Protective role of selenium on thyroid morphology in iodine-induced autoimmune thyroiditis in Wistar rats.

Excess iodine may induce and exacerbate autoimmune thyroiditis (AIT) in humans and animals. In order to assess the potential protective mechanisms of selenium (Se) in thyroid autoimmunity, the effects of inorganic Se (sodium selenite) administration on thyroid morphology and follicular cytology were investigated in adult Wistar rats with iodine-induced AIT. A total of 48 adult Wistar rats (24 females, 24 males) were allocated to one of four dietary regimens: C0, control; C1, only potassium iodine (KI); C2, concomitant KI and Se; C3, only KI initially, followed by Se administration. For AIT induction the rats were fed with 0.05% KI for 56 days. Se-treated rats received 0.3 mg/l sodium selenite in drinking water. Thyroid tissues were collected for pathologic diagnosis after 7 days in C0 group, 56 days in C1 and C2 groups, and 112 days in C3 group. In C1 group, moderate to severe thyroiditis was observed in 83% of males and 50% of female rats (P=0.223). In C3 group 16.7% of male rats developed moderate thyroiditis and none in C2 group, whereas no females were identified with moderate to severe thyroiditis in C2 or C3 group. Thus, the administration of Se was proven to have protective effects against thyroiditis cytology in both male and female Wistar rats.

Atelocollagen-based Hydrogels Crosslinked with Oxidised Polysaccharides as Cell Encapsulation Matrix for Engineered Bioactive Stromal Tissue.

Tissue stroma is responsible for extracellular matrix (ECM) formation and secretion of factors that coordinate the behaviour of the surrounding cells through the microenvironment created. It's inability to spontaneously regenerate makes it a good candidate for research studies such as testing various tissue engineered products capable of replacing the stroma in order to assure normal tissue regeneration and function. In this study, a bioactive stroma was obtained considering two main components: 1) the artificial ECM formed using atelocollagen-oxidized polysaccharides hydrogels in which the polysaccharide compound (oxidised gellan or pullulan) has the role of crosslinker and 2) encapsulated stromal cells (dermal fibroblasts, ovarian theca-interstitial and granulosa cells). The cell-hosting ability of the hydrogels is demonstrated by a good diffusion of globular proteins (albumin) while the fibrillar morphology proves to be optimal for cell adhesion. These structural properties and cytocompatibility of the components maintain good cell viability and cell encapsulation for more than 12 days. Nevertheless, the results indicate some differences favouring the gellan crosslinked hydrogels. Ovarian stromal cells functionality was maintained as indicated by hormone secretion, confirming cell-cell signalling in encapsulated and co-culture conditions. In vivo implantation shows the regenerative potential of the cell-populated hydrogels as they are integrated into the natural tissue. The possibility of cryopreserving the hydrogel-cell system, while maintaining both cell viability and hydrogel structural integrity underlines the potential of these ready-to-use hydrogels as bioactive stroma for multipurpose tissue regeneration.

Signaling pathways downstream of P2 receptors in human neutrophils.

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y(2) receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y(2) receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only approximately 75% loss of nucleotide-induced Ca(2+) mobilization indicating that nucleotides cause Ca(2+) mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca(2+) mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca(2+) increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca(2+) mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca(2+) increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca(2+) chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y(2) receptor can cause Ca(2+) mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores.

P2Y(2) receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca(2+)-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils.

Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor.

Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y2 receptors. However, P2Y2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca2+ increases in isolated human neutrophils with an EC50 of 1 microM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.

Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) is a potent activator of neutrophil degranulation. The intracellular signaling mechanisms involved in the potentiating effect of fibrinogen on fMLP-induced primary granule release from human neutrophils were investigated. Fibrinogen caused a significant leftward shift of the concentration-response curve of fMLP-induced elastase release. An antibody against Mac-1 (CD11b/CD18) prevented the potentiating effect of fibrinogen, suggesting that soluble fibrinogen potentiates fMLP-induced degranulating effect by a mechanism mediated by the integrin Mac-1. Fibrinogen enhanced fMLP-induced tyrosine phosphorylation in human neutrophils and markedly enhanced the phosphorylation of mitogen-activated protein kinases (MAPK) caused by fMLP. However, U0126, an inhibitor of p44/42 MAPK activation, or SB-203580, an inhibitor of p38 MAPK, did not alter the effect of fibrinogen on fMLP-induced elastase release. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) kinase inhibitor, and genistein, a nonspecific tyrosine kinase inhibitor, strongly inhibited fMLP-induced elastase release both in the presence and in the absence of fibrinogen. An Akt/PKB inhibitor failed to alter the potentiating effect of fibrinogen, suggesting that the effect of fibrinogen is mediated by Akt-independent pathways. Go6976, an inhibitor of classical PKC isoforms, caused a significant inhibition of fMLP-induced elastase release in the presence or absence of fibrinogen, while nonselective inhibitors of PKC, Ro 31-8220, GF-109203X, and staurosporine, caused potentiation of fMLP-induced elastase release. We conclude that fibrinogen potentiation of primary granule release induced by fMLP is mediated by the integrin CD11b/CD18 through pathways dependent on PI3K and tyrosine kinases, but other regulatory mechanisms may be also involved.