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LAM S5 is a polysulphated derivative of the glucan laminarian that inhibits basic fibroblast growth factor (bFGF) binding and the bFGF-stimulated proliferation of fetal bovine heart endothelial (FBHE) cells. This report demonstrates that LAM S5 has anti-angiogenic activity, as shown by inhibition of tubule formation by endothelial cells cultured on Matrigel and inhibition of vascularisation of the chick chorioallantoic membrane. In addition, LAM S5 caused a tumour growth delay of the murine RIF-1 tumour of 2.6 days (P = 0.01). Images Figure 2

Myocilin is a 55-57kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic betaB1-crystallin-MYOC mice have been developed that strongly overexpress myocilin in their eyes. The transgenic overexpression of myocilin results in an almost five-fold increase of secreted normal myocilin in the aqueous humor of betaB1-crystallin-MYOC mice. In the present study, we wanted to use betaB1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of myocilin. RNA was isolated from ocular tissues of betaB1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in betaB1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant myocilin. Although betaB1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant myocilin. Substantially higher amounts in ocular tissues of betaB1-crystallin-MYOC mice were found for connexin 46 and alphaB-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family.

The influence of the triterpenoid saponins 1-10 has been investigated on murine spleenocytes in the lymphocyte transformation test and on murine macrophages in an phagocytosis assay. The lymphocyte transformation test and the phagocytosis assay showed that the tested compounds have no stimulating effect. However, a significant inhibition of lymphocyte proliferation by the triterpenoid saponins 2, 6 and 10 was demonstrated.

l-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of l-carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the anti-proliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of l-carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC50 value), approx. 0.5 microgram/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of l-carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by l-carrageenan (IC50 value, 5.5 micrograms/ml) and the cells were arrested at the G1/S boundary. l-Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor alpha (TGF alpha) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody alpha IR3 enhanced the inhibitory activity of l-carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by l-carrageenan. l-Carrageenan had low anti-coagulant activity. l-Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.

Group 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (TH2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating ‘naive’ ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings., ILC2 metabolism has been largely unexplored. Di Santo and colleagues examine metabolic profiles from naive and cytokine-activated ILC2s and find that IL-33-triggered ILC2s rely on distinct metabolic pathways to sustain proliferation and function.

Journal Article; Research Support, Non-U.S. Gov't; CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p