Epigenetic immune cell counting in human blood samples for immunodiagnostics.

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 cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns.

The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.

Essential role of interleukin-6 in post-stroke angiogenesis.

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.

Evidence of intrathecal immunoglobulin synthesis in stroke: a cohort study.

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 fluid­specific immunoglobulin(IgG, IgM, and IgA) synthesis was significantly(P.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 CSF-specific immunoglobulin synthesis and stroke suggests a role in the development of cerebral ischemia and might constitute an immunologically defined stroke subgroup.

Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system.

The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC) technology in constitutive expression systems, it has been shown that integration site effects resulting in unwanted expression patterns can be largely eliminated. Here we describe a strategy to minimize unfavourable integration effects on conditional expression constructs based on a 75 kb genomic BAC fragment. This fragment was derived from a transgenic mouse line, termed LC-1, which carries the Tet-inducible genes luciferase and cre (Schönig et al. 2002). Animals of this mouse line have previously been shown to exhibit optimal expression properties in terms of tightness in the off state and the absolute level of induction, when mated to appropriate transactivator expressing mice. Here we report the cloning and identification of the transgenic LC-1 integration site which was subsequently inserted into a bacterial artificial chromosome. We demonstrate that this vector facilitates the efficient generation of transgenic mouse and rat lines, where the Tet-controlled expression unit is shielded from perturbations caused by the integration site.

Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis.

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly, treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis.

Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity.

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.

Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids.

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). In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21(Waf1/Cip1) at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21(Waf1/Cip1) preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21(Waf1/Cip1) and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21(Waf1/Cip1) deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21(Waf1/Cip1) suffices to protect neurons from apoptosis. We identify p21(Waf1/Cip1) as a novel antiapoptotic factor for postmitotic neurons and implicate p21(Waf1/Cip1) as the molecular target of neuroprotection by high-dose glucocorticoids.