Tirucallane triterpenoids from the stem bark of Araliopsis synopsis

Two new tirucallane triterpenoids, 21-methoxy-21,23-epoxy-tirucalla-7,24-dien-3α-ol (1) and 21-methoxy-21,23-epoxy-tirucalla-7,24-diene-1α,3α-diol (2), together with thirteen known compounds were isolated from the CH₂Cl₂ extract of the stem bark of Araliopsis synopsis. The structures of the compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data and comparison with previously known analogs. Compounds 1–10 were tested against bacteria, fungi and plant pathogen oomycetes by the paper disk agar diffusion assay resulting in missing to low activities corresponding with MICs > 1 mg/mL. However, compounds 5–10 exhibited high cytotoxic activity against the human Caucasian prostate adenocarcinoma cell PC-3 line, with IC₅₀ 8.5–12.5 μM compared to the standard Doxorubicin with IC₅₀ = 0.9 μM, while compounds 1, 3 and 4 showed low activity.     

Changes in neuronal DNA content variation in the human brain during aging

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.     

Erythropoietin mimetic compound AGEM400(HES) binds to the same receptor as erythropoietin but displays a different spectrum of activities

EPO mimetic peptides (EMPs) have a completely different structure than erythropoietin (EPO) or new generation recombinant erythropoiesis stimulating agents (ESAs) like Darbepoietin alfa (Aranesp) and continuous erythropoiesis stimulating agent (CERA). This study intended to compare the effects of a novel compound called AGEM400(HES), consisting of a dimeric EMP conjugated to hydroxyethyl starch (HES), to those of recombinant EPO. AGEM400(HES) efficiently stimulated erythropoiesis in vitro and efficiently stimulated survival of EPO-dependent cell line UT7/EPO. It also efficiently induced phosphorylation of signaling proteins in these models. However, AGEM400(HES) was shown to have weak or absent effects on survival of, and signaling in, three different EPO-responsive hematopoietic cell lines. In the latter models, when added in excess to moderate concentrations of EPO, AGEM400(HES) inhibited the activity of EPO in a fashion indicating receptor binding competition between EPO and AGEM400(HES). It was furthermore shown, using stably transfected BA/F3 cells, that the degree of responsiveness of a cell to AGEM400(HES) relative to its responsiveness to EPO, correlated with the level of EPO receptor surface expression. The findings presented raise intriguing possibilities because they imply that not all side-effects said to be associated with EPO must necessarily be elicited by AGEM400(HES) too.     

Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4⁺ T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4⁺ T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.     

Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Long-term immunity against actual poxviral HLA ligands as identified by differential stable isotope labeling

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.