IL-15 augments TCR-induced CD4⁺ T cell expansion in vitro by inhibiting the suppressive function of CD25High CD4⁺ T cells

Due to its critical role in NK cell differentiation and CD8(+) T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4(+) T cells. The increased levels of IL-15 found in several CD4(+) T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4(+) T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4(+) and CD8+ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4(+) T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15R alpha was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15R beta, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4(+) T cell suppression by a gradually expanding CD25(High)CD4(+) T cell subset that expresses Foxp3 and originates from CD4(+)CD25(+) Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology

Clinical procedure for colon carcinoma tissue sampling directly affects the cancer marker-capacity of VEGF family members

Background: mRNA levels of members of the Vascular Endothelial Growth Factor family (VEGF-A, -B, -C, -D, Placental Growth Factor/PlGF) have been investigated as tissue-based markers of colon cancer. These studies, which used specimens obtained by surgical resection or colonoscopic biopsy, yielded contradictory results. We studied the effect of the sampling method on the marker accuracy of VEGF family members. Methods: Comparative RT-qPCR analysis was performed on healthy colon and colon carcinoma samples obtained by biopsy (n = 38) or resection (n = 39) to measure mRNA expression levels of individual VEGF family members. mRNA levels of genes encoding the eicosanoid enzymes cyclooxygenase 2 (COX2) and 5-lipoxygenase (5-LOX) and of genes encoding the hypoxia markers glucose transporter 1 (GLUT-1) and carbonic anhydrase IX (CAIX) were included as markers for cellular stress and hypoxia. Results: Expression levels of COX2, 5-LOX, GLUT-1 and CAIX revealed the occurrence in healthy colon resection samples of hypoxic cellular stress and a concurrent increment of basal expression levels of VEGF family members. This increment abolished differential expression of VEGF-B and VEGF-C in matched carcinoma resection samples and created a surgery-induced underexpression of VEGF-D. VEGF-A and PlGF showed strong overexpression in carcinoma samples regardless of the sampling method. Conclusions: Sampling-induced hypoxia in resection samples but not in biopsy samples affects the marker-reliability of VEGF family members. Therefore, biopsy samples provide a more accurate report on VEGF family mRNA levels. Furthermore, this limited expression analysis proposes VEGF-A and PlGF as reliable, sampling procedure insensitive mRNA-markers for molecular diagnosis of colon cancer

Boosting in planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (PRRSV) and subsequent evaluation of their immunogenicity

Porcine reproductive and respiratory syndrome (PRRS) is a disease of swine, caused by an arterivirus, the PRRS virus (PRRSV). This virus infects pigs worldwide and causes huge economic losses. Due to genetic drift, current vaccines are losing their power. Adaptable vaccines could provide a solution to this problem. This study aims at producing in planta a set of antigens derived from the PRRSV glycoproteins (GPs) to be included in a subunit vaccine. We selected the GP3, GP4 and GP5 and optimized these for production in an Arabidopsis seed platform by removing transmembrane domains (Tm) and/or adding stabilizing protein domains, such as the green fluorescent protein (GFP) and immunoglobulin (IgG) 'Fragment crystallizable' (Fc) chains. Accumulation of the GPs with and without Tm was low, reaching no more than 0.10% of total soluble protein (TSP) in homozygous seed. However, addition of stabilizing domains boosted accumulation up to a maximum of 2.74% of TSP when GFP was used, and albeit less effectively, also the Fc chains of the porcine IgG3 and murine IgG2a increased antigen accumulation, to 0.96% and 1.81% of TSP respectively, while the murine IgG3 Fc chain did not. Antigens with Tm were less susceptible to these manipulations to increase yield. All antigens were produced in the endoplasmic reticulum and accordingly, they carried high-mannose N-glycans. The immunogenicity of several of those antigens was assessed and we show that vaccination with purified antigens did elicit the production of antibodies with virus neutralizing activity in mice but not in pigs

Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Background: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 mu g and less) and has a sound reproducibility. Conclusions: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner

Mycolates of Mycobacterium tuberculosis modulate the flow of cholesterol for bacillary proliferation in murine macrophages

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic <it>P. pastoris </it>yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.</p> <p>Results</p> <p>Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and <it>P. pastoris </it>cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from <it>Pichia </it>supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in <it>Pichia </it>more efficient than most other previously described trispecific or trivalent molecules produced in <it>E. coli</it>.</p> <p>Conclusion</p> <p>Bi- and tribody molecules are efficiently produced in <it>P. pastoris</it>. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of <it>P. pastoris </it>as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.</p

Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice

Introduction: Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. Methods: To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. Results: In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXR and LXR isoforms (LXR-/--/-) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXR-/--/- mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4(+) lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. Conclusions: In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production

pH-Degradable mannosylated nanogels for dendritic cell targeting

We report on the design of glycosylated nanogels via core-cross linking of amphiphilic non-water-soluble block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepared by reversible addition fragmentation (RAFT) polymerization. Self-assembly, pH-sensitive core-cross-linking, and removal of remaining PFP esters and protecting groups are achieved in one pot and yield fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor (MR) under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the MR that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunological applications involving DCs and macrophage subsets