NLRP3 Inflammasome Inhibitors for Antiepileptogenic Drug Discovery and Development.

Epilepsy is one of the most prevalent and serious brain disorders and affects over 70 million people globally. Antiseizure medications (ASMs) relieve symptoms and prevent the occurrence of future seizures in epileptic patients but have a limited effect on epileptogenesis. Addressing the multifaceted nature of epileptogenesis and its association with the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation requires a comprehensive understanding of the underlying mechanisms of these medications for the development of targeted therapeutic strategies beyond conventional antiseizure treatments. Several types of NLRP3 inhibitors have been developed and their effect has been validated both in in vitro and in vivo models of epileptogenesis. In this review, we discuss the advances in understanding the regulatory mechanisms of NLRP3 activation as well as progress made, and challenges faced in the development of NLRP3 inhibitors for the treatment of epilepsy.

Demographic Profile and Service-Connection Trends of Posttraumatic Stress Disorder and Traumatic Brain Injury in US Veterans Pre- and Post-9/11.

INTRODUCTION: This study seeks to understand the demographic changes in the active-duty service member profile, both prior to and following September 11, 2001 (9/11). The study analyzed diagnosis of posttraumatic stress disorder (PTSD) and traumatic brain injury (TBI) and measures of severity of those diagnoses as recorded in service-connection ratings (percent disability). METHODS: A retrospective cohort-study of military veterans who received care at Veterans Health Administration medical centers between December 1998 and May 2014 was conducted based on clinical data recorded and stored within the Corporate Data Warehouse. RESULTS: A cohort of 1,339,937 veterans received an inpatient or outpatient diagnosis of PTSD and/or TBI. The cohort was divided into 4 service period groups and 3 diagnosis categories. The service periods included pre-9/11 (n = 1,030,806; 77%), post-9/11 (n = 204,083; 15%), overlap-9/11 (n = 89,953; 7%), and reentered post-9/11 (n = 15,095; 1%). The diagnosis categories included PTSD alone (n = 1,132,356; 85%), TBI alone (n = 100,789; 7%) and PTSD+TBI (n = 106,792; 8%). Results of the post-9/11 group revealed significant changes, including (1) increase of veterans with PTSD+TBI; (2) increase of female veterans with PTSD+TBI; and (3) increase of severity level of diagnosed PTSD/TBI as evidenced by higher service-connected disability pensions at younger age in the post-9/11 group. Additionally, data revealed unequal distribution of veterans with PTSD+TBI across geographic areas. CONCLUSIONS: The veteran of the post-9/11 service period does not mirror the veteran of the pre-9/11 service period. Findings are valuable for policy making, allocation of resources, and for reconsidering the prevailing paradigm for treating veterans with PTSD and/or TBI.

Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage.

In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that γ-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that γ-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated γ-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.

X-ray and ultraviolet C irradiation-induced γ-H2AX and p53 formation in normal human periosteal cells in vitro: markers for quality control in cell therapy.

BACKGROUND AIMS: For successful cell transplantation therapy, the quality of cells must be strictly controlled. Unfortunately, to exclude inappropriate cells that possess structurally abnormal chromosomes, currently only karyotyping functions as an assessment. Unfortunately, this methodology is time-consuming and only effective for metaphasic cells. To develop a more efficient, inclusive and sensitive methodology, we examined the phosphorylation of histone H2AX and the p53 levels in normal human periosteal cells exposed to x-rays or other oxidative stressors. METHODS: Periosteal cells were obtained from human alveolar bone before being exposed to x-rays, ultraviolet C or hydrogen peroxide. The cell cycle, electric nuclear volume and CD44 expression were evaluated using flow cytometry, and the phosphorylated H2AX (γ-H2AX), p53, p21 and proliferating cell nuclear antigen (PCNA) levels were evaluated by Western blot analyses. RESULTS: Each oxidative stress dose-dependently arrested cell growth and partially induced premature cellular senescence. In parallel, each oxidative stress rapidly phosphorylated H2AX and stabilized p53, and intense stress sustained these high levels for at least 8 days. CONCLUSIONS: Intensive oxidative stress induces sustained high levels of γ-H2AX and p53, which force cells toward senescence or non-apoptotic cell death. Lower doses of oxidative stress induced more modest and transient increases in γ-H2AX and p53, and these cells eventually survive. However, because DNA is repaired without a template in the majority of these cells, G1 mutations accumulate. Therefore, we recommend that any cell population expressing elevated γ-H2AX and p53 levels be excluded from cell transplantation therapy.

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell-multilayered periosteal sheets in vitro.

BACKGROUND AIMS: Cultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell-extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. METHODS: Periosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. RESULTS: Real-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (ß1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of ß1 integrin was substantially downregulated in the stem cell medium-expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. CONCLUSIONS: Integrin α1ß1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets.

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.

Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo.

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199+10% FBS), periosteal sheets expanded with MesenPRO-RS™ medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.

Improved adhesion of human cultured periosteal sheets to a porous poly(L-lactic acid) membrane scaffold without the aid of exogenous adhesion biomolecules.

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 µm diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane.

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate.

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.

Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37).

Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)(2,7)]CGRP, and a relatively specific antagonist of CGRPR1, CGRP(8-37). [Cys(Acm)(2,7)]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP(8-37) potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools.

Spontaneous Ca2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca2+ pool. This report describes spontaneous Ca2+ oscillations dependent on two separate Ca2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+ were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+ (less than 1 microM)decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+ stores that was greater in the nuclear compartment (4.20 +/- 0.23 ratio units, n = 56) than cytoplasm (2.54 +/- 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+ transient in the cytoplasm (4.99 +/- 0.66, n = 17) than in the nucleus (2.67 +/- 0.29). Pre-treatment with Ca2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+ most diminished. Depletion of internal Ca2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+ influx.

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms.

Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca(2+) concentration ([Ca(2+)](int)) and membrane potential (E(m)) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca(2+)](int): a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC(50) of approximately 0.30 nM, and the maximal effect (initially approximately 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca(2+) mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca(2+) influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca(2+) discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca(2+) influx through L-type voltage-dependent Ca(2+) channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular E(m), with maximal effect at 20 nM and an EC(50) of 0.30 nM. This effect was attenuated with charybdotoxin (-20%) or glyburide (glibenclamide; -80%), suggesting that E(m) hyperpolarization is induced by both Ca(2+)-activated and ATP-sensitive K(+) channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells.

Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism.

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (K(D)=405+/-29 pM; 13,100+/-223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP(1)) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP(2)) receptor, [Cys(Acm)(2,7)]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP(1) receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP(2) receptor agonist than are typical CGRP(1) receptors. These receptor proteins were not exactly matched with the known components of a CGRP(1) receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP(1) receptor.

Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21WAF1/cip1 and prevents its inhibition of human oral epithelial cell proliferation.

We have previously demonstrated that porcine enamel matrix derivative (EMD) contains TGF-beta 1 (or a TGF-beta-like substance), and that EMD rapidly translocates smad2, which is an effector of the TGF-beta signaling pathway, into the nucleus and modulates the proliferation of both human gingival fibroblastic and oral epithelial cells in a cell type-specific manner. To investigate the involvement of TGF-beta in the growth modulatory action of EMD, two approaches have been used in the present study: i) a neutralizing anti-TGF-beta antibody to block EMD action, and ii) authentic porcine TGF-beta 1 to compare with EMD. Both in epithelial and fibroblastic cells, TGF-beta 1 closely mimicked EMD in nuclear accumulation of smad2, phosphorylation of MAP kinase family members, and consequent cell type-specific growth modulation. Anti-TGF-beta antibody, at levels which completely blocked TGF-beta 1-induced smad2 translocation, strongly blocked EMD-induced smad2 translocation. This antibody also blocked other actions of EMD in epithelial cells, i.e. p38-MAP kinase (p38-K) phosphorylation, p21WAF1/cip1 expression, and inhibition of DNA synthesis. In support of our previous proposal, these data suggest that TGF-beta 1 (or a TGF-beta-like substance), which is delivered as a principal bioactive factor in EMD, inhibits epithelial cell proliferation probably by a smad2-mediated, p21WAF1/cip1-dependent mechanism. However, the same neutralizing antibody failed to convincingly block EMD-induced fibroblastic proliferation, which suggests that EMD may contain additional unidentified mitogenic factor(s), which act in combination with TGF-beta to fully stimulate fibroblastic proliferation.