Our 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 SamplesWe 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 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 SamplesBalb/c donor hearts were transplanted into C57/BL6 recipients as previously described (Corry et al, 1973). Recipient mice were treated with 250g anti-CD40L mAb for tolerance induction on days 0, 2, and 4 as previously described (Jiang et al., 2011) or left untreated. On day 5 after transplantation graft infiltrating myeloid subsets were isolated using fluorescence activated cell sorting (FACS). Affymetrix Mouse Gene arrays were run in triplicate with the samples of interest. Raw CEL file data from Affymetrix Expression Console were background corrected, normalized, and summarized using RMA.
DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.
Treatment
View SamplesTo determine the molecular basis of gene regulation in pancreatic ductal epithelial cells, we developed methods for the isolation of this cell population during mouse development and normal adult homeostasis, as well as in conditions with ductal features (acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)). Our technique utilizes the specificity of Dolichos biflorus Agglutinin (DBA) lectin marking the entire normal ductal tree, including terminal intercalated ducts (putative sites of stem or progenitor cells) and ductal structures in ADM and PanIN. We used ferromagnetic-labeled DBA lectin to isolate ductal structures. Ductal cells were isolated under the following conditions: (1) Embryonic Development in wild type mice: E14.5, E15.5, E16.5, and postnatal day 1 (P1); (2) Injury and regeneration (pancreatitis) 0, 1, 3, 5 days following cerulein-induced acute pancreatitis. Cerulein is a cholecystokinin analog which produces a self-limited pancreatitis with injury and subsequent regeneration and repair, completed five days after insult; and (3) Pdx1-Cre;LSL-KrasG12D/+ mice aged 10 and 20 weeks that harbor PanIN lesions and a subset develop PDAC. Ductal/PanIN cells were isolated from these mice and appropriate control mice (Pdx1-Cre;Kras+/+).
The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis.
Age, Specimen part, Treatment, Time
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 Samples