MR delayed enhancement imaging findings in suspected acute myocarditis.

International audienceThe purpose of the study was to prospectively assess the clinical impact of routinely performed delayed enhancement imaging in suspected acute myocarditis. A two-centre prospective study was performed in patients with suspected acute myocarditis. The protocol included horizontal long axis, vertical long axis and short axis ciné MR and delayed enhancement imaging after Gd-DTPA infusion (0.2 mmol/kg). Sixty consecutive patients were enrolled (aged 49.4 +/- 17.8 years). MRI demonstrated delayed enhancement sparing the subendocardicardial layer in 51.6% of patients, concordant with the diagnosis of acute myocarditis; 16.7% of patients exhibited delayed enhancement involving the subendocardial layer with irregular margins, concordant with the diagnosis of acute myocardial infarction; 31.7% of patients had delayed enhancement imaging that was considered normal. Routine imaging to identify delayed enhancement provided crucial information in suspected acute myocarditis by reinforcing the diagnosis in 51.6% of patients and correcting a misdiagnosed acute myocardial infarction in 16.7% of patients

Identification of pVHL as a novel substrate for Aurora-A in clear cell renal cell carcinoma (ccRCC).

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer and is often characterized by mutations or deletions of the Von Hippel Lindau (VHL) tumour suppressor gene. Aurora gene family members are implicated in proper mitotic progression and spindle checkpoint function and play a crucial role in cancer progression. In the present study, we assessed the expression of Aurora-A in a cohort of 30 ccRCC with fully characterized VHL status (wt/wt or mut/del) and Fuhrman grade. Aurora-A transcript and protein levels were significantly increased in high Fuhrman grade tumours and in VHLwt/wt tumours. These results suggest that Aurora-A and VHL interact in the ccRCC. We demonstrated that the two proteins interact in vivo and identified the Ser72 on the sequence of VHL as the unique site phosphorylated by Aurora-A

Figure 6

<p>Putative function of the interaction between VHL and Aurora-A in the ccRCC.</p

Detection of Aurora-A and VHL in healthy and tumour tissues.

<div><p>A-Western blot analysis of protein extracts from ccRCC and matching healthy tissue samples. Total proteins from tumour (T) and healthy (N) tissue samples were extracted using the RIPA buffer and analysed by western blotting with the anti-Aurora-A polyclonal antibody (1:200) and the anti-pVHL monoclonal antibody (1:500). β-actin was used as loading control.</p> <p>B-Immunohistochemical analysis of Aurora-A and VHL expression in ccRCC and matching healthy tissue samples. Tissues samples were prepared for immunohistochemistry as described in Materials and Methods and analysed using the polyclonal antibody against Aurora-A (1:100) and the monoclonal antibody against pVHL (1:200). Microphotographs were acquired with a Leica™ DMRXA microscope equipped with a CoolSnapsHQ camera (Photometrics™) (bar 10µm).</p></div

pVHL is phosphorylated on Ser72 by Aurora-A <i>in vitro</i>.

<div><p>A- Purified recombinant 6xHis-tagged pVHL30 [VHL(His)₆] (lane 1) or mutant 6xHis-tagged pVHL30 S72A [VHLSA72(His)₆] ( (lane 2) were incubated in the presence of [γ-³²P] ATP and recombinant Aurora-A(His) 6. Samples were separated by SDS-PAGE and the gel was stained with Coomassie Blue (higher panel). pVHL phosphorylation status was analysed by autoradiography (lower panel). The asterisk (*) indicate the pVHL protein.</p> <p>B-Localization and sequence of the putative Aurora-A phosphorylation site on pVHL.</p></div

pVHL interacts with Aurora-A.

<div><p>A-HeLa cells were synchronized as described in Materials and Methods. Twenty µg of protein extracts were separated on a poly-acrylamide gel and VHL expression was assessed with the monoclonal anti-pVHL antibody (1:200). Loading was controlled with the anti-Actin antibody (1:200).</p> <p>B-In HeLa and RCC4 cells, Aurora-A and VHL co-localize at the centrosome and at the mitotic spindle extremities (anti-Aurora-A antibody, 1:200 and anti-pVHL antibody, 1:200).</p> <p>C-HeLa, RCC4 and 786-0 cell protein extracts were immunoprecipitated with the anti-pVHL polyclonal antibody (lane 2) or with the IgG (lane 3, negative control). Aurora-A and pVHL were both detected in the immunoprecipitates using a polyclonal antibody against Aurora-A (1:200) (upper panel) and a monoclonal antibody against pVHL (1:200) (lower panel). Lane 1 was corresponding to the total cell extract.</p></div

Statistical analysis of Aurora-A and pVHL levels and localization in ccRCCs according to their Fuhrman grade and <i>VHL</i> status.

<p>pVHL expression level (A) and localization (B and C) in ccRCCs were analysed relative to the Fuhrman grade of the tumours. Aurora-A expression level was assessed relative to the VHL status (D) and Fuhrman grade (E) of the tumours. Aurora-A expression according to the VHL cytoplasmic localization was also evaluated (F). The non-parametric Mann-Whitney test was used to evaluate the statistical relationships between the expression of Aurora-A and pVHL and the tumour parameters.</p