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Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR–RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8⁺ T cells determined by ELISpot (p?<?0.01). Moreover, CD8⁺ T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p?<?0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p?<?0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.     

Identification of novel potentially toxic oligomers formed in vitro from mammalian-derived expanded huntingtin exon-1 protein

Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.     

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.     

The lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTbetaR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTbetaR expression was found in all TEC subsets, but the highest levels were detected in MTS-15(+) thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTbetaR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEC(low)), as well as maintenance of MTS-15(+) fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta(-/-) and Ltbr(-/-) mice. Thus, we propose a broader role for LTalpha1beta2-LTbetaR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEC(low) for efficient induction of central tolerance.     

Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-rich mucilage

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.