Interleukin-8 (IL8/CXCL8) is present in decidua and trophoblast, which also expresses the IL8 receptors, CXCR1 and CXCR2. IL8 was shown to stimulate trophoblast migration. Matrix metalloproteinase (MMP)2, MMP9, and integrins a 5 b 1 and a 1 b 1 were found to play important roles in trophoblast invasion. We hypothesized that IL8 would increase this cell migration and invasion by HTR-8/SVneo cells through the activity of MMPs and integrins. Isolated first trimester of pregnancy cytotrophoblast (CT) and HTR-8/SVneo cell line were used. Migration was studied by monolayer wounding test, and invasion by Matrigel invasion test. The effects of IL8 on MMPs and integrin subunit expression were determined in HTR-8/SVneo cells by gelatin zymography and western blot respectively. The results that were obtained showed that exogenous IL8 stimulated HTR-8/SVneo cell migration and invasion. MMP2 and MMP9 levels were stimulated to 182% (P!0.01) and 134% (P!0.01) respectively. Integrin a 5 expression was increased to 119% (P!0.05) and integrin b 1 expression to 173% (P!0.001) of the control values. The data that were obtained show for the first time the sensitivity of the HTR-8/SVneo cells, in addition to isolated first trimester CT, to IL8. Exogenous IL8/CXCL8 increased trophoblast cell migration and invasion, which may be partly attributable to stimulation of MMP2 and MMP9 levels and an increase in integrins. HTR-8/SVneo cell viability and proliferation were also increased.

BackgroundInteractions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.Methods and FindingsFunction blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM±6.3, P<0.001) and to 66% of control (SEM±1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM±16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM±51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM±58, P<0.001) by CS-gal-1, and to 200% (SEM±24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.Conclusion and SignificanceThese findings qualify gal-1 as a member of human trophoblast cell invasion machinery.

SUMMARYWe have studied the effect of mycophenolate mofetil (MMF), a new drug used in prevention of transplant rejection, on differentiation, maturation and allostimulatory activity of human monocyte-derived dendritic cells (MDDC). MDDC were generated in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of MMF. MMF reduced the number of immature MDDC in culture, dose-dependently, by inducing apoptosis and inhibited their stimulatory activity on allogeneic lymphocytes. These changes correlated with down-regulation of costimulatory and adhesion molecules such as CD40, CD54, CD80 and CD86. No differences were observed in mannose receptor (MR)-mediated endocytosis, measured by the uptake of fluorescein isothiocyanate (FITC)-dextran. MDDC differentiated in the presence of MMF showed significantly reduced maturation upon stimulation with lipopolysaccharide, as judged by lower expresson of CD83 and co-stimulatory molecules, lower production of tumour necrosis factor (TNF)-a , IL-10, IL-12 and IL-18 as well as lower stimulation of alloreactive T cells including naive CD4 + CD45RA + T cells. In contrast, MDDC matured in the presence of MMF showed a more marked decrease in the FITC-dextran uptake than mature MDDC cultivated without MMF and the phenomenon correlated with downregulation of the MR expression. These results suggest that MMF impairs differentiation, maturation and function of human MDDC in vitro , which is an additional mechanism of its immunosuppressive effect.

Hot water extract (LN), partially purified polysaccharides (LP) and hot alkali extracted polysaccharides (LNa) obtained from fruiting bodies of the wild basidiomycete Laetiporus sulphureus were examined for their antioxidant activities. LNa was the most active antioxidant, as shown by the median effective concentrations (EC50 values) of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity (0.5 ± 0.2 mg/ml), reducing power (4.0 ± 0.3 mg/ml) and ferrous ion-chelating ability (1.5 ± 0.1 mg/ml). LNa contained the highest level of α-glucan (17.3 ± 1.2 g/100 g dw), whereas LP contained mostly β-glucans (66.8 ± 1.3 g/100 g dw). The prevalent monosaccharide in all extracts was glucose. The EC50 values of all three antioxidant activity assays were well-correlated with the α-glucan content. Strong and significant correlation was found between total phenolic compounds and DPPH scavenging ability and also reducing power. The three investigated extracts (at concentrations of 0.1-10 mg/ml) were not toxic to HTR-8/SVneo trophoblast cell line.

Invasive extravillous cytotrophoblast of the human placenta expresses galectins-1, -3, and -8 in vivo and in vitro. This study aimed to investigate the potential role of galectin-3 in cell migration and invasion, using recombinant human galectin-3 (rhgalectin-3), small molecule galectin inhibitor I47, and galectin-3 silencing. HTR-8/SVneo cell migration was stimulated by rhgalectin-3 and reduced by I47, which could be neutralised by rhgalectin-3. Inhibitor specificity and selectivity for the galectins expressed in extravillous trophoblast were validated in solid phase assays using recombinant galectin-1, -3, -8, confirming selectivity for galectin-3. HTR-8/SVneo cell migration and invasion, and invasion by isolated trophoblast cells in primary culture were significantly reduced in the presence of I47, which could be restored by rhgalectin-3. Upon HTR-8/SVneo cell treatment with galectin-3 siRNA both LGALS3 and galectin-3 protein were dramatically decreased. Silencing of galectin-3 induced significant reduction in cell migration and invasion, which was restored by rhgalectin-3. The influence on known mediators of cell invasion, MMP2 and -9, and integrins α1, α5, and β1 was followed in silenced cells, showing lower levels of MMPs and a large reduction in integrin subunit β1. These results show that galectin-3 acts as a pro-invasive autocrine/paracrine factor in trophoblast in vitro.