Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Fossilization and degradation of archaeal intact polar tetraether lipids in ODP Site 201-1229 sediments

Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX86. GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL-GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death but it has been suggested that some of these IPL-GDGTs can, just like the CL-GDGTs, be preserved over geological timescales. Here, we examined IPL-GDGTs in deeply buried (0.2-186 mbsf, ~2.5 Myr) sediments from the Peru Margin. Direct measurements of the most abundant IPL-GDGT, IPL-crenarchaeol, specific for Thaumarchaeota, revealed depth profiles which differed per head group. Shallow sediments (<1 mbsf) contained IPL-crenarchaeol with both glycosidic- and phosphate headgroups, as also observed in thaumarchaeal enrichment cultures, marine suspended particulate matter and marine surface sediments. However, hexose, phosphohexose-crenarchaeol is not detected anymore below 6 mbsf (~7 kyr), suggesting a high lability. In contrast, IPL-crenarchaeol with glycosidic head groups is preserved over time scales of Myr. This agrees with previous analyses of deeply buried (>1 m) marine sediments, which only reported glycosidic and no phosphate-containing IPL-GDGTs. TEX86 values of CL-GDGTs did not markedly change with depth, and the TEX86 of IPL-derived GDGTs decreased only when the proportions of monohexose- to dihexose-GDGTs changed, likely due to the enhanced preservation of the monohexose GDGTs. Our results support the hypothesis that in situ GDGT production and differential IPL degradation in sediments is not substantially affecting TEX86 paleotemperature estimations based on CL GDGTs and indicate that likely only a small amount of IPL-GDGTs present in deeply buried sediments is part of cell membranes of active Archaea. The amount of archaeal biomass in the deep biosphere based on these IPLs may have been substantially overestimated.

Fabrication and characterization of SSZ tape cast electrolyte-supported solid oxide fuel cells

A 10 mol%Sc₂O₃, 1 mol%CeO₂ stabilized-ZrO₂ (SSZ) powder was successfully prepared using the sol-gel method. Subsequent SSZ electrolyte pellets were prepared by tape casting technique and sintered at 1400 °C, 1450 °C, 1500 °C, 1550 °C and 1600 °C. These were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). SSZ showed a pure cubic phase after sintering, the grain size of SSZ increased with the increase of sintering temperature. The SSZ sintered at 1550 °C showed the highest ion conductivity. The maximum power densities of Ni-SSZ/SSZ/La_0.8Sr_0.2MnO_3-δ (LSM)-SSZ single cells sintered at 1550 °C were 0.18, 0.36, 0.51 and 0.72 W cm^-2 at 650, 700, 750 and 800 °C, respectively. The polarization resistance (R_p) of the single cell attained 0.201 Ω cm² at 800 °C.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Up-regulation Of Dnam-1 And Nkp30, Associated With Improvement Of Nk Cells Activation After Long-term Culture Of Mononuclear Cells From Patients With Ovarian Neoplasia.

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.

Nanomolar Levels Of Pahs In Extracts From Urban Air Induce Mapk Signaling In Hepg2 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Ghost Cells In Pilomatrixoma, Craniopharyngioma, And Calcifying Cystic Odontogenic Tumor: Histological, Immunohistochemical, And Ultrastructural Study.

Pilomatrixoma, craniopharyngioma, and calcifying cystic odontogenic tumor are the main entities presenting ghost cells as an important histological feature, in spite their quite different clinical presentation; it seems that they share a common pathway in the formation of these cells. The aim of this study is to examine and compare the characteristics of ghost and other cells that form these lesions. Forty-three cases including 21 pilomatrixomas, 14 craniopharyngiomas, and eight calcifying cystic odontogenic tumors were evaluated by immunohistochemistry for cytokeratins, CD138, β-catenin, D2-40, Glut-1, FAS, CD10 and also by scanning electron microscopy. The CKs, CD138, β-catenin, Glut-1, FAS, and CD10 were more often expressed by transitional cells of craniopharyngioma and calcifying cystic odontogenic tumor, compared with pilomatrixoma. Basaloid cells of pilomatrixoma showed strong positivity for CD138 and CD10. Differences on expression pattern were identified in transitional and basal cells, as ghost cells were negative for most antibodies used, except by low expression for cytokeratins. By scanning electron microscopy, the morphology of ghost cells were similar in their fibrillar cytoplasm, but their pattern varied from sheets in pilomatrixoma to small clusters in craniopharyngioma and calcifying cystic odontogenic tumor. Mechanisms involved in formation of ghost cells are unknown, but probably they follow different pathways as protein expression in the basal/transitional cells was not uniform in the three tumors studied.

Antibody Recognition Of Plasmodium Falciparum Infected Red Blood Cells By Symptomatic And Asymptomatic Individuals In The Brazilian Amazon.

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Hippocampal Dysplasia With Balloon Cells: Case Report And Discussion On Classification.

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Platinum Blue Staining Of Cells Grown In Electrospun Scaffolds.

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.