We have applied a new software to analyse a human naive single-chain antibody (scFv) library, comprehensively revealing the diversity of antibody variable complementarity-determining regions (CDRs) and their families.
A novel DNAseq program for enhanced analysis of Illumina GAII data: a case study on antibody complementarity-determining regions.
No sample metadata fields
View SamplesIn this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration.
Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells.
Disease, Disease stage
View SamplesOur group showed that DC-instrinsic C3ar1/C5ar1 signals are required for TLR-initiated DC maturation in vivo. To more broadly analyze how local complement signaling affects DC maturation process in response to TLR9 stimulation, WT or C3ar1-/-C5ar1-/- mice were stimulated with CpG (i.v. 100 micrograms) or vehicle control. 4hrs later, splenic CD11c+DCs were isolated and RNAs from the cells were purified for microarray analyses.
TLR-Induced Murine Dendritic Cell (DC) Activation Requires DC-Intrinsic Complement.
Specimen part
View SamplesAnalysis of gene expression (RNAseq) from isolated kidney macrophages injetced i.v. with PBS Overall design: C57BL/6J mice were injected i.v. with PBS. One hour after injection, kidney macrophages were isolated (sorted by FACS) for gene expression analysis.
Immune Monitoring of Trans-endothelial Transport by Kidney-Resident Macrophages.
Cell line, Subject
View SamplesTo provide a more detailed survey of adaptive changes in the physiology of P. aeruginosa (PA) during long-term infection of the cystic fibrosis (CF) lung, we performed a comparative proteome and transcriptome analysis of a set of isogenic sequential non-mutator and mutator isolates from three selected CF patients. Recently, we showed that during CF lung persistence PA mutators converge to a virulence-attenuated phenotype. In this study, we demonstrate that besides virulence-associated traits (VATs) the adaptation process of PA predominantly comprises metabolic pathways. In end-stage mutator strains, transcripts of genes encoding VATs, chemotaxis, degradation of aromatic compounds and several two-component regulatory systems were decreased. In contrast, several transcripts of genes or proteins involved in metabolism of fatty acids, nucleotides, amino acids and the generation of energy were increased. Of particular interest is the increased expression level of genes involved in (i) the anaerobic arginine-deiminase pathway, (ii) the anaerobic respiration such as nitrate-uptake protein OprF, redox-active azurin and cytchrome c551 peroxidase, (iii) the micro-aerobic respiration such as high oxygen-affinity cytochrome oxidase cbb3 (iv) the tricarboxylic acid cycle (TCA), glyoxylate shunt and anaplerotic carboxylation reactions to oxaloacetate. Strikingly, an increased transcription of the anaerobic regulator gene anr correlates with the up-regulation of ANR-dependent genes. In conclusion, these changes in transcriptome and proteome indicate an adaptive shift towards constitutive expression of genes of metabolic pathways obviously required for growth under micro-aerobic and nutritional conditions of suppurative CF lung tissue. Finally, these results provide us with new targets for antimicrobial agents and biomarkers reflecting adaptation of PA towards progressive CF lung disease.
Stage-specific adaptation of hypermutable Pseudomonas aeruginosa isolates during chronic pulmonary infection in patients with cystic fibrosis.
No sample metadata fields
View SamplesHuman ILCs are classically categorized into five subsets; cytotoxic CD127-CD94+ NK cells and non-cytotoxic CD127+CD94-, ILC1s, ILC2s, ILC3s and LTi cells. Here, we identify a novel subset within the CD127+ ILC population, characterized by the expression of the cytotoxic marker CD94. These CD94+ ILCs strongly resemble conventional ILC3s in terms of phenotype, transcriptome and cytokine production, but are highly cytotoxic. IL-15 was unable to induce differentiation of CD94+ ILCs towards mature NK cells. Instead, CD94+ ILCs retained RORγt, CD127 and CD200R expression and produced IL-22 in response to IL-15. Culturing non-cytotoxic CD127+ ILC1s or ILC3s with IL-12 induced upregulation of CD94 and cytotoxic activity, effects that were not observed with IL-15 stimulation. Thus, human helper ILCs can acquire a cytotoxic program without differentiating into NK cells.
Identification of human cytotoxic ILC3s.
Specimen part, Subject
View SamplesWe report that WT1 transcriptional repressor protein BASP1 interacts with oestrogen receptor alpha (Era), and interaction which in enhanced in the presence of Tamoxifen. We utilised RNASeq to identify common BASP1 and ERa target genes as well as Tamoxifen responsive genes that are altered in the absence of BASP1. Overall design: Total mRNA sequencing analysis of MCF7 cells treated with either siRNA against BASP1 or negative control siRNA, with and without Tamoxifen treatment. Each experiment was performed in triplicate.
BASP1 interacts with oestrogen receptor α and modifies the tamoxifen response.
No sample metadata fields
View SamplesDietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the Caenorhabditis elegans HNF4a-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span. Overall design: Young adult worms before bearing eggs inside were collected. N2 serves as the control of wild type. 3 biological replicates included in this experiment.
Dietary restriction induced longevity is mediated by nuclear receptor NHR-62 in Caenorhabditis elegans.
Subject
View SamplesBronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of anti-microbial effectors. To investigate the role of epithelial cells upon infection of airway pathogens, we stimulated BEAS-2B cells for 4 h with UV-inactivated bronchial pathogens including Staphylococcus aureus, Pseudomonas aeruginosa and Respiratory Syncitial Virus (RSV) that among other receptors can strongly activate TLR2, TLR4 and TLR3, respectively.
Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells.
No sample metadata fields
View SamplesMultiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Four histopathological patterns of MS have been described. Pattern II is characterized by infiltrating macrophages and T-cells and by antibody and complement deposition. Transcriptome analysis of three patern II demyelinating brain lesions from a multiple sclerosis patient using RNA sequencing demonstrated the presence of mRNA transcripts for genes specific of activated macrophages, T and B cells as well as genes coding for immunoglobulins, complement proteins and some pattern II associated proteins, providing additional evidence supporting pattern II demyelination. Overall design: Examination of 3 different demyelinating lesions identified by Immunohistopathology.
Central role of Th2/Tc2 lymphocytes in pattern II multiple sclerosis lesions.
No sample metadata fields
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