Anti-proliferative and pro-apoptotic effects of lichen-derived compound protolichesterinic acid are not mediated by its lipoxygenase-inhibitory activity.

Lipoxygenases (LOXs) and their products are involved in several biological functions and have been associated with carcinogenesis. Protolichesterinic acid (PA), a lichen metabolite, inhibits 5- and 12-LOX and has anti-proliferative effects on various cancer cell lines. Here, PA was shown to inhibit proliferation of multiple myeloma cells, RPMI 8226 and U266, and pancreatic cancer cells AsPC-1. Apoptosis was induced only in multiple myeloma cells. Cell-cycle associated changes in expression and sub-cellular localization of 5- and 12-LOX were not affected by PA but increased cytoplasmic localisation was found to accompany morphological changes at later stages. Assessment by mass spectrometry showed that PA entered the pancreatic cancer cells. However, effects on LOX metabolites were only evident after treatment with concentrations exceeding those having anti-proliferative effects and no effects were measurable in the myeloma cells. We conclude that the anti-proliferative and pro-apoptotic effects of PA are not mediated directly through inhibition of LOX activity.

Predictors of sustained maternal milk feeds in extremely preterm infants.

OBJECTIVE: To investigate the predictors of maternal milk feeds (MMFs) in extremely preterm (EPT) infants during neonatal stay. STUDY DESIGN: Maternal characteristics, obstetrical data and infant characteristics were correlated to MMFs in 97 EPT infants during the first 6 weeks of life and at hospital discharge. RESULT: High MMFs (>90%) at second week predicted sustained MMFs the first 6 weeks of life; nonuniversity education and non-Nordic origin were unfavorable predictors. The proportion of MMFs the first 6 weeks of life and maternal age were positively associated with MMFs at discharge, whereas overweight was an unfavorable predictor. High MMFs at second week, assisted reproduction technology and employment were predictive factors for exclusive MMFs at discharge. CONCLUSION: High MMFs at week 2 promote sustained MMFs in EPT infants and exclusive MMFs at discharge. Mothers who are either young, overweight, non-Nordic or without university education may need special interventions to establish successful lactation.

Human cytomegalovirus infection induces leukotriene B4 and 5-lipoxygenase expression in human placentae and umbilical vein endothelial cells.

INTRODUCTION: Human cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). METHODS: Seven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. RESULTS: HCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae. HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). CONCLUSION: The presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease.

Structural characterisation of a complex heteroglycan from the cyanobacterium Nostoc commune.

An alkali-extractable O-methylated ribofuranose-containing heteroglycan, Nc-5-s, was isolated from wild-growing field colonies of the cyanobacterium Nostoc commune collected in Iceland, using ethanol fractionation and anion-exchange chromatography. The average molecular weight was estimated to be 1500 kDa. Structural characterisation of the heteroglycan was performed by high-field NMR spectroscopy (1D proton, 2D-COSY, 2D-NOESY, 2D-TOCSY, (1)H (13)C-HSQC, HMBC, H2BC and HSQC-NOESY) as well as monosaccharide analysis after methanolysis by GC and supported by linkage analysis by GC-MS. According to the data obtained, the structure of Nc-5-s is composed of repeating units of 1, 1a, 1b and 2 and 2a in approximate molar ratio of (10:25:50:5:10).

In vitro and in vivo immunomodulating effects of traditionally prepared extract and purified compounds from Cetraria islandica.

Cetraria islandica (Iceland moss) has been used for centuries in folk medicine in many countries against a number of conditions, including inflammatory conditions, mainly as an aqueous extract. C. islandica contains many compounds, such as polysaccharides and secondary metabolites, some of which have established biological activity. However, very little is known about their effect on the immune system. Human monocyte-derived immature dendritic cells were cultured with an aqueous extract from C. islandica quantified with regard to the polysaccharides lichenan and isolichenan and secondary metabolites protolichesterinic and fumarprotocetraric acids. The purified compounds were also tested individually. Their effect on the maturation of the dendritic cells was assessed by measuring secretion of IL-10 and IL-12p40 and expression of surface molecules. In addition, the effect of the aqueous extract on antigen-induced arthritis in rats was investigated. The aqueous extract caused upregulated secretion of both IL-10 and IL-12p40, with IL-10 secretion being more prominent. Lichenan had similar effects, whereas isolichenan and the secondary metabolites were inactive, suggesting that the effect observed by the aqueous extract was mainly mediated by lichenan. Significantly less arthritis was observed for rats treated by the aqueous extract, administered subcutaneously, compared with rats treated with saline alone. These results suggest that the aqueous extract of C. islandica has anti-inflammatory effect, possibly by changing the cytokine secretion bias from IL-12p40 towards IL-10.

Effects of lichen heteroglycans on proliferation and IL-10 secretion by rat spleen cells and IL-10 and TNF-alpha secretion by rat peritoneal macrophages in vitro.

Four polysaccharides Pc-1, Pc-2, Pc-3 and Pc-4 were isolated from water and alkali extracts of the lichen Peltigera canina using ethanol fractionation, gel filtration and preparative HP-GPC. The monosaccharide composition was determined by methanolysis and GC and showed mannose and galactose as the predominating structural units. The mean M(r) was determined by HP-GPC. The heteroglycans were tested for in vitro immunomodulating activities and showed mitogenic activity in rat spleen cell proliferation assay and stimulated IL-10 secretion. In rat peritoneal macrophages, the heteroglycans stimulated TNF-alpha secretion, but not IL-10 secretion. These results indicate that the polysaccharides influence cells of the immune system both from the innate and the adaptive systems.

Immunologically active O6-branched (1-->3)-beta-glucan from the lichen Thamnolia vermicularis var. subuliformis.

A lentinan-type gel-forming beta-glucan, Ths-2, has been isolated in about 1.5% yield from the alkali extract of the lichen Thamnolia vermicularis var. subuliformis, using ethanol fractionation, dialysis and gel filtration. The mean Mr of Ths-2 was determined by GP-HPLC to be 67 kD, and the optical rotation was measured to be -14 degrees. The structure of Ths-2 was further elucidated by methylation analysis by GC-MS, 1H- and 13C-NMR spectroscopy and selective enzymatic hydrolysis with exo-(1 --> 3)-beta-D-glucanase followed by analysis of oligosaccharides by HPAEC-PAD. Ths-2 was found to be consisting of a (1 --> 3)-beta-D-glucopyranosyl main chain with branches of a (1 --> 6) linked glucopyranosyl unit on every third unit of the main chain. Similar polysaccharide structures have been described from fungi, but this is the first report of a lentinan-type (1 --> 3)-beta-D-glucan from a lichen species. The immunomodulating activity of Ths-2 was tested in an in vitro anti-complementary assay, and proved to be strongly active.

Rhamnopyranosylgalactofuranan, a new immunologically active polysaccharide from Thamnolia subuliformis.

A complex polysaccharide, Ths-3, consisting mainly of rhamnopyranosyl and galactofuranosyl units, has been isolated from the water extract of the lichen Thamnolia subuliformis using ethanol fractionation, dialysis, ion-exchange chromatography, gel filtration and preparative GP-HPLC. The mean M(r) of Ths-3 was determined to be 1450 kD, and the monosaccharide composition is gal/rha/glc/xyl/man in the ratio of 40:31:13:10:6. The structure of Ths-3 was further elucidated by methylation analysis by GC-MS and NMR spectroscopy and found to be basically composed of (1-->3)-linked beta-D-galactofuranosyl units with branches on C6, and rhamnosyl units being predominantly (1-->2)-linked with branches on C3 and C4, while some units are (1-->3)-linked. Glucose, mannose and galactofuranose are found as terminal units and glucose and mannose are also (1-->4)-linked, while xylose is only present as terminal units. The trisaccharide xylglcglc was detected after partial hydrolysis of the polysaccharide. The immunomodulating activity of Ths-3 was tested in an in vitro phagocytosis assay and the classical anticomplementary assay, and proved to be active in both tests. The authors suggest the trivial name thamnolan for Ths-3.