Cytokine Mix Research Articles

Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells

Airway goblet cell hyperplasia (GCH)—detectable by mucin staining—and abnormal macrophage infiltrate are pathological features present in many chronic respiratory disorders. However, it is unknown if both factors are associated. Using in-vivo and in-vitro models, we investigated whether macrophages are related with GCH and changes in mucin immunophenotypes. Lung sections from Sprague-Dawley rats treated for 48 h with one intra-tracheal dose of PBS or LPS (n=4–6 per group) were immunophenotyped for rat-goblet cells, immune, and proliferation markers. Human monocyte-derived macrophages (MDM) were pre-treated with or without LPS, immunophenotyped, and their supernatant, as well as cytokines at levels equivalent to supernatant were used to challenge primary culture of normal human bronchus epithelial cells (HBEC) in air–liquid interface, followed by MUC5B and MUC5AC mucin immunostaining. An association between increased bronchiolar goblet cells and terminal-bronchiolar proliferative epithelial cells confirmed the presence of GCH in our LPS rat model, which was related with augmented bronchiolar CD68 macrophage infiltration. The in-vitro experiments have shown that MUC5AC phenotype was inhibited when HBEC were challenged with supernatant from MDM pre-treated with or without LPS. In contrast, TNF-α and interleukin-1β at levels equivalent to supernatant from LPS-treated MDM increased MUC5AC. MUC5B was induced by LPS, supernatant from LPS-treated MDM, a mix of cytokines including TNF-α and TNF-α alone at levels present in supernatant from LPS-treated MDM. We demonstrated that macrophages are related with bronchiolar GCH, and that they induced MUC5B and inhibited MUC5AC in HBEC, suggesting a role for them in the pathogenesis of airway MUC5B-related GCH.

Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage

Analysis of cryopreserved peripheral mononuclear cells (PBMC) is important for evaluating new vaccines in immune based therapies and in pathogenesis studies. To ensure comparable assay results from different laboratories and points of time, collaborative research in multicenter trials needs reliable and reproducible cryopreservation protocols that maintain cell viability and functionality. Current cryomedia consist largely of fetal bovine serum (FBS), a natural mix of growth factors, cytokines, and undefined compounds. Standardized procedures are not possible, as FBS can affect the antigen-specific T-cell response, the most important parameter in functionality assays. Also, worldwide sample exchange is complicated by the strict import restrictions on FBS, because of transfection risk.After establishing a serum-free cryopreservation protocol that maintains cell viability, recovery and antigen-specific T-cell response of PBMC comparably to FBS-based cryomedia (Germann et al., 2011), the aim of this study was the complete avoidance of animal proteins and products in combination with efficient cryopreservation.As long-term stability of the cryopreservation process is crucial for retrospective evaluation of samples at different points of time, PBMC were analyzed after storage for maximal four weeks and again after approximately six months.The cryopreservation efficiency of the protein-free and fully chemically defined cryomedium was comparable to FBS-medium after storage for few weeks and several months. Directly after thawing, this medium yielded viabilities over 97% and recovery values over 84%. Also, the specific T-cell functionality was preserved. Additionally, short-term and six month cryopreservation gave comparable results. The fully chemically defined medium presented here will increase standardization and reproducibility of analysis in multicenter-studies or in retrospective evaluation.

Cytokines, glucose and angiotensin II (ANG II) and the expression of Connexin (Cx) 37, 40 and 43 in cultured microvascular endothelial cells

Endothelial connexins emerge as molecules that strongly affect the conductive behavior of the endothelial cell layer and are involved in renin release in the kidney. Since many of the cardiovascular disease states are associated with micro‐inflammation and with increased activity of ANG II or with increased glucose, we hypothesized that these could affect expression levels of the connexins 37, 40 and 43. Immortalized microvascular endothelial cells (HMEC‐1) were exposed to Ang II (100 nM), a combination of IL6 (20 IU/ml), IFN (4 ng/ml) and TNFa (20 ng/ml) or to 25 mM Glucose for 4 hours in 50% and 100% confluent HMEC‐1 layers. Then, cells were harvested, RNA extracted and reverse transcribed and subjected to qPCR using pre‐designed primers for Cx37, Cx40 and Cx43 using GAPDH as control. Confluency by itself increased Cx37 expression, but did not affect Cx40 and Cx43 expression. The cytokine mix increased expression of 50% and 100% confluent cells 13‐ and 16‐fold (both P<0.05) for Cx40 and 2‐fold (P<0.05) and 2‐fold (NS) for Cx37. Neither ANG II or high glucose affected Cx expression. The present data indicate that inflammatory factors can affect the gene expression of Cx40 in particular. This might point toward a role for Cx40 in coupling inflammation to microvascular function and to renin release.

Effect of Silymarin and Harpagoside on Inflammation Reaction of BEAS-2B Cells, on Ciliary Beat Frequency (CBF) of Trachea Explants and on Mucociliary Clearance (MCC)

Silymarin and harpagoside are derived from drugs which are used for their protective effects against hepatotoxicity and inflammatory processes. Both are now investigated with respect to the respiratory tract. They were able to reduce the release of the inflammatory cytokine RANTES (regulated on activation, normal T cells expressed and secreted) from BEAS-2B cells in a concentration-dependent manner when stimulated by a cytokine mix (10 ng/mL of TNF- α and IFN- γ). This effect was not due to a possible toxic effect (control experiments using LDH release as a marker). Silymarin but not harpagoside was able to increase ciliary beat frequency. Effects were comparable to positive controls (isoprenaline and salbutamol). Silymarin also increases mucociliary clearance. In conclusion, silymarin should be further investigated for its clinical use in distinct respiratory diseases.

Schizandra arisanensisextract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells

To explore the bioactivity of an ethanolic extract of Schizandra arisanensis (SA-Et) and isolated constituents against interleukin-1β and interferon-γ-mediated β cell death and abolition of insulin secretion. By employing BRIN-BD11 cells, the effects of SA-Et administration on cytokine-mediated cell death and abolition of insulin secretion were evaluated by a viability assay, cell cycle analysis, and insulin assay. The associated gene and protein expressions were also measured. In addition, the bioactivities of several peak compounds collected from the SA-Et were tested against cytokine-mediated β cell death. Our results revealed that SA-Et dose-dependently ameliorated cytokine-mediated β cell death and apoptosis. Instead of suppressing inducible nitric oxide synthase/nitric oxide cascade or p38MAPK activity, suppression of stress-activated protein kinase/c-Jun NH2-terminal kinase activity appeared to be the target for SA-Et against the cytokine mix. In addition, SA-Et provided some insulinotropic effects which re-activated the abolished insulin exocytosis in cytokine-treated BRIN-BD11 cells. Finally, schiarisanrin A and B isolated from the SA-Et showed a dose-dependent protective effect against cytokine-mediated β cell death. This is the first report on SA-Et ameliorating cytokine-mediated β cell death and dysfunction via anti-apoptotic and insulinotropic actions.

Peripheral benzodiazepine receptor protein expression in cells treated with alcohol and cytokines; a study on alcoholism

Alcoholism is a complex disease that impacts the cnS through tolerance, dependence, brain damage and neurological and cognitive deficits. chronic exposure to ethanol brings neurodegeneration and an increase in the level of an outer mitochondrial membrane protein, called the peripheral benzodiazepine receptor, PBr for short. In this study, western blotting and immunocytochemical methods were used to detect the effects of ethanol and cytokine treatments on astrocytes, astrocytomas, and microglia. Although ethanol seemed to have led to a decrease in PBr in the astrocytes and microglia, it did produce an increase in astrocytomas. Additionally, LPS and cytokine mix seemed to have at least physically altered the appearance of these ethanol-exposed cells.

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Astrocytes are activated in most chronic neurodegenerative diseases associated with inflammatory events such as Parkinson's disease or Alzheimer's disease, but also in stroke. Due to an aging population worldwide, research efforts in these areas are likely to expand in the future. This will entail an increased demand for appropriate experimental models. We introduce here the new immortalized mouse astrocyte cell line IMA 2.1 as an alternative to currently used primary astrocyte cultures. IMA 2.1 were directly compared with primary mouse astrocytes with respect to their response to proinflammatory stimuli, expression of typical astrocyte markers, and to the cell line's capacity to metabolize the parkinsonian toxin MPTP to its toxic metabolite MPP+. Under inflammatory conditions, mimicked with the addition of a cytokine mix, IMA 2.1 responded similarly to primary astrocytes with mRNA upregulation, expression of iNOS and COX-2, and the release of various inflammatory mediators. Analysis of astrocytic markers indicated that IMA 2.1 represent a relatively early, GFAP-negative stage of astrocyte development. Moreover, conversion of MPTP by monoamine oxidase-B proceeded in IMA at least as quickly as in primary cells. For all endpoints investigated, the cell line IMA 2.1, derived from a single clone, delivered reproducible results over a period of several years and allowed upscaling of experiments due to its easy handling compared with primary cells.

Anti-VEGFC Treatment Reduces the Leukemic Outgrowth of Primary CD34+ Pediatric Acute Myeloid Leukemia Cells

Abstract 1425Previously, it was demonstrated that exogenous addition of vascular endothelial growth factor C (VEGFC) increased the leukemic cell viability, reduced apoptosis via activation of Bcl-2, and decreased chemotherapy induced apoptosis via its receptor FLT-4 (Further revert to as VEGFR3) (Dias et al. Blood 2002). Furthermore, it was shown that VEGFC promotes angiogenesis by induction of COX-2 through VEGFR3 activation in THP-1 cells (Chien et al. Carcinogenesis 2005). We have previously found that endogenous VEGFC expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia (AML), both in vitro as well as in vivo (de Jonge et al. Clinical Cancer Research 2008). In addition, high VEGFC mRNA expression is strongly associated with reduced complete remission and overall survival in adult as well as pediatric AML (de Jonge et al. Blood 2010). It was thought that the leukemic blast population is organized as a hierarchy, whereby leukemia initiating cells (LICs) reside at the top of this hierarchy, and it is only these cells that have the capacity to engraft in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The LIC is thought to be enriched in the CD34+ leukemic cell fraction and is shown to expand in vitro using a myeloid cytokine mix of IL-3, TPO, and G-CSF in colony forming cell (CFC) assays and long-term culture-initiating cell (LTC-IC) assays (Guan et al. Exp. Hematol. 2002, van Gosliga et al. Exp. Hematol. 2007). Moreover, LTC-IC assays performed in limiting dilution detect the in vitro outgrowth potential of stem-like cells that reside underneath the stromal cell layer. In this study, we set out to investigate the potential of anti-VEGFC treatment as an inhibitor of the outgrowth of LICs within the CD34+ fraction of primary AML samples. First, we determined the possibility of an autocrine loop for VEGFC in AML. Pediatric AML cell (n=7) derived VEGFC levels were found to be 1.4-fold increased (P =.008) compared to secreted VEGFC levels from normal bone marrow (NBM) cells (n=4). Pediatric AML blast cells showed KDR (further revert to as VEGFR2) membrane expression in 44 out of 50 patient samples (varying 8–99% of the total blast population), whereas on NBM cells VEGFR2 expression was below 5%. VEGFR3 expression was below 5% on both leukemic blasts and NBM cells. We evaluated the effect of anti-VEGFC (VGX-100, kindly provided by Vegenics, used at a concentration of 30 μg/ml) treatment on the CD34+ isolated compartment of pediatric AML bone marrow samples. Anti-VEGFC treatment reduced the outgrowth potential of AML derived CD34+ cells (n=2) with >25% in CFC assays. Interestingly, morphological analysis revealed a 3-fold enhanced formation of macrophages. LTC-IC assays demonstrated a (15% to 50%) decrease in the long-term growth of CD34+ isolated AML cells in 3 out of 4 patient samples. Morphological characterization of the suspension cells suggested a shift in development along the myelomonocytic lineage after two weeks of anti-VEGFC treatment. With FACS analysis, these cells showed a higher number of cells stained positive for CD11b, and CD14, and lower numbers where positive for CD34. Anti-VEGFC treated LTC-IC assays in limiting dilution demonstrated a (44% and 74%) reduction in the outgrowth potential of long-term cultured CD34+ isolated AML cells and blocked the erythroid colony formation in 2 out of 3 patient samples. Anti-VEGFC treatment did not have an effect on the outgrowth of CD34+ sorted NBM cells in the various assays (n=2). In conclusion, anti-VEGFC treatment of the CD34+ isolated fraction from primary pediatric AML samples showed a reduction of AML outgrowth. Differentiating cells are skewed to the myelomonocytic lineage upon anti-VEGFC treatment. We hypothesize that deprivation of VEGFC in primary CD34+ AML cell cultures results in enhanced leukemic cell death and abates an important proliferation signal for AML cells. Yet, further investigations are warranted. [Display omitted] Disclosures:Baldwin:Circadian Technologies Limited: Employment. Robert:Circadian Technologies Limited: Employment, Membership on an entity's Board of Directors or advisory committees.

GFAP‐independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells

The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent-stem-cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two-step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte-associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin-4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF-α, IL1-β, IFN-γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand-induced apoptosis and produced NO and IL-6. This was preceded by NF-kB activation, and up-regulation of relevant mRNAs. Also GFAP-negative astrocytes were fully inflammation-competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia-free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.

Regulation of VEGF expression in human retinal cells by cytokines: Implications for the role of inflammation in age‐related macular degeneration

Chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN-γ+ TNF-α+IL-1β) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10- and 9-fold increase in VEGF-A and VEGF-C expression, respectively. The microarray results were validated by quantitative RT-PCR and secretion of VEGFs proteins. IL-1β is the most potent in inducing VEGFs secretion followed by IFN-γ and TNF-α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF-κB and JAK-STAT pathway, but not HIF-1α, Sp-1, Sp-3, and STAT-3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF-A and VEGF-C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF-1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF-A and VEGF-C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD.

Effect of Rifampin on Production of Inflammatory Mediators in HepG2 Liver Epithelial Cells

Rifampin, a potent antibacterial agent, is one of the main drugs used in the treatment of mycobacterial infections. Hepatotoxicity is a well-documented adverse event. The aim of this study was to investigate the effect of rifampin on the production of inflammatory mediators in human epithelial HepG2 liver cells in the absence or presence of proinflammatory cytokines. Incubation of HepG2 cells with a cytokine mix plus rifampin was associated with a significant dose-dependent increase in the production of nitric oxide compared to incubation with the cytokine mix alone (P < 0.05) as well as with an increase in inducible nitric oxide synthase protein and mRNA expression. Rifampin significantly increased the secretion of interleukin 8 (IL-8) in both untreated cells (P < 0.001) and cytokine-treated cells (P < 0.006). An array screening assay revealed that rifampin stimulated the production of IL-1β and gamma interferon-induced protein-10 (IP-10) in untreated cells and increased the secretion of RANTES in cytokine-treated cells. Together, these results indicate that rifampin may exert proinflammatory effects on liver cells.

Pharmacological Cholinergic Blockade Stimulates Inflammatory Cytokine Production and Lymphocytic Infiltration in the Mouse Lacrimal Gland

To investigate the effects of cholinergic blockade on inflammatory cell infiltration and cytokine production in the mouse lacrimal gland (LG). C57BL/6 mice were untreated (UT) or received subcutaneous injections of either scopolamine hydrobromide (SCOP; 0.5 mg/0.2 mL) or saline (SAL) four times daily for 2 or 5 days (2D, 5D). This was followed by a 7-day rest period in separate groups. Tear volume (cotton thread) and tear epidermal growth factor (EGF, by ELISA) concentrations were measured. Extraorbital LGs were surgically excised and sectioned or lysed for gene expression analysis. Immunohistochemistry evaluated immunophenotype of infiltrating cells. Expression of EGF and T helper (Th)-1, -2, and -17-associated cytokines in LGs was evaluated by real-time PCR. Goblet cell density was evaluated in periodic acid Schiff-stained conjunctival sections. Tear volume and EGF protein levels were significantly reduced in SCOP5D mice compared with controls, indicating that cholinergic blockade decreased LG secretory function. LGs of SCOP2D and SCOP5D mice showed an increased density of CD4(+), CD11c+, CD11b+, and myeloperoxidase+ cells compared with UT controls. At day 5, these cells were significantly elevated compared with SAL-treated counterparts. Elevated levels of IL-17A, IL-17R, IFN-γ, IL-12Rβ1, IL-2, IL-13, IL-6, IL-1β, and TNF-α transcripts were noted in SCOP2D mice and IFN-γ, TGF-β1, and IL-18R transcripts in SCOP5D mice. Pharmacological blockade of lacrimal secretion induced a significant CD4(+) infiltration in the LG, mimicking Sjögren's syndrome. The mRNA expression profile revealed elevations of a mix of inflammatory cytokines and Th-1-associated factors.

TLR2 Hypersensitivity of Astrocytes as Functional Consequence of Previous Inflammatory Episodes

Precedent inflammatory episodes may drastically modify the function and reactivity of cells. We investigated whether priming of astrocytes by microglia-derived cytokines alters their subsequent reaction to pathogen-associated danger signals not recognized in the quiescent state. Resting primary murine astrocytes expressed little TLR2, and neither the TLR2/6 ligand fibroblast-stimulating lipopeptide-1 (FSL1) nor the TLR1/2 ligand Pam(3)CysSK(4) (P3C) triggered NF-κB translocation or IL-6 release. We made use of single-cell detection of NF-κB translocation as easily detectable and sharply regulated upstream indicator of an inflammatory response or of c-Jun phosphorylation to measure restimulation events in astrocytes under varying conditions. Cells prestimulated with IL-1β, with a TLR3 ligand, with a complete cytokine mix consisting of TNF-α, IL-1β, and IFN-γ, or with media conditioned by activated microglia responded strongly to FSL1 or P3C stimulation, whereas the sensitivity of the NF-κB response to other pattern recognition receptors was unchanged. This sensitization to TLR2 ligands was associated with an initial upregulation of TLR2, displayed a "memory" window of several days, and was largely independent of the length of prestimulation. The altered signaling led to altered function, as FSL1 or P3C triggered the release of IL-6, CCL-20, and CXCL-2 in primed cells, but not in resting astrocytes. These data confirmed the hypothesis that astrocytes exposed to activated microglia assume a different functional phenotype involving longer term TLR2 responsiveness, even after the initial stimulation by inflammatory mediators has ended.

Inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating JAK/STAT pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ + TNF-α + IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Regulation of VEGFs expression in human retinal cells by cytokines: implications for the role of inflammation in age‐related macular degeneration

Chronic inflammation has been proposed to be associated with the pathogenesis of age‐related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in loss of vision. The objective of this study is to investigate the effects of inflammatory mediators on VEGFs expression in retinal cells. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IC) mix (IFN‐γ + TNF‐α + IL‐1β) on global gene expression (Affymetrix GeneChip) profiles in HRPE cells, revealed 10 and 9 fold increase in the expression of VEGF‐A and VEGF‐C respectively. The microarray results were validated by HRPE secretion of VEGFs proteins. IL‐1β is the most potent inducer of VEGFs secretion followed by IFN‐γ and TNF‐α. Combination of any of these cytokines was more effective in enhancing VEGFs secretion in a dose dependent manner. NFκB and JAK‐STAT pathway, but not HIF‐1α, Sp‐1, Sp‐3 and STAT‐3, transcription factors were involved in IC action. VEGFs secretion by HCHF cells was also significantly enhanced by IC. Our results show that three pro‐inflammatory cytokines induce the secretion of VEGFs by HRPE and HCHF cells. These findings indicate that in AMD the presence of pro‐inflammatory cytokines in chronic inflammation promote choroidal and retinal neovascularization.

Expression of iNOS is reduced by an inhibitor of fatty acyl CoA synthetase.

ObjectiveTo test the hypothesis that fatty acyl CoA derivatives of fatty acids modulate cytokine stimulated iNOS expression in C6 astrocytes.RationaleTriacsin C, a fungal metabolite, inhibits fatty acyl CoA synthetase in many cell types. The compound inhibits fatty acid induced iNOS expression and apoptosis in isolated islets of Langerhans (Shimabukuro et al 1996), suggesting that fatty acyl CoA derivatives regulate iNOS expression.MethodsC6 cells were stimulated with a cytokine mix consisting of TNFα (60 ng/mL), ILβ (2 ng/mL) and IFNγ (100 units/mL) in the presence or absence of triacsin C. Expression of iNOS was estimated by measuring iNOS protein by immunoblotting. In vivo iNOS activity was estimated by measuring media nitrite (Greiss reaction) and in vitro iNOS activity was measured by conversion of [14C]arginine to [14C]citrulline in the absence of added calcium.ResultsIn C6 cell homogenates, triacin C inhibited FACS activity in a concentration dependent manner, ranging from 31.9 ± 1.5% at 300 nM to 71.1 ± 3.8% at 5 μM. The effect of 5 μM triacsin C on cytokine‐stimulated media nitrite, catalytic activity and immunoreactive protein were measured at 18 and 24 hours: % of Cytokine Stimulated Activity Media Nitrite Catalytic Activity Immunoreactivity 18 hours 62.5 ± 6.9% 103 ± 11.1% 25.1 ± 13.2% 24 hours 80.6 ± 1.39%* 373 ± 72%* 36.6 ± 5.81% = p&lt; 0.05 as compared to 18 hours by Student's ttest; n=3 independent experiments for each parameter, each done in duplicate or triplicate. ConclusionsThese results suggest that triacsin C inhibits cytokine stimulated NO formation, and that iNOS protein is significantly less in triacsin C treated cells. Surprisingly, not all of the immunoreactive iNOS protein seems to be catalytically active. The data suggest that triacsin C may delay cytokine‐stimulated expression of iNOS and thus its overall activity in C6 cells.

Age-related T-cell cytokine profile parallels corneal disease severity in Sjogren's syndrome-like keratoconjunctivitis sicca in CD25KO mice

IL-2ralpha (CD25)(-/-) mice develop autoimmunity and lymphoproliferative disorders, including SS-like disease. The objective of this study was to evaluate the severity of corneal epithelial disease and T-cell cytokine profile in the ocular surface tissues of CD25KO mice. CD25KO mice were evaluated at 8, 12 and 16 weeks of age. Corneal epithelial smoothness and corneal permeability were measured. Phenotype of infiltrating lymphocytes was evaluated by immunohistochemistry. Th-1, -2 and -17 associated factors were measured by real-time PCR in cornea and conjunctiva and by Luminex immunobead assay in tears. Compared with 8-week-old wild-type (WT) mice, CD25KO mice of the same age had significantly greater corneal irregularity and a significant increase in the number of CD4(+) and CD8(+) T cells infiltrating the conjunctiva. CD25KO mice had significantly higher levels of IL-6, TGF-beta1, IL-23R, IL-17A, IL-17F, IL-21, CCL20, IL-10, GATA-3 and IFN-gamma mRNA transcripts in their cornea and conjunctiva than WT mice at 8 weeks. IL-17A and IL-17F mRNA transcripts peaked at 12 weeks, whereas IFN-gamma spiked at 16 weeks in CD25KO mice. Increased expression of IL-17A and IL-17F at 12 weeks in CD25KO mice was accompanied by a worsening of corneal surface parameters and an increase of CD4(+) T cell infiltrating the cornea. Disruption of IL-2 signalling in CD25KO mice results in age-dependent SS-like autoimmune lacrimal-keratoconjunctivitis. A mix of Th-1 and Th-17 cytokines was detected. The peak severity of corneal epithelial disease corresponded to the peak of IL-17 expression.

Chronische Entzündungen – Rolle am Tumorgeschehen

Die Erkenntnis, dass ein Zusammenhang zwischen chronischen Entzündungen und einem erhöhten Krebsrisiko besteht, wurde bereits vor mehr als 150 Jahren vom Berliner Mediziner Rudolph Virchow beschrieben. Heute wird vermutet, dass ca. 10-15 % aller humanen Krebserkrankungen auf chronische Entzündungsprozesse zurückzuführen sind, wobei die Ursachen sowohl auf humanpathogene Keime (z. B. Helicobacter pylori) als auch auf anorganische/organische Substanzen/Faktoren (Asbestfasern, Magensäure, Zigarettenrauch) zurückgeführt werden können. Chronisch entzündetes Gewebe stellt ein potenziell transformierendes Milieu dar, da es reich an Wachstumsfaktoren, Zytokinen und Chemokinen ist, welche die proliferatorische Aktivität von Zielzellen triggern, sowie an radikalischen Sauerstoff-/Stickstoffverbindungen, die bei hoher Konzentration Schädigungen der DNS bewirken. Durch die Faktor vermittelte bzw. inhärente Zellteilungsaktivität der Zielzellen, kombiniert mit mutagen wirkenden radikalischen Sauerstoff-/Stickstoffverbindungen, kann es zur Akkumulation von chromosomalen Aberrationen und nachfolgend zur malignen Transformation der Zielzellen kommen. Entgegen der alten Lehrmeinung, dass Krebs seinen Ursprung in somatischen Zellen hat, ist heute bekannt, dass die Ursprungszellen für maligne Transformationen Stammzellen (Gewebestammzellen bzw. rekrutierte Knochenmarkstammzellen) bzw. deren Progenitorzellen sind, aus denen die tumorinitiierenden Krebsstammzellen hervorgehen. So konnte z. B. anhand eines etablierten Mausmodells zur Analyse des durch H. felis induzierten Magenadenokarzinoms gezeigt werden, dass das neoplastische Gewebe aus rekrutierten Knochenmarkstammzellen hervorgegangen ist. Neben ihrer Rolle in der Karzinogenese sind chronische Entzündungsreaktionen darüber hinaus maßgeblich an der Progression von Tumorerkrankungen beteiligt. Fatal hierbei ist die symbiotische Beziehung zwischen tumorassoziierten Makrophagen (TAM) und Tumorzellen. TAM sind an sämtlichen Prozessen beteiligt, die eine erhöhte Malignität der Tumorerkrankung nach sich ziehen. Hierzu zählen die Neoangiogenese, die Proliferation sowie die Invasion und Metastasierung von Tumorzellen wie auch die Suppression von zytotoxischen T-Zellen.

HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells

Lentivectors (LVs) have attracted considerable interest for their potential as a vaccine delivery vehicle. In this study, we evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of human immunodeficiency virus (HIV) (LV-Gag) as a potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity is achieved through pseudotyping the vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the DC-SIGN protein. A single immunization by this vector induces a durable HIV Gag-specific immune response. We investigated the antigen-specific immunity and T-cell memory generated by a prime/boost vaccine regimen delivered by either successive LV-Gag injections or a DNA prime/LV-Gag boost protocol. We found that both prime/boost regimens significantly enhance cellular and humoral immune responses. Importantly, a heterologous DNA prime/LV-Gag boost regimen results in superior Gag-specific T-cell responses as compared with a DNA prime/adenovector boost immunization. It induces not only a higher magnitude response, as measured by Gag-specific tetramer analysis and intracellular IFN-gamma staining, but also a better quality of response evidenced by a wider mix of cytokines produced by the Gag-specific CD8(+) and CD4(+) T cells. A boosting immunization with LV-Gag also generates T cells reactive to a broader range of Gag-derived epitopes. These results demonstrate that this DC-directed LV immunization is a potent modality for eliciting anti-HIV immune responses.

In vitro cytokine responses of peripheral blood mononuclear cells from healthy dogs to distemper virus, Malassezia and Toxocara

Naïve CD4+ T cells may differentiate into a number of subsets including T helper 1 (Th1) Th2, Th3, Th17 and T regulatory (Treg) cells depending on the type of antigen they encounter. These CD4+ families have been defined based on the array of cytokines they produce and the effects they have on adaptive immune responses. CD4+ subsets are cross regulatory and at times cooperative. The study of these adaptive immune modulators has revealed the important role that cytokines play in mounting effective as well as detrimental immune responses to pathogens. Examining the cytokine responses of lymphocytes in culture can provide important understanding of how immune responses to pathogens are orchestrated. For this purpose the in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from healthy dogs was examined in response to stimulation with antigens from a common canine virus (canine distemper virus, CDV), a commensal skin yeast of dogs ( Malassezia pachydermatis) and a common canine helminth ( Toxocara canis ( T. canis)). Cell culture supernatants were removed from antigen stimulated and unstimulated control PBMC after 4, 24, 48 and 72 h and the concentration of Th1 type cytokines (IL-2, IFN-γ, TNF-α) and Th2 type cytokines (IL-4, IL-5, IL-10) was determined using sandwich ELISA assays. CDV induced low levels of cytokine production initially with a predominance of IL-10 at 24 h and a balanced response at 48 h of incubation. Malassezia antigen stimulated an early type 2 cytokine response with dramatic production of IL-4 at 24 h of incubation compared to the other stimulants examined. By 48 h of incubation, however, the cytokine mix in response to Malassezia had also moved toward a Th1 type response. T. canis induced early production of Th2 type cytokines with IL-5 predominating; however, with longer incubation (48–72 h) there was a switch to a balanced Th1/Th2 response. In conclusion, the cytokines produced in vitro by canine PBMC in response to prototypical Th1 and Th2 type pathogens were not clearly polarized and shifted over time. While the in vitro study of PBMC cytokine responses cannot be directly extrapolated to in vivo responses to the same antigens, the results do highlight the dynamic and fluctuating nature of cytokine production.