RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing.

Neurological manifestation of Churg-Strauss syndrome (CSS) is common and usually consists of peripheral neuropathy due to small-vessel vasculitis, while cerebral manifestation is less frequent. We report the case of a 77-year-old woman with multiple cerebral infarctions and hypereosinophilia. The presence of hypereosinophilia, asthma, sinusitis and vasculitis led to the diagnosis of CSS. As cerebral infarctions occurred monophasically and an elevation of heart enzymes was present, we assumed cardiac involvement and multiple cerebral infarctions due to cardiac embolism. Treatment with high-dose IV methylprednisone and cyclophosphamide pulses led to significant improvement. This case illustrates multiple cerebral infarctions in CSS. CSS should always be considered in patients with hypereosinophilia and stroke.

In microfracture, subchondral progenitors enter the cartilage defect and form cartilage repair tissue. We hypothesize that synovial fluid (SF) from rheumatoid arthritis (RA) donors affects chondrogenesis of human subchondral progenitors stimulated with transforming growth factor-beta3 (TGFB3), whereas SF from normal and osteoarthritis (OA) donors do not. Human progenitors from subchondral cortico-spongious bone (pool of n = 4) were cultured in micromasses under serum-free conditions and were stimulated with 10 ng/mL TGFB3 and with 5% SF from normal, OA, and RA donors (pool of n = 7, each). Histological staining of proteoglycan and immunostaining of type II collagen showed that progenitors stimulated with SF from RA donors show significantly reduced cartilage matrix formation compared to progenitors treated with TGFB3 or with SF from normal and OA donors (n = 3, each). Gene expression analysis of typical chondrocyte marker genes and genes encoding matrix modifying enzymes showed that SF from OA and RA donors influence the onset of TGFB3-mediated chondrogenesis (pool of 20 micromasses), but had no effect on the gene expression profile after prolonged culture in micromasses. These results suggest that SF from RA patients may impair the chondrogenic development of mesenchymal progenitors in microfracture, whereas osteoarthritic SF may has no negative effect on the cartilage matrix formation.(c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

BACKGROUND AND PURPOSE: It has recently been proposed that fluid-attenuated inversion recovery (FLAIR) imaging may serve as a surrogate marker for time of symptom onset after stroke. We assessed the hypothesis that FLAIR imaging could be used to decide if an MRI was performed within 4.5 hours from symptom onset or later. METHODS: All consecutive patients with presumed stroke who underwent an MRI within 12 hours after known symptom onset were included regardless of stroke subtype and severity between May 2008 and May 2009. Blinded to time of symptom onset, 2 raters judged the visibility of lesions on FLAIR. Apparent diffusion coefficient values, lesion volume on diffusion-weighted imaging, and relative signal intensity of FLAIR lesions were determined. RESULTS: In 94 consecutive patients with stroke, we found that median time from symptom onset for FLAIR-positive patients (189 minutes; interquartile range, 110 to 369 minutes) was significantly longer compared with FLAIR-negative patients (103 minutes; interquartile range, 75 to 183 minutes; P=0.011). Negative FLAIR had a sensitivity of 46% and a specificity of 79% for allocating patients to a time window of less than 4.5 hours. FLAIR positivity increased with diffusion-weighted imaging lesion volume (P<0.001) but showed no correlation with apparent diffusion coefficient values (P=0.795). There was no significant correlation between relative signal intensity and time from symptom onset (Spearman correlation coefficient -0.152, P=0.128). CONCLUSIONS: Based on our findings, we cannot recommend the use of FLAIR visibility as an estimate of time from symptom onset within the first 4.5 hours.

Autologous human serum is used in cartilage repair and may exert its effect by the recruitment of mesenchymal stem and progenitor cells (MSC). Aim of our study was to analyze the chemokine profile of human serum and to verify chemotactic activity of selected chemokines on MSC. Human MSC were isolated from iliac crest bone marrow aspirates. Chemotactic activity of human serum made from whole blood and pharma grade serum was tested in 96-well chemotaxis assays and chemokine levels were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on MSC was tested dose dependently using chemotaxis assays. Human serum derived from whole blood significantly attracted human MSC, while pharma grade serum did not recruit MSC. Human chemokine antibody array analysis showed that the level of chemokines CXCL-3, 5, 7-8, 10-12, 16; CCL- 2, 5, 11, 13, 16-20, 24-25, 27; as well as XCL-1 was elevated (fold change >1.5) in serum derived from whole blood compared to nonrecruiting pharma grade serum. Chemotaxis assays showed that the chemokines IP-10/CXCL-10 and I-TAC/CXCL-11 significantly recruit human MSC. PARC/CCL-18, HCC-4/CCL-16, CTACK/CCL-27, and Lymphotactin/XCL-1 showed no chemotactic effect on MSC. Therefore, human serum derived from whole blood contains chemokines that may contribute to serum-mediated recruitment of human mesenchymal progenitors from bone marrow.

ABSTRACT: BACKGROUND: The mismatch between diffusion weighted imaging (DWI) lesion and perfusion imaging (PI) deficit volumes has been used as a surrogate of ischemic penumbra. This pathophysiology-orientated patient selection criterion for acute stroke treatment may have the potential to replace a fixed time window. Two recent trials - DEFUSE and EPITHET - investigated the mismatch concept in a multicenter prospective approach. Both studies randomized highly selected patients (n=74/n=100) and therefore confirmation in a large consecutive cohort is desirable. We here present a single-center approach with a 3T MR tomograph next door to the stroke unit, serving as a bridge from the ER to the stroke unit to screen all TIA and stroke patients. Our primary hypothesis is that the prognostic value of the mismatch concept is depending on the vessel status. Primary endpoint of the study is infarct growth determined by imaging, secondary endpoints are neurological deficit on day 5-7 and functional outcome after 3 months. METHOD: S 1000Plus is a prospective, single centre observational study with 1200 patients to be recruited. All patients admitted to the ER with the clinical diagnosis of an acute cerebrovascular event within 24 hours after symptom onset are screened. Examinations are performed on day 1, 2 and 5-7 with neurological examination including National Institute of Health Stroke Scale (NIHSS) scoring and stroke MRI including T2*, DWI, TOF-MRA, FLAIR and PI. PI is conducted as dynamic susceptibility-enhanced contrast imaging with a fixed dosage of 5 ml 1M Gadobutrol. For post-processing of PI, mean transit time (MTT) parametric images are determined by deconvolution of the arterial input function (AIF) which is automatically identified. Lesion volumes and mismatch are measured and calculated by using the perfusion mismatch analyzer (PMA) software from ASIST-Japan. Primary endpoint is the change of infarct size between baseline examination and day 5-7 follow up. DISCUSSION The aim of this study is to describe the incidence of mismatch and the predictive value of PI for final lesion size and functional outcome depending on delay of imaging and vascular recanalization. It is crucial to standardize PI for future randomized clinical trials as for individual therapeutic decisions and we expect to contribute to this challenging task. TRIAL REGISTRATION clinicaltrials.gov NCT00715533.

BACKGROUND AND PURPOSE: Perfusion imaging is widely used in stroke, but there are uncertainties with regard to the choice of arterial input function (AIF). Two important aspects of AIFs are signal-to-noise ratio and bolus-related signal drop, ideally close to 63%. We hypothesized that distal branches of the middle cerebral artery (MCA) provide higher quality of AIF compared with proximal branches. METHODS: Over a period of 3 months, consecutive patients with suspected stroke were examined in a 3-T MRI scanner within 24 hours of symptom onset. AIFs were selected manually in M1, M2, and M3 branches of the MCA contralateral to the suspected ischemia. Signal-to-noise ratio and bolus-related signal drop were analyzed. Perfusion maps were created for every patient and mean values at the insular level as well as relative ranges were compared. RESULTS: Mean age of 132 included patients (53 females) was 67.3 years (SD, 14.9) and median National Institutes of Health Stroke Scale was 3 (interquartile range [IQR] 0 to 6). For further analyses, 4 patients were excluded due to discontinuation of scanning or insufficient bolus arrival (signal drop <15%). Median signal-to-noise ratio was highest in M3 branches (36.41; IQR, 29.29 to 43.58). Median signal-to-noise ratio in M2 branches was intermediate (27.54; IQR, 20.78 to 34.00) and median signal-to-noise ratio in M1 was low (12.40; IQR, 9.11 to 17.15). Using AIFs derived from M1 and M2 branches of the MCA median signal drop was 77%(IQR, 72% to 82%) and 78%(IQR, 73% to 83%), respectively. Signal drop was significantly reduced when AIF was selected in M3 branches with a median of 72%(IQR, 63% to 77%; P<0.01). Highest variability of 3456 perfusion maps was found in those derived from M1. CONCLUSIONS: The level of AIF selection in the MCA has a major impact on reliability and even quantitative parameters of perfusion maps. For better comparison of perfusion maps, the AIF should be defined by selection of distal branches of the MCA contralateral to the suspected ischemia. In future trials involving perfusion imaging, the MCA segment used for the AIF should be specified.

In this study, we analysed the frequency, morphological patterns and clinical characteristics of cerebral ischaemia in bacterial meningitis. We sought to determine predictors for the development of vasculopathy and ischaemic infarction in patients with bacterial meningitis. Consecutive adult patients admitted between March 1998 and February 2009 to a neurological intensive care unit at a university hospital in Germany with the diagnosis of bacterial meningitis were included in the study. Standard criteria were used to define bacterial meningitis. From 68 patients with bacterial meningitis, six patients suffered from cerebral ischaemia (8.8%). In our cohort, reduced level of consciousness on admission (p = 0.01) and lower white blood cell (WBC) count in cerebrospinal fluid (CSF)(p = 0.012) were associated with development of ischaemic cerebrovascular complications. The short-term outcome of all patients was poor (median modified Rankin scale 4.5). In patients presenting with reduced level of consciousness on admission and/or low WBC count in CSF early cerebral imaging including MR angiography or CT angiography are warranted to detect impending cerebrovascular complications.

The interdependence of both transport and metabolism on the disposition of drugs has recently gained heightened attention in the literature, and has been termed the "interplay of transport and metabolism". Such "interplay" is observed when inhibition of biliary clearance of a drug results in an "apparent" increase in the metabolic clearance of the drug or vice-versa. In this manuscript, we derived and explored through simulations a physiological-based pharmacokinetic model that integrates both transport and metabolism and explains the "apparent" dependence of hepatic clearance on both these processes. In addition, we show that the phenomenon of hepatic "transport-metabolism interplay" is as a result of using the plasma concentration as a point of reference when calculating metabolic or biliary clearance, and this interplay is maximal when the drug is actively transported into the hepatocytes (i.e. hepatocyte sinusoidal influx clearance is greater than the sinusoidal efflux clearance). When the hepatic drug concentration is used as a reference point to calculate metabolic or biliary clearance, this interplay ceases to exist. A mechanistic understanding of this interplay phenomenon can be used to explain the somewhat paradoxical results that may be observed in drug-drug interaction studies when a drug is cleared by both metabolism and biliary excretion. That is, when one of these two pathways is inhibited, the other pathway appears to be induced or activated. This interplay results in an increase in hepatic drug concentrations and therefore has implications for the hepatic efficacy and toxicity of a drug.

Several reports have recently demonstrated a detrimental role of Toll-like receptors (TLR) in cerebral ischemia, while there is little information about the endogenous ligands which activate TLR-signaling. The myeloid related proteins-8 and -14 (Mrp8/S100A8; Mrp14/S100A9) have recently been characterized as endogenous TLR4-agonists, and thus may mediate TLR-activation in cerebral ischemia. Interestingly, not only TLR-mRNAs, but also Mrp8 and Mrp14 mRNA were found to be induced in mouse brain between 3 and 48h after transient 1h focal cerebral ischemia/reperfusion. Mrp-protein was expressed in the ischemic hemisphere, and co-labelled with CD11b-positive cells. To test the hypothesis that Mrp-signaling contributes to the postischemic brain damage we subjected Mrp14-deficient mice, which also lack Mrp8-protein-expression, to focal cerebral ischemia. Mrp14-deficient mice had significantly smaller lesion volumes when compared to wildtype littermates (130 +/- 16 mm(3) vs. 105 +/- 28 mm(3)) at two days after transient focal cerebral ischemia (1h), less brain swelling, and a reduced macrophage/microglia cell count in the ischemic hemisphere. We conclude that upregulation and signaling of Mrp-8 and-14 contribute to neuroinflammation and the progression of ischemic damage.