Processing-dependent and -independent pathways for recognition of iodinated contrast media by specific human T cells

One to three percent of patients exposed to intravenously injected iodinated contrast media (CM) develop delayed hypersensitivity reactions. Positive patch test reactions, immunohistological findings, and CM-specific proliferation of T cells in vitro suggest a pathogenetic role for T cells. We have previously demonstrated that CM-specific T cell clones (TCCs) show a broad range of cross-reactivity to different CM. However, the mechanism of specific CM recognition by T cell receptors (TCRs) has not been analysed so far.

Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?

Curcumin exerts its anti-inflammatory activity via inhibition of nuclear factor κB. Oropharyngeal epithelia and residing bacteria closely interact in inflammation and infection. This in vitro model investigated the effects of curcumin on bacterial survival, adherence to, and invasion of upper respiratory tract epithelia, and studied its anti-inflammatory effect. We aimed to establish a model, which could offer insights into the host-pathogen interaction in cancer therapy induced mucositis.

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-gamma and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-gamma and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Stimulation of human T cells with sulfonamides and sulfonamide metabolites

Exposure to sulfonamides is associated with a high incidence of hypersensitivity reactions. Antigen-specific T cells are involved in the pathogenesis; however, the nature of the antigen interacting with specific T-cell receptors is not fully defined.

Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells

http://www.ncbi.nlm.nih.gov/pubmed/20381326

Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Dimethyloxalylglycine stabilizes HIF-1α in cultured human endothelial cells and increases random-pattern skin flap survival in vivo

The goal of this study was to evaluate in vitro and in vivo the effects of up-regulation of the proangiogenic hypoxia inducible factor (HIF)-1α induced by dimethyloxalylglycine on endothelial cell cultures and on skin flap survival.

Impact of testosterone on the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro

Postmenopausal hormone therapy (HT) increases local estrogen formation in breast tissue. The enzymatic substrates depend on transmembrane anion transporting polypeptides (OATPs) to reach intracellular enzymes. The aim of this study was to investigate the effect of testosterone (T) on the expression of OATP-1A2, OATP-2B1, and OATP-3A1 in malignant (MCF-7, BT-474) and non-malignant (HBL-100) breast cells in vitro.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.

Evidence for chemokine-mediated coalescence of preformed flotillin hetero-oligomers in human T-cells

We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also targeted to the uropod of T-cells and neutrophils. Biochemical studies suggest formation of flotillin homo- and hetero-oligomers in other cell types, but so far knowledge is lacking on in situ flotillin organization in leukocytes. We have now analyzed flotillin organization in human T-cells using fluorescence resonance energy transfer (FRET). Coexpressed C-terminally tagged flotillin-1-mCherry and flotillin-2-enhanced green fluorescent protein (EGFP) show significant FRET when analyzed in intact human T-cells in the absence and presence of chemokine. In contrast, little FRET was observed between coexpressed flotillin-1-mCherry and flotillin-1-EGFP before or after chemokine addition, indicating predominant formation of heterodimers and/or -oligomers. Interestingly coexpression of untagged flotillin-2 strongly enhanced FRET between differently tagged flotillin-1 molecules in resting and chemokine-stimulated cells, indicating that close contacts of flotillin-1 molecules only occur in flotillin-2-containing hetero-oligomers. Comparable results were obtained for tagged flotillin-2. We further show that disruption of the actin network, depletion of intracellular calcium, and inhibition of phospholipase C all result in suppression of chemokine-induced polarization and flotillin cap formation, but do not abolish FRET between tagged flotillin-1 and -2. Our results support predominant formation of flotillin-1 and -2 hetero-oligomers in resting and chemokine-stimulated human T-cells which may importantly contribute to structuring of the uropod.