Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.     

An unusual case of vaccine-associated paralytic poliomyelitis

The present article reports a case involving an immunocompetent, previously well child who, despite two previous doses of inactivated poliovirus vaccine, developed severe flaccid paralysis consistent with polio after receiving oral polio vaccine.     

Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

Bcl-2 interacts with the inositol 1,4,5-trisphosphate receptor (InsP₃R) and thus prevents InsP₃-induced Ca²⁺ elevation that induces apoptosis. Here we report that Bcl-2 binds dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a protein kinase A (PKA)-activated and calcineurin (CaN)-deactivated inhibitor of protein phosphatase 1 (PP1). Bcl-2 docks DARPP-32 and CaN in a complex on the InsP₃R, creating a negative feedback loop that prevents exaggerated Ca²⁺ release by decreasing PKA-mediated InsP₃R phosphorylation. T-cell activation increases PKA activity, phosphorylating both the InsP₃R and DARPP-32. Phosphorylated DARPP-32 inhibits PP1, enhancing InsP₃R phosphorylation and Ca²⁺ release. Elevated Ca²⁺ activates CaN, which dephosphorylates DARPP-32 to dampen Ca²⁺ release by eliminating PP1 inhibition to enable it to dephosphorylate the InsP₃R. Knocking down either Bcl-2 or DARPP-32 abrogates this feedback mechanism, resulting in increased Ca²⁺ elevation and apoptosis. This feedback mechanism appears to be exploited by high levels of Bcl-2 in chronic lymphocytic leukemia cells, repressing B-cell receptor-induced Ca²⁺ elevation and apoptosis.Periodic elevations of intracellular Ca²⁺ serve as second messengers regulating mitochondrial metabolism, cell cycle entry, and cell survival (1, 2). Ca²⁺ signals are generated when inositol 1,4,5-trisphosphate receptors (InsP₃Rs) open in response to InsP₃, thus transferring Ca²⁺ from the endoplasmic reticulum into the cytoplasm and mitochondria. InsP₃R-mediated Ca²⁺ release is highly regulated, as excessive Ca²⁺ release causes cellular dysfunction and death (3). A number of proteins bind to InsP₃Rs and regulate channel opening, thus preventing excessive Ca²⁺ release (4). Among these is the antiapoptotic protein B-cell lymphoma-2 (Bcl-2) (5).The Bcl-2 homology domain 4 (BH4) domain of Bcl-2 mediates its interaction with InsP₃Rs (6, 7). This domain also binds the Ca²⁺/calmodulin-activated serine/threonine (Thr) protein phosphatase calcineurin (CaN) (8). The BH4 domain binds within the regulatory and coupling domain of the InsP₃R, located between the InsP₃ binding site near the N terminus and the channel domain near the C terminus (6). Protein kinase A (PKA) phosphorylates serine (Ser) 1589 and Ser1755 within this domain, increasing InsP₃-mediated channel opening and Ca²⁺ release (9). Bcl-2 is reported to regulate InsP₃R phosphorylation at Ser1755 and to thereby govern InsP₃R-mediated Ca²⁺ release in murine embryonic fibroblasts, although the mechanism is not yet identified (10).Bcl-2 plays an important role in regulating T-cell development and selection, processes that involve Ca²⁺ signaling and apoptosis regulation (11, 12). A 20-amino acid peptide (InsP₃R-derived peptide, or IDP) corresponding to the Bcl-2 binding site on the InsP₃R inhibits Bcl-2–InsP₃R interaction, thus eliminating Bcl-2’s control over InsP₃R-mediated Ca²⁺ elevation (6, 7). This peptide induces marked Ca²⁺ elevation and Ca²⁺-mediated apoptosis in primary chronic lymphocytic leukemia (CLL) cells and in B-cell lymphoma lines, indicating that Bcl-2–InsP₃R interaction contributes to the apoptosis-resistance characteristic of these lymphoid malignancies (13, 14).Here we report that Bcl-2 interacts with dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a potent inhibitor of protein phosphatase 1 (PP1) that is activated by PKA-mediated phosphorylation at Thr 34 and deactivated by CaN-mediated dephosphorylation at this site (15). Our findings indicate that Bcl-2 docks DARPP-32 and CaN on the InsP₃R, creating a negative feedback loop that responds to InsP₃R-mediated Ca²⁺ release by inhibiting InsP₃R phosphorylation at Ser1755, thereby preventing excessive Ca²⁺ elevation capable of inducing cell death. Our findings implicate this Bcl-2–CaN–DARPP-32 feedback mechanism in regulating Ser1755 phosphorylation and apoptosis in primary human CLL cells.     

Olfactory Ensheathing Cells Promote Differentiation of Neural Stem Cells and Robust Neurite Extension

Aims

The goal of this study was to gain insight into the signaling between olfactory ensheathing cells (OECs) and neural stem cells (NSCs). We sought to understand the impact of OECs on NSC differentiation and neurite extension and to begin to elucidate the factors involved in these interactions to provide new targets for therapeutic interventions.

Materials and Methods

We utilized lines of OECs that have been extremely well characterized in vitro and in vivo along with well studied NSCs in gels to determine the impact of the coculture in three dimensions. To further elucidate the signaling, we used conditioned media from the OECs as well as fractioned components on NSCs to determine the molecular weight range of the soluble factors that was most responsible for the NSC behavior.

Results

We found that the coculture of NSCs and OECs led to robust NSC differentiation and extremely long neural processes not usually seen with NSCs in three dimensional gels in vitro. Through culture of NSCs with fractioned OEC media, we determined that molecules larger than 30 kDa have the greatest impact on the NSC behavior.

Conclusions

Overall, our findings suggest that cocultures of NSCs and OECs may be a novel combination therapy for neural injuries including spinal cord injury (SCI). Furthermore, we have identified a class of molecules which plays a substantial role in the behavior that provides new targets for investigating pharmacological therapies.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.