IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities

IFN-alpha is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-alpha can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-alpha can differentiate into DCs (IFN-alpha-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-alpha, IL-1beta, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-alpha neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-gamma secretion and expansion of CD8(+) T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-alpha following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-alpha at the early steps of immune response to pathogens or in autoimmune diseases.     

The harpacticoid copepod Tisbe holothuriae is resistant to the insidious effects of polyunsaturated aldehyde-producing diatoms

Numerous species of diatoms liberate oxylipins including polyunsaturated aldehydes (PUAs) in response to cellular damage such as may occur during grazing. PUAs are cyto- and genotoxic and negatively disrupt reproductive processes in copepods, their principal grazers, although experimental evidence would suggest that the grazer response may be species specific. The reproduction of the benthic harpacticoid copepod Tisbe holothuriae was compared over two generations. Copepods were reared using four diet treatments: PUA-producing diatom strains Skeletonema marinoi (Adriatic Sea Isolate FE6) and Melosira nummuloides (CCAP 1048/6); and non-PUA-producing diatom strains Phaeodactylum tricornutum (CCAP 1052/A) and S. marinoi (Seasalter (Walney) Ltd). Life tables were generated for each treatment using measured reproductive parameters and the net reproductive rate (R₀) calculated. No significant differences were observed between the individual reproductive parameters of T. holothuriae fed PUA-producing diatoms compared to those fed non-PUA-producing diatoms although diets of P. tricornutum resulted in some decreases in individual reproductive parameters in the second generation. There were no significant differences in the R₀ values between the four tested diets. These observations indicate that T. holothuriae exhibits a tolerance of known PUA-producing diatom diets that has not been similarly demonstrated in pelagic calanoid copepods. Harpacticoid copepods may have a greater capacity to detoxify diatom oxylipins than their planktonic calanoid counterparts.     

Raman spectroscopy predicts the link between claw keratin and bone collagen structure in a rodent model of oestrogen deficiency

Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.     

Glider and Vision: Two New Families of Miniature Inverted-Repeat Transposable Elements in Xenopus Laevis Genome

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the α-tropomyosin (α-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated α-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284 bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the laevis genome. Every MITEs elements but two described in our study are found either in 5′ or in 3′ regulatory regions of genes suggesting a potential role in gene regulation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of separate structural features that affect rate and cation concentration dependence of self-cleavage by the Neurospora VS ribozyme

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.     

A DEVELOPMENTAL STUDY OF GUTTULINOPSIS VULGARIS (ACRASIALES)

The cellular slime mold, Guttulinopsis vulgaris E. W. Olive, is a common and widely distributed species. Its myxamoebae have lobose pseudopodia and aggregate, not by streaming, but by movement singly or in small groups into the aggregation center. The resulting shield-shaped pseudoplasmodium gives rise to one (or possibly more on occasion) whitish, simple or compound sorocarp. Sorocarps are 95–465 μ in height, typically with relatively short, stout stalks and with mucilaginous spore heads that measure 75–350 μ broad. The spores are mostly irregular, with flattened or concave sides, and are surrounded by a thin spore wall. They measure 3.8–9.2 μ diam., or 2.6–7.8 × 3.9–9.2 μ. During sorocarp development the myxamoebae become grouped within membranous compartments both in the sorocarp and pseudoplasmodium. In the upper part of the sorocarp they develop almost entirely into spores, while in the lower stalk region of the sorocarp and in the pseudoplasmodium, some myxamoebae develop into spores while others degenerate. The phylogenetic position of Guttulinopsis is unclear, but the genus is probably not closely related to the Dictyosteliaceae. This investigation has been supported by National Science Foundation Grants G–44263 and GB-501. The writer is grateful to Miss Carmen Stoianovitch for her assistance.     

Hepatitis C virus fails to activate NF-κB signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.     

Human CD28 and CTLA-4 Ig superfamily genes are located on chromosome 2 at bands q33–q34

CD28 is a cell surface molecule present on most peripheral T cells which has been implied in the amplification of the T-cell response in vitro. Using in situ hybridization on human prometaphase cells, we have found that the human CD28 gene maps to chromosome 2 at bands q33–q34, as shown previously for the CTLA-4 gene. CD28 and CTLA-4 are both members of the Ig superfamily, where they define a subgroup of membrane-bound single V domains. Their chromosomal proximity and their close structural relationship suggest that these two genes could be the result of the duplication of a common evolutionary precursor and may share some functional properties. Address correspondence and offprint requests to: M. Lafage-Pochitaloff.