Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1

High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis.

Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-gamma mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-beta mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.

Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.