Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α(2) β(1)

Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.

Expression of Connexins in Normal and Neoplastic Canine Bone Tissue

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and I multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

Connexin36, an Essential Element in the Rod Pathway, Is Highly Expressed in the Essentially Rodless Retina of Gallus gallus

Electrical coupling provided by connexins (Cx) in gap junctions (GJ) plays important roles in both the developing and the mature retina. In mammalian nocturnal species, Cx36 is an essential component in the rod pathway, the retinal circuit specialized for night, scotopic vision. Here, we report the expression of Cx36 in a species (Gallus gallus) that phylogenetic development endows with an essentially rodless retina. Cx36 gene is very highly expressed in comparison with other Cxs previously described in the adult retina, such as Cx43, Cx45, and Cx50. Moreover, real-time PCR, Western blot, and immunofluorescence all revealed that Cx36 expression massively increased over time during development. We thoroughly examined Cx36 in the inner and outer plexiform layers, where this protein was particularly abundant. Cx36 was observed mainly in the off sublamina of the inner plexiform layer rather than in the on sublamina previously described in the mammalian retina. In addition, Cx36 colocalized with specific cell markers, revealing the expression of this protein in distinct amacrine cells. To investigate further the involvement of Cx36 in visual processing, we examined its functional regulation in retinas from dark-adapted animals. Light deprivation markedly up-regulates Cx36 gene expression in the retina, resulting in an increased accumulation of the protein within and between cone synaptic terminals. In summary, the developmental regulation of Cx36 expression results in particular circuitry-related roles in the chick retina. Moreover, this study demonstrated that Cx36 onto- and phylogenesis in the vertebrate retina simultaneously exhibit similarities and particularities. J. Comp. Neurol. 512:651-663, 2009. (C) 2008 Wiley-Liss, Inc.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K(ATP) channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC(50) of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca(2+)](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca(2+)](i) imaging.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats

In addition to reducing blood pressure, hydralazine can reduce the production of inflammatory cytokines and reduce the expression of leukocyte adhesion molecules. Differences in leukocyte behavior and leukocyte adhesion molecule expression in spontaneously hypertensive rats (SHR) compared to normotensive rats have been reported. However, whether hydralazine can reduce leukocyte migration in vivo in hypertension and in normotension remains unknown. To address this question, male SHR and Wistar rats were treated for 15 days with hydralazine at a dose of similar to 3.5 mg/kg or similar to 14 mg/kg in their drinking water. The numbers of rollers and adherent and migrated cells were determined by direct vital microscopy, and blood pressure was assessed by tail plethysmography. In addition, following treatment with the higher dose, immunohistochemistry was used to measure the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cells, while flow cytometry was used to evaluate the expression of leukocyte CD18 and L-selectin. Hydralazine reduced leukocyte adherence and migration in SHR either at the higher, that reduced blood pressure levels, or lower dose, which did not reduce it. Reduced ICAM-1 expression might be involved in the reduced migration observed in SHR. In Wistar rats, only at the higher dose hydralazine reduced blood pressure levels and leukocyte migration. Reduced P-selectin expression might be involved. We therefore conclude that hydralazine reduces leukocyte migration by different mechanisms in SHR and Wistar rats, specifically by reducing ICAM-1 expression in the former and P-selectin expression in the latter. (c) 2008 Elsevier B.V. All rights reserved.

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation. (C) 2007 Elsevier Ltd. All rights reserved.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Pivotal Role for Platelet-Activating Factor Receptor in CD36 Expression and oxLDL Uptake by Human Monocytes/Macrophages

The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined. Copyright (C) 2011 S. Karger AG, Basel

Human monocytes but not dendritic cells are killed by blocking of autocrine cyclooxygenase activity

Dendritic cells (DCs), in peripheral tissues, derive mostly from blood precursors that differentiate into DCs under the influence of the local microenvironment. Monocytes constitute the main known DC precursors in blood and their infiltration into tissues is up-regulated during inflammation. During this process, the local production of mediators, like prostaglandins (PGs), influence significantly DC differentiation and function. In the present paper we show that treatment of blood adherent mononuclear cells with 10 mu M indomethacin, a dose achieved in human therapeutic settings, causes monocytes` progressive death but does not affect DCs viability or cell surface phenotype. This resistance of DCs was observed both for cells differentiated in vitro from blood monocytes and for a population with DCs characteristics already present in blood. This phenomenon could affect the local balance of antigen-presenting cells, influence the induction and pattern of immune responses developed under the treatment with non-steroidal anti-inflammatory drugs and, therefore, deserves further investigation. (C) 2009 Elsevier Inc. All rights reserved.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Perpendicular exchange bias in IrMn/Pt/[Co/Pt](3) multilayers with cone magnetization

The perpendicular exchange bias and magnetic anisotropy were investigated in IrMn/Pt/[Co/Pt](3) multilayers through the analysis of in-plane and out-of-plane magnetization hysteresis loops. A phenomenological model was used to simulate the in-plane curves and the effective perpendicular anisotropies were obtained employing the area method. The canted state anisotropy was introduced by taking into account the first and second uniaxial anisotropy terms of the ferromagnet with the corresponding uniaxial anisotropy direction allowed to make a nonzero angle with the film`s normal. This angle, obtained from the fittings, was of approximately 15 degrees for IrMn/[Co/Pt](3) film and decreases with the introduction of Pt in the IrMn/Pt/[Co/Pt](3) system, indicating that the Pt interlayer leads to a predominant perpendicular anisotropy. A maximum of the out-of-plane anisotropy was found between 0.5 and 0.6 nm of Pt, whereas a maximum of the perpendicular exchange bias was found at 0.3 nm. These results are very similar to those obtained for IrMn/Cu/[Co/Pt](3) system; however, the decrease of the exchange bias with the spacer thickness is more abrupt and the enhacement of the perpendicular anisotropy is higher for the case of Cu spacer as compared with that of Pt spacer. The existence of a maximum in the perpendicular exchange bias as a function of the Pt layer thickness was attributed to the predominance of the enhancement of exchange bias due to more perpendicular Co moment orientation over the exponential decrease of the ferromagnetic/antiferromagnetic exchange coupling and, consequently, of the exchange-bias field. (C) 2011 Elsevier B.V. All rights reserved.

Temperature Dependence of the Intergranular Critical Current Density in Uniaxially Pressed Bi(1.65)Pb(0.35)Sr(2)Ca(2)Cu(3)O(10+delta) Samples

We have studied the normal and superconducting transport properties of Bi(1.65)Pb(0.35)Sr(2)Ca(2)Cu(3)O(10+delta) (Bi-2223) ceramic samples. Four samples, from the same batch, were prepared by the solid-state reaction method and pressed uniaxially at different compacting pressures, ranging from 90 to 250 MPa before the last heat treatment. From the temperature dependence of the electrical resistivity, combined with current conduction models for cuprates, we were able to separate contributions arising from both the grain misalignment and microstructural defects. The behavior of the critical current density as a function of temperature at zero applied magnetic field, J (c) (T), was fitted to the relationship J (c) (T)ae(1-T/T (c) ) (n) , with na parts per thousand 2 in all samples. We have also investigated the behavior of the product J (c) rho (sr) , where rho (sr) is the specific resistance of the grain-boundary. The results were interpreted by considering the relation between these parameters and the grain-boundary angle, theta, with increasing the uniaxial compacting pressure. We have found that the above type of mechanical deformation improves the alignment of the grains. Consequently the samples exhibit an enhance in the intergranular properties, resulting in a decrease of the specific resistance of the grain-boundary and an increase in the critical current density.

Optical Properties and Charge-Transfer Excitations in Edge-Functionalized All-Graphene Nanojunctions

We investigate the optical properties of edge-fiinctionalized graphene nanosystems, focusing on the formation of junctions and charge-transfer excitons. We consider a class of graphene structures that combine the main electronic features of graphene with the wide tunability of large polycyclic aromatic hydrocarbons. By investigating prototypical ribbon-like systems, we show that, upon convenient choice of functional groups, low-energy excitations with remarkable charge-transfer character and large oscillator strength are obtained. These properties can be further modulated through an appropriate width variation, thus spanning a wide range in the low-energy region of the UV-vis spectra. Our results are relevant in view of designing all-graphene optoelectronic nanodevices, which take advantage of the versatility of molecular functionalization, together with the stability and the electronic properties of graphene nanostructures.