Cytokines In Cell Culture Supernatants Research Articles

Synthesis of diosgenin analogues as potential anti-inflammatory agents

We herein report the synthesis of diosgenin analogues from commercially available diosgenin as the starting material. The structures of newly synthesised compounds were confirmed by 1H NMR, 13C NMR and mass spectrometry. All analogues were evaluated for in-vitro anti-inflammatory profile against LPS-induced inflammation in primary peritoneal macrophages isolated from mice by quantification of pro-inflammatory (TNF-α, IL-6 and IL-1β) cytokines in cell culture supernatant using the ELISA technique followed by in-vitro cytotoxicity study. Among the synthesised analogues, analogue 15 [(E) 26-(3′,4′,5′-trimethoxybenzylidene)-furost-5en-3β-acetate)] showed significant anti-inflammatory activity by inhibiting LPS-induced pro-inflammatory cytokines in a dose-dependent manner without any cytotoxicity. Efficacy and safety of analogue 15 were further validated in an in-vivo system using LPS-induced sepsis model and acute oral toxicity in mice. Oral administration of analogue 15 inhibited the pro-inflammatory cytokines in serum, attenuated the liver and lung injury and reduced the mortality rate in sepsis mice. Acute oral toxicity study showed that analogue 15 is non-toxic at higher dose in BALB/c mice. Molecular docking study revealed the strong binding affinity of diosgenin analogues to the active site of the pro-inflammatory proteins. These findings suggested that analogue 15 may be a useful therapeutic candidate for the treatment of inflammatory diseases.

Killed but Metabolically Active Mycobacterium bovis bacillus Calmette-Guérin Retains the Antitumor Ability of Live bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin is the most effective treatment for high risk noninvasive bladder cancer. Although bacillus Calmette-Guérin immunotherapy clearly decreases recurrence and progression rates, side effects are common and infection with the bacillus has been described. For these reasons it is necessary to find safer alternatives to the live bacillus. We explored the possibility of using killed but metabolically active bacillus Calmette-Guérin. T24, J82 and RT4 bladder tumor cell lines were cultured with live and irradiation or heat treated bacillus Calmette-Guérin Connaught. We measured the inhibition of cell proliferation and the production of cytokines in cell culture supernatants. Peripheral mononuclear blood cells were also infected and the production of different cytokines in cell culture supernatants was analyzed. Peripheral blood mononuclear cell and cell culture supernatants activated by mycobacteria were then cultured with T24 cells to analyze whether they showed cytotoxic activity. Compared to the other bacillus Calmette-Guérin treatments, γ irradiated bacillus Calmette-Guérin showed activity similar to that of the live bacillus for inhibiting tumor growth and inducing cytokine production. Irradiated bacillus Calmette-Guérin showed metabolic activity and, thus, was considered killed but metabolically active. This is the treatment that most accurately preserved the mycobacterial structure. Killed but metabolically active bacillus Calmette-Guérin induced cytokine production by infected peripheral mononuclear blood cells. Mycobacteria activated peripheral blood mononuclear cell and cell supernatants showed cytotoxic activity against tumor cells, retaining the antitumor capacity of the live bacillus. Our results suggest that killed but metabolically active bacillus Calmette-Guérin could be considered a safer immunotherapy alternative to treatment with the live bacillus.

Aging is associated with circulating cytokine dysregulation

PurposeAging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC). MethodsCirculating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation. ResultsSubjects were divided into three different groups according to age: 28 young HC (<30years, 26.2±2.4), 24 middle age HC (30–60years, 44.7±8.4) and 21 elderly HC (>60years, 70.6±7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile. ConclusionAging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.

CD101 Expression and Function in Normal and Rheumatoid Arthritis-affected Human T Cells and Monocytes/Macrophages

It was recently reported that CD101 surface expression discriminates potency among CD4+CD25+ FoxP3+ regulatory T cells in the mouse. We investigated whether CD101 may also have a role in the suppressor function of regulatory T cells in humans given that the latter population may affect the autoimmune response in patients with rheumatoid arthritis (RA). Sorted T cells and monocyte/macrophage cell populations were analyzed by flow cyto metry using conjugated antibodies specific for cell-surface markers. T cell proliferation assays were conducted by [(3)H]thymidine incorporation and CD8(high) cytotoxicity measurements by Cyto-Scan-LDH cytotoxicity assays. ELISA were used to measure cytokines in cell culture supernatants and Western blotting was performed for profiling mitogen-activated protein (MAP) kinase activation using specific antiphospholipid antibodies. CD101 expression coincided with PMA-induced monocyte/leukocyte lineage differentiation. CD8(high)CD101- T cells exhibited greater cytotoxic activity than CD8(high)CD101+ T cells, while no difference was observed between CD4CD25(high)CD101+ and CD4CD25(high)CD101- Treg inhibitory activity through responder T cells. LPS-induced proinflammatory cytokine production and p38 MAP kinase activation were made possible by ligation of CD101 with an anti-CD101 antibody F(ab')(2) fragment. These results suggested a modulatory/coregulatory function of CD101 in the human immune system, in contrast to murine models, in which CD101 surface expression discriminates potency among FoxP3+ regulatory T cells. Cytotoxic CD8(high)CD101+ T cells were markedly less cytotoxic than CD8(high) T cells negative for the CD101 antigen and were conspicuously downregulated in patients with RA, suggesting a possible role for CD101 expression and function in the control of certain manifestations of RA pathology.

Using luminex (xMAP) technology to assay for inflammatory cytokines in cell culture supernatants from daudi and ovcar-3 cells treated with interferon

Using luminex (xMAP) technology to assay for inflammatory cytokines in cell culture supernatants from daudi and ovcar-3 cells treated with interferon

Circulating cytokines in active polymyalgia rheumatica

Objective:To characterise the circulating cytokine profile and the cellular source of circulating cytokines in polymyalgia rheumatica (PMR).Methods:The study included 34 patients with active untreated PMR and 17 age-matched healthy controls...

Mycotoxin-induced depletion of intracellular glutathione and altered cytokine production in the human alveolar epithelial cell line A549

Mould exposure has been associated with asthma and other inflammatory airway conditions. However, cellular effects of inhaled mould components are not well understood. We hypothesised that host defence mechanisms, such as production of cytokines (TGFβ1, IL-6 and IL-8) and the intracellular antioxidant glutathione (GSH), could be adversely affected by different concentrations of mycotoxins. We studied the effects of citrinin and gliotoxin on lipopolysaccharide (LPS)-stimulated alveolar epithelial cells (A549). Cytokines in cell culture supernatants were analysed by ELISA and levels of GSH were measured by colorimetric (absorbance) determination. We found that GSH decreased in a dose- and time-dependent manner when cells were exposed to citrinin in particular. TGFβ1 was moderately reduced at low mycotoxin concentrations but elevated at higher sub-toxic concentrations. A tendency for an inverse relationship between TGFβ1 and GSH levels was observed. IL-6 and IL-8 were not significantly reduced at non-toxic mycotoxin concentrations. Thus, reduced epithelial GSH and TGFβ1 levels combined with elevated IL-6 and IL-8 levels may result in increased pro-inflammatory activity during mycotoxin exposure. We suggest that this mechanism can contribute to inflammation in mould exposure.

Recombinant factor VIII and factor VIII-von Willebrand factor complex do not present danger signals for human dendritic cells

Several lines of evidence have shown that antibody responses to coagulation factor VIII (FVIII) in patients with hemophilia A depend on the help of activated CD4(+) T cells. The primary activation of CD4(+) T cells requires interaction with mature dendritic cells (DCs) that present antigenic peptides in the context of MHC class II and express costimulatory molecules. Maturation of DCs requires danger signals provided by exogenous or endogenous stimuli such as pathogen-derived products or inflammatory cytokines. We asked the question whether FVIII itself, FVIII complexed with von Willebrand factor (VWF) or thrombin-activated FVIII contain danger signals for human DCs that induce the upregulation of costimulatory molecules or the expression of proinflammatory cytokines necessary for effective activation of CD4(+) T cells. Human peripheral monocytes were differentiated into DCs. FVIII, thrombin-activated FVIII, VWF, VWF-FVIII, lipopolysaccharide (LPS), LPS + FVIII, LPS + VWF or LPS + FVIII-VWF were added either on day 0 or on day 5 of differentiation cultures. Differentiation markers, cytokines in cell culture supernatants and the capacity of DCs to stimulate autologous and allogeneic T cells were analysed after seven days of differentiation cultures. Our results indicate that neither FVIII, thrombin-activated FVIII, VWF nor a complex of FVIII and VWF modulate the maturation of human DCs or their capacity to stimulate autologous or allogeneic T cells. We conclude that neither of these proteins present danger signals to human DCs.

Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants.

An increasing volume of data suggests a relationship between cytokine levels in human body fluids and disease pathogenesis. Traditionally, many individual assays would be performed to measure the large number of known cytokines and determine their associations with disease. A new technique for the simultaneous measurement of multiple cytokines in cell culture supernatants by fluorescent microsphere-based flow cytometry was adapted to human sera. Multiplexed sandwich immunoassays for eight cytokines were developed by coupling cytokine-specific capture antibodies to beads with different emission spectra. The binding of biotinylated detection antibodies bound with a streptavidin-conjugated fluorochrome was analyzed. Recovery of "spiked" cytokines, sensitivity, and variability of the assays were evaluated. In addition, the results of the bead assays were compared with the results of commercial enzyme-linked immunosorbent assays (ELISAs) that used the same antibody pairs. Correlations of the bead assays and the ELISAs were 0.974 (n = 18) for supernatant samples and 0.859 (n = 28) for serum samples. High, false-positive values observed with some sera, assumed to be produced by heterophilic antibodies, were reduced by preincubation with a cocktail of animal sera. Fluorescent bead-based immunoassays can be used to quantitate multiple cytokines in human sera and contribute to an understanding of the role of cytokines in disease processes. This methodology is applicable to many combinations of purified analytes and high-affinity antibodies. Published 2001 Wiley-Liss, Inc.