Ambivalent effects of interleukin-6 on the pathogenesis of ischaemic stroke have been reported. However, to date, the long-term actions of interleukin-6 after stroke have not been investigated. Here, we subjected interleukin-6 knockout (IL-6(-/-)) and wild-type control mice to mild brain ischaemia by 30-min filamentous middle cerebral artery occlusion/reperfusion. While ischaemic tissue damage was comparable at early time points, IL-6(-/-) mice showed significantly increased chronic lesion volumes as well as worse long-term functional outcome. In particular, IL-6(-/-) mice displayed an impaired angiogenic response to brain ischaemia with reduced numbers of newly generated endothelial cells and decreased density of perfused microvessels along with lower absolute regional cerebral blood flow and reduced vessel responsivity in ischaemic striatum at 4 weeks. Similarly, the early genomic activation of angiogenesis-related gene networks was strongly reduced and the ischaemia-induced signal transducer and activator of transcription 3 activation observed in wild-type mice was almost absent in IL-6(-/-) mice. In addition, systemic neoangiogenesis was impaired in IL-6(-/-) mice. Transplantation of interleukin-6 competent bone marrow into IL-6(-/-) mice (IL-6(chi)) did not rescue interleukin-6 messenger RNA expression or the early transcriptional activation of angiogenesis after stroke. Accordingly, chronic stroke outcome in IL-6(chi) mice recapitulated the major effects of interleukin-6 deficiency on post-stroke regeneration with significantly enhanced lesion volumes and reduced vessel densities. Additional in vitro experiments yielded complementary evidence, which showed that after stroke resident brain cells serve as the major source of interleukin-6 in a self-amplifying network. Treatment of primary cortical neurons, mixed glial cultures or immortalized brain endothelia with interleukin 6-induced robust interleukin-6 messenger RNA transcription in each case, whereas oxygen-glucose deprivation did not. However, oxygen-glucose deprivation of organotypic brain slices resulted in strong upregulation of interleukin-6 messenger RNA along with increased transcription of key angiogenesis-associated genes. In conclusion, interleukin-6 produced locally by resident brain cells promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection.

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3 T, CD8 T, CD4 T, and FOXP3 regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21 Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21 Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.

The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.Initiation of DNA replication on the chromosomes of eukaryotic cells requires the prior assembly of the pre-replicative complex (pre-RC), establishing the licensed chromatin state. Of the multiple proteins participating in the stepwise formation of the pre-RC, the initiator origin recognition complex (ORC) makes the first chromatin contacts. In Saccharomyces cerevisiae, the heterohexameric ORC binds to distinct sequence elements in the context of small, modular origins of DNA replication. These are occupied by ORC throughout the cell cycle. Thus, while the initial ORC-DNA interaction can define the position of origins on the chromosomes, it is an unlikely candidate to determine the timing of pre-RC formation, which is confined to the M and G 1 phases of the cell cycle. Instead, this process is mainly controlled by changes in cyclindependent kinase (CDK) activity during these cell cycle phases and is part of the safeguard mechanisms against unscheduled DNA synthesis (4,6,15,16,28).In contrast to yeast, origins in metazoans are less well defined and their distribution along chromosomes is subject to tissue-specific and developmental control. No conserved DNA sequence motifs that would indicate a sequence-specific DNA binding by initiator proteins have been identified within replication initiation regions. For these reasons, alternative modes of origin selection are discussed (13,18,30).It is nonetheless assumed that the position of metazoan origins is determined by nonrandom ORC-chromatin interactions. To what extent these interactions persist after origin firing has not been extensively studied. The same holds true for the issue of reoccupation of the additional potential ORC binding site emerging immediately after DNA replication initiation. It is clear, though, that ORC has to be chromatin bound at the latest toward the end of mitosis for the subsequent pre-RC assembly steps to occur. When exactly ORC associates with chromatin and, especially, whether ORC is bound to metaphase chromosomes have not been firmly establishe...

Background: Immune mechanisms are included in stroke pathophysiologic factors, but the frequency and role of intrathecal antibodies is unclear and diagnostic tests are not routinely performed on cerebrospinal fluid (CSF). Objective: To determine the frequency of intrathecal immunoglobulin synthesis in a well-characterized cohort of patients who experienced "noninflammatory" acute stroke. Design: Retrospective cohort study. Setting: University hospital neurology department. Patients: Patients (n = 318) with stroke who were undergoing lumbar puncture during diagnostic workup and 79 control patients. Results: Cerebrospinal fluid-specific immunoglobulin (IgG, IgM, and IgA) synthesis was significantly (P Ͻ .001) more frequent after stroke (24.8%) compared with the incidence in age-and sex-matched controls (2.5%). Furthermore, 31.3% of stroke patients demonstrated blood-brain barrier dysfunction and 18.1% displayed pleocytosis. Conclusion: The strong association between CSFspecific immunoglobulin synthesis and stroke suggests a role in the development of cerebral ischemia and might constitute an immunologically defined stroke subgroup.