ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord

During antenatal development, the operation and maturation of mammalian spinal networks strongly depend on the activity of ventral horn GABAergic interneurons that mediate excitation first and inhibition later. Although the functional consequence of GABA actions may depend on maturational processes in target neurons, it is also likely that evolving changes in GABAergic transmission require fine-tuning in GABA release, probably via certain intrinsic mechanisms regulating GABAergic neuron excitability at different embryonic stages. Nevertheless, it has not been possible, to date, to identify certain ionic conductances upregulated or downregulated before birth in such cells. By using an experimental model with either mouse organotypic spinal cultures or isolated spinal cord preparations, the present study examined the role of the ERG current (IK(ERG) ), a potassium conductance expressed by developing, GABA-immunoreactive spinal neurons. In organotypic cultures, only ventral interneurons with fast adaptation and GABA immunoreactivity, and only after 1 week in culture, were transformed into high-frequency bursters by E4031, a selective inhibitor of IK(ERG) that also prolonged and made more regular spontaneous bursts. In the isolated spinal cord in which GABA immunoreactivity and m-erg mRNA were colocalized in interneurons, ventral root rhythms evoked by NMDA plus 5-hydroxytryptamine were stabilized and synchronized by E4031. All of these effects were lost after 2 weeks in culture or before birth in coincidence with decreased m-erg expression. These data suggest that, during an early stage of spinal cord development, the excitability of GABAergic ventral interneurons important for circuit maturation depended, at least in part, on the function of IK(ERG)

Complement protein C1q stimulates hyaluronic acid degradation via gC1qR/HABP1/p32 in malignant pleural mesothelioma

Complement component C1q can act as a pro-tumorigenic factor in the tumor microenvironment (TME). The TME in malignant pleural mesothelioma (MPM) is rich in C1q and hyaluronic acid (HA), whose interaction enhances adhesion, migration and proliferation of malignant cells. HA-bound C1q is also capable of modulating HA synthesis. Thus, we investigated whether HA-C1q interaction would affect HA degradation, analyzing the main degradation enzymes, hyaluronidase (HYAL)1 and HYAL2, and a C1q receptor candidate. We first proceeded with the characterization of HYALs in MPM cells, especially HYAL2, since bioinformatics survival analysis revealed that higher HYAL2 mRNA levels have an unfavorable prognostic index in MPM patients. Interestingly, Real-Time quantitative PCR, flow cytometry and Western blot highlighted an upregulation of HYAL2 after seeding of primary MPM cells onto HA-bound C1q. In an attempt to unveil the receptors potentially involved in HA-C1q signaling, a striking co-localization between HYAL2 and globular C1q receptor/HABP1/p32 (gC1qR) was found by immunofluorescence, surface biotinylation and proximity ligation assays. RNA interference experiments revealed a potentially regulatory function exerted by gC1qR on HYAL2 expression, since C1QBP (gene for gC1qR) silencing unexpectedly caused HYAL2 downregulation. In addition, the functional blockage of gC1qR by a specific antibody hindered HA-C1q signaling and prevented HYAL2 upregulation. Thus, C1q-HA interplay is responsible for enhanced HYAL2 expression, suggesting an increased rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments in the MPM TME. Our data support the notion of an overall tumor-promoting property of C1q. Moreover, the overlapping localization and physical interaction between HYAL2 and gC1qR suggests a potential regulatory effect of gC1qR within a putative HA-C1q macromolecular complex

Effect of methylene blue photodynamic therapy on human neutrophil functional responses.

Photodynamic therapy (PDT) has become an emerging novel therapeutic approach for treating localized microbial infections, particularly those sustained by multidrug-resistant strains. Given the irreplaceable role played by professional phagocytes in limiting infections, such as polymorphonuclear neutrophils, any newly designed antimicrobial therapeutic approach must not interfere with their function. The present investigation presents a detailed analysis of the effect of PDT on the viability and several functional responses of human polymorphonuclear neutrophils loaded with methylene blue (MB), one of the more commonly used photosensitizers in antimicrobial PDT. Taking advantage of the use of a specifically-designed optical LED array for illuminating MBloaded human polymorphonuclear neutrophils, a number of cell functions have been assayed under miniaturized, strictly controlled and reproducible experimental conditions. The major findings of this study are the following: (1) MB-PDT increases human neutrophils adhesion and does not modify myeloperoxidase release; (2) MB-PDT markedly enhances reactive oxygen species generation that is independent of superoxide-forming phagocytic oxidase and very likely ascribable to LED-dependent excitation of accumulated methylene blue; (3) MB-PDT almost abolishes human neutrophils candidacidal activity by hindering the engulfing machinery. This in vitro study may represent a valuable reference point for future research on PDT applications for treating localized microbial infections. 1. Introduction Two main therapeutic approaches are nowadays available to counteract infectious diseases, i.e. (i) eliminate the microbes causing the infection and, (ii) potentiate the immune response of the affected patient. In some instances, due to the pressing need for quickly containing the ongoing infection, both therapeutic interventions are adopted a

\u201cAlginate/Hydroxyapatite Biocomposite For Bone Ingrowth: A Trabecular Structure With High And Isotropic Connectivity\u201d

Alginate/hydroxyapatite composite scaffolds were developed using a novel production design. Hydroxyapatite (HAp) was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid hydrolyzing D-gluconic acid \u3b4-lactone (GDL) for the direct release of calcium ions from HAp. Hydrogels were then freeze-casted to produce a three-dimensional isotropic porous network. Scanning electron microscopy (SEM) observations, confocal laser scanning microscopy (CLSM) and microcomputed tomography (\u3bc-CT) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 and 300 \u3bcm and over 88% porosity. Proliferation assay and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Overall, these combined results indicate that the novel alginate based composites efficiently support the adhesion and proliferation of cells showing at the same time adequate structural and physical-chemical properties for being used as scaffolds in bone tissue engineering strategies

179 C1q stimulates hyaluronic acid degradation via gC1qR/HABP1/p32 in malignant pleural mesothelioma

Background: C1q exerts pro-tumorigenic functions in the tumour microenvironment (TME), independently of complement activation [1]. In malignant pleural mesothelioma (MPM), HA-bound C1q is able to increase tumor cell adhesion, migration and proliferation, but also to enhance the production of pro-inflammatory and pro-metastatic HA fragments due to HAS3 upregulation [2]. An increasingly activated HA metabolism is associated with cancer progression. Here, we investigated HA-bound C1q contribution in HA degradation via modulation of hyaluronidases (HYAL1, HYAL2) and the involvement of globular C1q receptor/HABP1/p32 (gC1qR), as a receptor of both HA and C1q. Methods: Primary cell isolation, zymography, immunohistochemistry, Real-Time quantitative PCR (RT-qPCR), immunofluorescence, Western blot, surface biotinylation assay, flow cytometry, proximity ligation assay (PLA), RNA interference, bioinformatics analysis. Results: After initial characterization of HYALs in MPM primary cells, we focused on HYAL2, since GEPIA bioinformatics analysis revealed an unfavorable prognostic index in MPM patients with higher HYAL2 mRNA levels. Upon seeding MPM cells onto HA-bound C1q, HYAL2 upregulation was highlighted by RT-qPCR, flow cytometry and Western blot. In the attempt to determine receptors involved in HA-C1q signaling, a striking membrane and intracellular co-localization between HYAL2 and gC1qR was found by immunofluorescence, surface biotinylation assay and PLA. RNA interference of C1QBP (gene for gC1qR) unveiled a promising regulatory effect of gC1qR on HYAL2 expression, determining an unexpected HYAL2 downregulation. Moreover, functional blockage of gC1qR by a specific antibody hampered HA-C1q signaling and impeded HYAL2 upregulation. Conclusions: HA-C1q interplay can act as a tumor-promoting signaling complex by enhancing HYAL2 expression, suggesting a consequent higher rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments. Furthermore, we demonstrated a novel regulatory function of gC1qR in the TME due to its involvement in the modulation of HA metabolism. The regulation of HA metabolism is crucial in view of its key role in tumor progression and its connection with most of the hallmarks of cancer

Autocrine activation of nicotinic acetylcholine receptors contributes to Ca(2+) spikes in mouse myotubes during myogenesis

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca(2)(+) ([Ca(2)(+)](i)) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine-like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca(2)(+)](i) spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves