Alumina-zirconia composites functionalized with laminin-1 and laminin-5 for dentistry: effect of protein adsorption on cellular response.

The present paper describes a study on laminin interaction with the surface of two alumina-zirconia composites with different percentages of ZrO2, both with submicrometric grain size. As major molecules within the basement membrane (BM), laminins are important protein fragments for epithelial cell adhesion and migration. On the other hand, alumina-zirconia composites are very attractive materials for dental applications due to their esthetic and mechanical properties. X-Ray photoelectron spectroscopy and atomic force microscopy were used to study the adsorption of two types of laminin, laminin-1 (Ln-1) and laminin-5 (Ln-5), onto the ceramics surfaces. The in vitro cell response was determined by intracellular phosphorylation of major kinases. Ceramics samples functionalized with laminins showed better cellular activation than untreated specimens; furthermore, cellular activation was found to be greater for the composite with higher percentage in zirconia when functionalized with Ln-5, whereas the adsorption of Ln-1 resulted in a greater activation for the alumina-rich oxide.

Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-κB signaling in resident macrophages.

Human mesenchymal stem/progenitor cells (hMSCs) repair tissues and modulate immune systems but the mechanisms are not fully understood. We demonstrated that hMSCs are activated by inflammatory signals to secrete the anti-inflammatory protein, TNF-α-stimulated gene 6 protein (TSG-6) and thereby create a negative feedback loop that reduces inflammation in zymosan-induced peritonitis. The results demonstrate for the first time that TSG-6 interacts through the CD44 receptor on resident macrophages to decrease zymosan/TLR2-mediated nuclear translocation of the NF-κB. The negative feedback loop created by MSCs through TSG-6 attenuates the inflammatory cascade that is initiated by resident macrophages and then amplified by mesothelial cells and probably other cells of the peritoneum. Because inflammation underlies many pathologic processes, including immune responses, the results may explain the beneficial effects of MSCs and TSG-6 in several disease models.

Effect of hyperbaric oxygen preconditioning on spleen lymphocytes and cell adhesion molecules after skin transplantation in mice.

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.

Preclinical absorption, distribution, metabolism and excretion (ADME) characterization of ICAM1988, an LFA-1/ICAM antagonist, and its prodrug.

Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min(-1) kg(-1), respectively. In mass balance studies using [(14)C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.

In vitro migration of melanoblasts requires matrix metalloproteinase-2: implications to vitiligo therapy by photochemotherapy.

It is known that the migration of melanocyte precursors (melanoblasts) from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial to the repigmentation of vitiliginous skin treated with photochemotherapy (PUVA), but such migratory cells must penetrate extracellular matrix tissue barriers in vivo. To test the hypothesis that matrix metalloproteinases (MMPs) are required for this process, we determined whether cultured melb-a cells, an immortal line of melanoblasts isolated from neonatal mouse epidermis, express and secrete MMPs and whether a synthetic metalloproteinase inhibitor, GM6001 (Galardin), inhibits their migratory behavior in vitro. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine the patterns of MMP expression by melanoblasts at the mRNA and protein levels, respectively. The proteolytic activities of MMPs secreted into the culture medium were assessed by gelatin zymography. The capacity of melanoblasts to migrate on fibronectin, laminin or laminin-5 substrates was estimated using Transwell migration assays. The results show that MMP2, MMP9 and MT1-MMP transcripts are expressed by these melanoblasts, but only MMP2 is secreted and activated in the extracellular environment. Although the therapeutic efficacy of PUVA in stimulating repigmentation of vitiliginous skin might derive from direct effects of UVA and/or 8-methoxypsoralen (8MOP), recent studies have shown that keratinocyte-derived factors induced by ultraviolet radiation, especially alpha-melanocyte stimulating hormone (alpha MSH), play a major role in regulating melanocyte function. Therefore, we also examined whether 8MOP and/or alphaMSH are involved in the up-regulation of MMP2 expression in melanoblasts. Western blotting and zymographic analyses revealed that MMP2 synthesis and secretion were induced by 8MOP and/or by alpha MSH. This induction of MMP2 resulted in significant increases of migration by melanoblasts on laminin or on laminin-5 substrates, while concomitant treatment with GM6001 blocked that induced migration. Taken together, these results suggest the importance of MMP2 in melanoblast migration and in the response to PUVA therapy.

A putative receptor mediating cell-density sensing in Dictyostelium.

When Dictyostelium cells starve, they begin secreting a glycoprotein called conditioned medium factor (CMF). When there is a high density of starved cells, as indicated by a high concentration of CMF, the cells begin expressing some genes and aggregate using pulses of cAMP as a chemoattractant. CMF regulates gene expression via a G protein-independent pathway, whereas CMF regulates cAMP signal transduction via a G protein-dependent pathway. To elucidate receptors mediating cell density sensing, we used CMF-Sepharose to isolate membrane proteins that bind CMF. We identified a 50-kDa protein, CMFR1, that is sensitive to trypsin treatment of whole cells. We obtained partial amino acid sequence of CMFR1 and isolated the cDNA encoding it. The derived amino acid sequence has no significant similarity to known proteins and has two or three predicted transmembrane domains. Expression of CMFR1 in insect cells caused an increase in CMF binding. Repression of CMFR1 in Dictyostelium by gene disruption resulted in a approximately 50% decrease of the CMF binding and a loss of CMF-induced G protein-independent gene expression. The G protein-dependent CMF signal transduction pathways appear to be functional in cmfr1 cells, suggesting that cells sense the density-sensing factor CMF using two or more different receptors.

TSG-6, a glycoprotein associated with arthritis, and its ligand hyaluronan exert opposite effects in a murine model of inflammation.

TSG-6 is an arthritis-associated hyaluronan binding protein whose production in synovial cells, chondrocytes, fibroblasts and mononuclear cells is stimulated by TNF-alpha and IL-1. The purpose of this study was to gain insights into the role of TSG-6 and its functional interactions with hyaluronan in inflammation. In the murine air pouch model of carrageenan/IL-1-induced inflammation TSG-6 showed a dramatic inhibitory effect on the cellular infiltration of the inflammatory site by neutrophilic PMN, while hyaluronan enhanced cellular infiltration. There was no indication of a neutralizing or cooperative effect when TSG-6 and hyaluronan were injected together. The potent antiinflammatory effect of TSG-6 along with its induction by proinflammatory cytokines suggests that TSG-6 is part of a negative feedback loop in the control of the inflammatory response.

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons.

Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN beta) and immune (IFN gamma) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN beta than to IFN gamma and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN beta plus IFN gamma was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN beta and IFN gamma differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN beta plus IFN gamma can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN beta or IFN gamma, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.