival proportions of mice from both strains before sacrifice at 16 weeks. 129S2/SvPasCrl mortality was significantly increased (p = 0.045) and necropsy revealed kidney dilation due to urolithiasis. <b>B</b>, Infrared cartography of 129S2/SvPasCrl kidney slices revealed the presence of focal cystine aggregates in renal tissues (cystine tubular casts). <b>C</b>, Renal function as assessed by enzymatic serum creatinine dosage was not significantly impaired in 129S1/SvPasCrl mice (n = 6) compared to C57BL/6J mice (n = 9) at 16 weeks (p = 0.11). <b>D–F</b>, Macrophage infiltrate in kidney tissues was assessed by morphometric analyses of F4-80 immunostaining and was increased in 129S2/SvPasCrl mice in comparison with C57BL/6J (n = 5/group, p = 0.046). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">Figures 3E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">3F</a> are representative of macrophage infiltrates in C57BL/6 and 129S1/SvPasCrl mice respectively (magnification x200+ zoom). <b>G</b>, Fibrosis assessed by sirius red morphometric analysis did not evidence significant fibrosis amount in both strains (percentage of fibrotic area, p = NS).</p

Identification of a functional missense mutation in a highly conserved sequence of the <i>Slc3a1</i> gene in 129S2/SvPasCrl mouse.

<p><b>A</b>, Mutation of G by A nucleotide at position 1232 in 129S2/SvPasCrl mouse (c.1232G>A). <b>B</b>, In position 383, the glutamine is substituted by a lysine in the extracellular part of rBAT protein in 129S2/SvPasCrl mouse. The sequence is highly conserved among mammal species. <b>C</b>, 129S1/Sv mice from the Jackson laboratory express rBAT at the brush border of proximal tubular cells. <b>D</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 10 weeks were affected by urolithiasis whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.047). <b>E</b>, One hundred percent of 129S2/SvPasCrl mice presented cystine crystals in urine whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.008). <b>F</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice and 129S2/SvPas mice (n = 5/group). Cysteine (reduced form of cystine) was significantly higher in 129S2/SvPasCrl mice urine (p = 0.008). Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

From arterial stiffness to kidney graft microvasculature: Mortality and graft survival within a cohort of 220 kidney transplant recipients - Fig 2

<p>Unadjusted Kaplan-Meier event curves for all-cause of mortality (a) or kidney graft loss (b) according to M3 CF-PWV threshold of 10.6 m/s.</p