Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis

Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell-cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.     

Cocaine administration induces human splenic constriction and altered hematologic parameters

Cocaine is a potent vasoconstrictor that hasbeen shown to alter hemoglobin, hematocrit, and red blood cell countsin both animals and humans. The present study evaluated whether cocaine administration induces splenic constriction in men and whether spleen-volume changes temporally correlate with altered hematologic parameters. Spleen volume was assessed at baseline and after cocaine administration (0.4 mg/kg) by using magnetic resonance imaging. A groupof five healthy men, aged 31 ± 2 (SE) yr and reporting occasionalcocaine use (13 ± 5 lifetime exposures), participated. Cocainereduced spleen volume by 20 ± 4%(P < 0.03) 10 min after drugadministration. Spleen volume returned to normal (101 ± 3% baseline) within 35 min after cocaine administration, indicating thatthe reduction is a transient phenomenon. In subjectsadministered cocaine from whom blood samples were obtained(n = 3), cocaine increased hemoglobinlevels, hematocrit, and red blood cell count to 104.5 ± 0.9, 105.6 ± 1.2, and 106.5 ± 1.0% of baseline levels, respectively(P < 0.03), but it did not alterwhite blood cell and platelet counts. Placebo administration(n = 5) did not alter hematologicparameters. These results suggest that cocaine induces splenicconstriction in humans, and this may contribute to temporally concordant hematologic parameter changes. These events may help topreserve or increase tissue oxygenation in periods of high oxygendemand and/or increased vascular resistance.

     

Pathways governing G1/S transition and their response to DNA damage

The ability to self-replicate is a fundamental feature of life, reflected at the cellular level by a highly regulated process initiated in G1 phase via commitment to a round of DNA replication and cell division. Here we briefly highlight recent advances in understanding the molecular pathways which govern the decision of mammalian somatic cells to enter S phase, and the so-called cell cycle checkpoints which guard the G1/S transition and S phase progression against potentially deleterious effects of genotoxic stress. Particular emphasis is put on the emerging parallel yet cooperative pathways of retinoblastoma protein (pRB)–E2F and Myc, their convergence to control the activity of the cyclin-dependent kinase 2 (Cdk2) at the G1/S boundary, as well as the two waves of checkpoint responses at G1/S: the rapid pathway(s) leading to Cdc25A degradation, and the delayed p53–p21 cascade, both silencing the Cdk2 activity upon DNA damage.     

Src-mediated Post-translational Regulation of Endoglin Stability and Function Is Critical for Angiogenesis

Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif (⁶¹²YIY⁶¹⁴) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following ⁶¹²YIY⁶¹⁴ phosphorylation. Interestingly, Src phosphorylation of endoglin-⁶¹²YIY⁶¹⁴ was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of ⁶¹²YIY⁶¹⁴ phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.     

A new analytical material-enhanced laser desorption ionization (MELDI) based approach for the determination of low-mass serum constituents using fullerene derivatives for selective enrichment

60]fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as material-enhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel high-speed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new "top-down" strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation.     

Untapped potential: exploiting fungi in bioremediation of hazardous chemicals

Fungi possess the biochemical and ecological capacity to degrade environmental organic chemicals and to decrease the risk associated with metals, metalloids and radionuclides, either by chemical modification or by influencing chemical bioavailability. Furthermore, the ability of these fungi to form extended mycelial networks, the low specificity of their catabolic enzymes and their independence from using pollutants as a growth substrate make these fungi well suited for bioremediation processes. However, despite dominating the living biomass in soil and being abundant in aqueous systems, fungi have not been exploited for the bioremediation of such environments. In this Review, we describe the metabolic and ecological features that make fungi suited for use in bioremediation and waste treatment processes, and discuss their potential for applications on the basis of these strengths.     

Viral escape from the neutralizing antibody response: the lymphocytic choriomeningitis virus model

In addition to CD8+ cytotoxic T lymphocyte (CTL) responses, neutralizing antibodies contribute substantially to the long-term immune control of noncytopathic viruses, as demonstrated during infection with the lymphocytic choriomeningitis virus (LCMV). The high virus load during the initial phase of an infection and the ability of this RNA virus to spontaneously acquire mutations are important prerequisites for escaping an ongoing immune response. In this context, LCMV escape from the humoral response by single point mutations in neutralizing envelope protein determinants may occur, particularly during conditions of CTL deficiency, leading to virus persistence.