Cytokines Granulocyte-macrophage Colony-stimulating Factor Research Articles

Wnt Signaling in Chronic Rhinosinusitis with Nasal Polyps.

The signaling pathways that sustain the disease process of chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly understood. We sought to determine the expression levels of Wnt signaling genes in CRSwNP and to study the role of the Wnt pathway in inflammation and epithelial remodeling in the nasal mucosa. Microarrays and real time-quantitative polymerase chain reaction comparing gene expression in matched NPs and inferior turbinates revealed that WNT2B, WNT3A, WNT4, WNT7A, WNT7B, and FZD2 were up-regulated and that FZD1, LRP5, LRP6, and WIF1 were down-regulated in NPs. Immunolabeling showed robust expression of Wnt ligands, nuclear β-catenin, and Axin-2 in NP tissue, suggesting that Wnt/β-catenin signaling is activated in NPs. We used primary human nasal epithelial cell (HNEpC) cultures to test the functional consequences of Wnt pathway activation. Monolayer HNEpCs treated with recombinant human WNT (rhWNT) 3A, but not with rhWNT4, had altered epithelial morphology and decreased adhesion, without loss of viability. We found that neither rhWNT3A nor rhWNT4 treatment induced proliferation. The expression and release of inflammatory cytokines IL-6 and granulocyte-macrophage colony-stimulating factor were increased after rhWNT3A exposure of HNEpCs. When differentiated at an air-liquid interface, rhWNT3A- and WNT agonist-, but not rhWNT4-treated HNEpCs, had abnormal epithelial architecture, failed to undergo motile ciliogenesis, and had defective noncanonical Wnt (planar cell polarity) signaling. On the basis of these results, we propose a model in which Wnt/β-catenin signaling sustains mucosal inflammation and leads to a spectrum of changes consistent with those seen during epithelial remodeling in NPs.

Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of Tcells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression invivo, we generated a transgenic mouse line allowing theinduction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment.

CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

Introduction:Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease.Methods:We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools.Results:Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope.Conclusions:A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients.

TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner.

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is involved in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis. However, the environmental cues promoting differentiation of GM-CSF producing T cells are unclear. Herein, we performed a broad experimental screening of cytokines and data-driven analysis assessing their ability to induce human GM-CSF+ CD4+ T cells and their subpopulations. TGF-β was discovered to induce GM-CSF production independently of proliferation and IL-2 signaling including STAT5. In contrast, IL-6 and IL-23 decreased GM-CSF production. On the population level, GM-CSF induction was highly correlated with expression of FOXP3 across cytokine stimulations but not with that of IL-17. However, on single-cell level GM-CSF and IFN-γ expression were most correlated, independently of the cytokine environment. Importantly, under low sodium conditions in the medium or upon stimulation with plate-bound instead of bead-bound anti-CD3 and anti-CD28 antibodies, the effects of TGF-β on GM-CSF, but not on FOXP3, were reversed. Our analysis indicates a novel role for TGF-β in generating GM-CSF+ subsets of human CD4+ T cells. These results are important for understanding of autoimmune disease and therapeutic considerations.

Common airborne fungi induce species-specific effects on upper airway inflammatory and remodelling responses

Whilst the exact cause of chronic rhinosinusitis (CRS) remains elusive, it is clear that both inflammation and remodelling are key disease processes. Environmental fungi have been linked to airway inflammation in CRS; however, their role in the pathogenesis of this condition remains controversial. The current consensus suggests that whilst fungi may not be directly causative, it is likely that CRS patients have deficits in their innate and potentially acquired immunity, which in turn may modify their ability to react to fungi. This study used a nasal polyp explant tissue stimulation model to study the inflammatory and remodelling responses related to challenge with common airborne fungal species. Ex vivo nasal polyp tissue from six well phenotyped CRSwNP patients undergoing functional endoscopic sinus surgery was stimulated with 1, 10 and 100 μg/ml of Alternaria alternata, Aspergillus niger, Cladosporium sphaerospermum and Penicillium notatum and compared with unchallenged polyp tissue as control. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α); and pro-remodelling cytokines transforming growth factor-b1 (TGF-b1), and basic fibroblast growth factor (bFGF) in the polyp supernatant. Aspergillus niger stimulation increased pro-inflammatory cytokines TNF-α, GM-CSF and IL-6 whilst having little effect on the remodelling cytokines bFGF and TGF-b1. In contrast, stimulation with Cladosporium sphaerospermum, Alternaria alternata and Penicillium notatum reduced pro-inflammatory cytokines TNF-α and IL-6, but induced a dose-dependent increase in remodelling cytokines TGF-b1 and bFGF. This study shows that common airborne fungi induce species-specific effects on the upper airway inflammatory and remodelling responses. These findings provide further immunological evidence of a disease-modifying role for fungi in CRS.

Evaluation of the potential therapeutic benefits of macrophage reprogramming in multiple myeloma

AbstractTumor-associated macrophages (TAM) are important components of the multiple myeloma (MM) microenvironment that support malignant plasma cell survival and resistance to therapy. It has been proposed that macrophages (MØ) retain the capacity to change in response to stimuli that can restore their antitumor functions. Here, we investigated several approaches to reprogram MØ as a novel therapeutic strategy in MM. First, we found tumor-limiting and tumor-supporting capabilities for monocyte-derived M1-like MØ and M2-like MØ, respectively, when mixed with MM cells, both in vitro and in vivo. Multicolor confocal microscopy revealed that MM-associated MØ displayed a predominant M2-like phenotype in the bone marrow of MM patient samples, and a high expression of the pro-M2 cytokine macrophage migration inhibitory factor (MIF). To reprogram the protumoral M2-like MØ present in MM toward antitumoral M1-like MØ, we tested the pro-M1 cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) plus blockade of the M2 cytokines macrophage colony-stimulating factor or MIF. The combination of GM-CSF plus the MIF inhibitor 4-iodo-6-phenyl-pyrimidine achieved the best reprogramming responses toward an M1 profile, at both gene and protein expression levels, as well as remarkable tumoricidal effects. Furthermore, this combined treatment elicited MØ-dependent therapeutic responses in MM xenograft mouse models, which were linked to upregulation of M1 and reciprocal downregulation of M2 MØ markers. Our results reveal the therapeutic potential of reprogramming MØ in the context of MM.

Invited commentary

Dr Edwards and colleagues have detailed the in vivo angiogenic potential of an engineered stromal cell-derived factor-1α (SDF) in a rat arterial ligation model. SDF is a powerful chemoattractant of endothelial progenitor cells (EPCs), which represent a cornerstone of the response to ischemia and angiogenesis. Several studies have highlighted the therapeutic role of SDF to increase migration of EPCs and activation of EPCs, to improve healing of diabetic wounds, and to improve angiogenesis and ventricular physiology in cardiac models. A major hurdle to translation of SDF clinically has been the susceptibility to protease degradation and a complex tertiary structure that limits cost-effective production. Gene delivery approaches to overcome these issues add orders of magnitude to the complexity of potential SDF therapies in terms of vector toxicity and regulation of gene expression. The distinctive aspect of this study by Edwards et al is the use of an engineered SDF analog (ESA) peptide that mimics the physiologic properties of recombinant SDF but at a fraction of the size. The authors found that administration of ESA resulted in increased vascular endothelial growth factor expression, capillary density, and limb perfusion over a clinically relevant interval of 14 days. A significant limitation of this study was the administration of the cytokine granulocyte-macrophage colony-stimulating factor (GMCSF) to upregulate bone marrow EPCs in all study groups. Although GMCSF alone has not been shown to improve perfusion without a chemotactic agent such as SDF, the prospective administration of GMCSF clinically represents a significant additional therapeutic for eventual clinical translation. If future studies can demonstrate that ESA mediates angiogenesis independent of GMCSF, this would significantly improve the appeal of engineered SDF. Also, as noted by the authors, they used a model of acute rather than the intended clinical application of chronic ischemia. Potentially, mechanisms of EPC recruitment and the effect of SDF therapies might differ between these two scenarios. Although angiogenic therapies are unlikely to replace current macrovascular open and endovascular surgical approaches, they will be especially useful in scenarios where distal outflow has been compromised by conditions such as diabetes or chronic distal embolism, for instance. Broadly, these therapies should improve distal tissue perfusion and offer additional applications in coronary ischemia. The findings of this study are compelling and represent a meaningful step toward the frontier of angiogenic therapy in peripheral vascular disease. Biochemically engineered stromal cell-derived factor 1-alpha analog increases perfusion in the ischemic hind limbJournal of Vascular SurgeryVol. 64Issue 4PreviewDespite promising therapeutic innovation over the last decade, peripheral arterial disease remains a prevalent morbidity, as many patients are still challenged with peripheral ischemia. We hypothesized that delivery of engineered stromal cell-derived factor 1-alpha (ESA) in an ischemic hind limb will yield significant improvement in perfusion. Full-Text PDF Open Archive

Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

Interleukin (IL)-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD) vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A]) to stimulate peripheral blood mononuclear cells (PBMCs) of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Direct effects of airborne PM2.5 exposure on macrophage polarizations

BackgroundExposure of atmospheric particulate matter with an aerodynamic diameter less than 2.5μm (PM2.5) is epidemiologically associated with illnesses. Potential effects of air pollutants on innate immunity have raised concerns. As the first defense line, macrophages are able to induce inflammatory response. However, whether PM2.5 exposure affects macrophage polarizations remains unclear. MethodsWe used freshly isolated macrophages as a model system to demonstrate effects of PM2.5 on macrophage polarizations. The expressions of cytokines and key molecular markers were detected by real-time PCR, and flow cytometry. The specific inhibitors and gene deletion technologies were used to address the molecular mechanisms. ResultsPM2.5 increased the expression of pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα). PM2.5 also enhanced the lipopolysaccharide (LPS)-induced M1 polarization even though there was no evidence in the change of cell viability. However, PM2.5 significantly decreased the number of mitochondria in a dose dependent manner. Pre-treatment with NAC, a scavenger of reactive oxygen species (ROS), prevented the increase of ROS and rescued the PM2.5-impacted M1 but not M2 response. However, mTOR deletion partially rescued the effects of PM2.5 to reduce M2 polarization. ConclusionsPM2.5 exposure significantly enhanced inflammatory M1 polarization through ROS pathway, whereas PM2.5 exposure inhibited anti-inflammatory M2 polarization through mTOR-dependent pathway. General significanceThe present studies suggested that short-term exposure of PM2.5 acts on the balance of inflammatory M1 and anti-inflammatory M2 macrophage polarizations, which may be involved in air pollution-induced immune disorders and diseases. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity.

AimTo determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF).MethodsMaghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA.Resultsγ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 μg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h.ConclusionsThe cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.

Common airborne fungi induce species-specific effects on upper airway inflammatory and remodelling responses.

Whilst the exact cause of chronic rhinosinusitis (CRS) remains elusive, it is clear that both inflammation and remodelling are key disease processes. Environmental fungi have been linked to airway inflammation in CRS; however, their role in the pathogenesis of this condition remains controversial. The current consensus suggests that whilst fungi may not be directly causative, it is likely that CRS patients have deficits in their innate and potentially acquired immunity, which in turn may modify their ability to react to fungi. This study used a nasal polyp explant tissue stimulation model to study the inflammatory and remodelling responses related to challenge with common airborne fungal species. Ex vivo nasal polyp tissue from six well phenotyped CRSwNP patients undergoing functional endoscopic sinus surgery was stimulated with 1, 10 and 100 μg/ml of Alternaria alternata, Aspergillus niger, Cladosporium sphaerospermum and Penicillium notatum and compared with unchallenged polyp tissue as control. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α); and pro-remodelling cytokines transforming growth factor-b1 (TGF-b1), and basic fibroblast growth factor (bFGF) in the polyp supernatant. Aspergillus niger stimulation increased pro-inflammatory cytokines TNF-α, GM-CSF and IL-6 whilst having little effect on the remodelling cytokines bFGF and TGF-b1. In contrast, stimulation with Cladosporium sphaerospermum, Alternaria alternata and Penicillium notatum reduced pro-inflammatory cytokines TNF-α and IL-6, but induced a dose-dependent increase in remodelling cytokines TGF-b1 and bFGF. This study shows that common airborne fungi induce species-specific effects on the upper airway inflammatory and remodelling responses. These findings provide further immunological evidence of a disease-modifying role for fungi in CRS.

Epidermal growth factor receptor signalling regulates granulocyte-macrophage colony-stimulating factor production by airway epithelial cells and established allergic airway disease.

Airway epithelial cells (AEC) are increasingly recognized as a major signalling centre in the pathogenesis of allergic asthma. A previous study demonstrated that epithelial growth factor receptor (EGFR) signalling in AEC regulated key features of allergic airway disease. However, it is unclear what mediators are regulated by EGFR signalling in AEC, although the production of the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is EGFR dependent in keratinocytes. To determine whether EGFR signalling regulates GM-CSF production by human AEC downstream of the clinically relevant mediators house dust mite (HDM) and interleukin (IL)-17A and in a mouse model of established allergic asthma. EGFR inhibitors were used to determine whether EGFR signalling regulates GM-CSF production by cultured human AEC in response to HDM and IL-17A. The roles of EGFR ligands, p38 mitogen-activated protein kinase (MAPK) and tumour necrosis factor-alpha (TNF-α) converting enzyme (TACE) were also assessed. To determine whether EGFR regulates GM-CSF as well as key asthma characteristics invivo, mice were chronically exposed to HDM to establish allergic airway disease and then treated with the EGFR inhibitor Erlotinib. EGFR inhibition reduced HDM and IL-17A induced GM-CSF production in a dose-dependent manner in cultured human AEC. GM-CSF production also required amphiregulin, p38 MAPK signalling and protease/TACE activity. In mice with established allergic airway disease, EGFR inhibition reduced levels of GM-CSF and TNF-α, as well as airway hyperreactivity, cellular inflammation, smooth muscle thickening and goblet cell metaplasia without changes in IgE and Th1, Th2 and Th17 cytokines. Results link HDM, IL-17A, amphiregulin, EGFR and GM-CSF in a mechanistic pathway in AEC and demonstrate that EGFR regulates GM-CSF production and the severity of established disease in a clinically relevant asthma model. These results identify the EGFR→GM-CSF axis as a target for therapeutic development.

Uninephrectomy in Rats on a Fixed Food Intake Potentiates Both Anorexia and Circulating Cytokine Subsets in Response to LPS

Recent human studies have suggested that mild reduction in kidney function can alter immune response and increase susceptibility to infection. The role of mild reduction in kidney function in altering susceptibility to bacterial lipopolysaccharide (LPS) responses was investigated in uninephrectomized rats compared to Sham-operated controls rats 4 weeks after surgery. Throughout the 4 weeks, all rats were maintained under mild food restriction at 90% of ad libitum intake to ensure the same caloric intake in both groups. In comparison to Sham, uninephrectomy (UniNX) potentiated LPS-induced anorexia by 2.1-fold. The circulating anorexigenic cytokines granulocyte-macrophage colony stimulating factor, interferon-γ, tumor necrosis factor-α, and complement-derived acylation-stimulating protein were elevated after LPS in UniNX animals compared to Sham animals. Interleukin(IL)1β and IL6 pro-inflammatory cytokines were transiently increased. Anti-inflammatory cytokines IL4 and IL10 did not differ or had a tendency to be lower in UniNX group compared to Sham animals. LPS-induced anorexia was associated with increased anorexigenic neuropeptides mRNA for pro-opiomelanocortin, corticotrophin-releasing factor, and cocaine–amphetamine-regulated transcript in the hypothalamus of both Sham and UniNX groups, but at higher levels in the UniNX group. Melanocortin-4-receptor mRNA was markedly increased in the UniNX group, which may have contributed to the enhanced anorexic response to LPS of the UniNX group. In summary, UniNX potentiates pro-inflammatory cytokine production, anorexia, and selected hypothalamic anorexigenic neuropeptides in response to LPS.

Nanoparticles enhance the ability of human neutrophils to exert phagocytosis by a Syk-dependent mechanism

BackgroundSome reports indicate that NPs are ingested by cells via different mechanisms, including phagocytosis. In contrast, the direct role of NPs on the phagocytic process is not well documented. The aim of this study was to determine if titanium dioxide (TiO2), zinc oxide (ZnO) and cerium dioxide (CeO2) NPs, could alter the ability of neutrophils to exert phagocytosis. MethodsFreshly isolated human neutrophils were incubated with NPs and their ability to phagocytose opsonized sheep red blood cells (SRBCs) or fluorescent latex beads (LBs) was assessed by optical and fluorescence microscopy, respectively. Syk activation was assessed by western blot experiments and a pharmacological approach with piceatannol, a Syk inhibitor, was used to determine its role in NPs-induced neutrophils. The cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) was used as a positive control. ResultsAll tested NPs enhanced the ability of neutrophil to phagocytose SRBCs and LBs. Syk was activated in NPs-induced neutrophils as evidenced by its increased tyrosine phosphorylation level vs controls and the ability of NPs-induced phagocytosis was reversed by piceatannol. ConclusionsWe found that the tested NPs enhanced phagocytosis, although at different degree, and this occurred by a Syk-dependent mechanism. General significanceThis is the first study demonstrating that NPs, by themselves, can directly enhance FcR-mediated (opsonized SRBCs) and complement-mediated (LBs) phagocytosis. Moreover, as part of their mode of action, we determined that NPs can act similarly to GM-CSF leading to Syk activation involved in phagocytosis. This has to be taken under consideration for future nanobiology and nanomedicine studies.

The human rhabdomyosarcoma cell line TE671 – Towards an innovative production platform for glycosylated biopharmaceuticals

The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-translational modifications, in particular glycosylation, is a major issue. Glycans can influence ligand binding, serum half-life as well as biological activity or product immunogenicity. Aiming to establish a novel production platform for recombinant glycoproteins, the human TE671 cell line was investigated. Since the initial analysis of cell membrane proteins showed a promising glycosylation of TE671 cells for biotechnological purposes, we focused on the recombinant expression of two model glycoproteins of therapeutical relevance. The optimization of the cell transfection procedure and serum-free expression succeeded for the human serine protease inhibitor alpha-1-antitrypsin (A1AT) and the hematopoietic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF). N-glycan analyses of both purified proteins by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided first fundamental insights into the TE671 glycosylation potential. Besides protein specific pattern, strong distinctions – in particular for N-glycan fucosylation and sialylation – were observed depending on the medium conditions of the respective TE671 cell cultivations. The cell line’s ability to synthesize complex and highly sialylated N-glycan structures has been shown. Our results demonstrate the TE671 cell line as a serious alternative to other existing human expression systems.

The effect of dimethyl fumarate on gene expression and the level of cytokines related to different T helper cell subsets in peripheral blood mononuclear cells of patients with psoriasis.

Fumaric acid esters such as dimethyl fumarate (DMF) have proven to be effective in the treatment of psoriasis. In view of the role of Th17 in the pathogenesis of psoriasis, the present study was conducted to investigate the effects of DMF on Th1, Th2, and Th17 responses in patients. Peripheral blood mononuclear cells (PBMCs) were isolated from psoriasis patients and healthy individuals and were cultured in the presence or absence of phytohemagglutinin and DMF. The cell supernatants were removed to measure cytokine secretion, and the lymphocytes were used for real-time polymerase chain reaction to establish gene expression. An increase in gene expression of interferon-γ (IFN-γ), as a marker for Th1 activity, and interleukin-17 (IL-17), granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-22 representing the Th17 subset in the PBMCs of patients in comparison with those of control subjects was observed. Culture of PBMCs from psoriasis patients and controls in the presence of DMF decreased IFN-γ and increased IL-4 gene expression in both groups. Treatment with DMF could significantly decrease IL-17, GM-CSF, and IL-22 mRNA levels in the PBMCs of patients. Decreased release of IFN-γ and GM-CSF cytokine secretion after DMF treatment was also observed in PBMC cultures of patients and controls. These data show the effectiveness of DMF in modulating Th17 cells in addition to Th1/Th2 cells and reflect one of the underlying mechanisms of action of DMF in psoriasis. These findings may also support the possible benefits of using fumarate in the treatment of other autoimmune diseases in the pathogeneses of which Th1 and Th17 cells play major roles.

A GMCSF and IL7 fusion cytokine leads to functional thymic-dependent T-cell regeneration in age-associated immune deficiency.

The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3−CD4−CD8− triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44intCD25− double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8+ T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.

Umbilical cord blood as a source of nerve and stem cells

The aim of the study was to assess proliferative ability of the stem cells in the umbilical cord blood and their potential to differentiate in in vitro culture. The material consisted of 14 samples of umbilical cord blood collected from the umbilical cord vein. Mononuclear cells were isolated using the method of density gradient medium. Next, CD34 cells were isolated from the interphase with the use of the VarioMACS sorter and anti-CD34 antibodies. Long-term cultures were conducted on Iscove's modified Dulbecco medium (IMDM) with addition of granulocyte-macrophage colony stimulating factor (GM-CSF) and nerve growth factor (NGF). Qualitative identification was performed using the May-Grunwald-Giemsy staining method, taking photographs with a confocal microscope, and with the immunoenzymatic method. In our research, CD34+ stem cells constituted 1.16% of the mononuclear cells, and after centrifugation in medium 0.37% of leukocytes in whole umbilical cord blood. Even after 60 days of culture without addition of the growth factors, CD34+ cells were present in the fraction of adherent cells. After stimulation with GM-CSF and NGF a part of the umbilical cord blood cells were transformed into nerve cells (presence of neuron-specific enolase was shown) and into cells morphologically similar to fibroblast and dendritic cells. After stimulation with GM-CSF and NGF cytokines, the umbilical cord blood cells proliferate in long-term medium, transform into nerve cells and into cells similar to fibroblast and dendritic cells.

Overexpression of granulocyte macrophage colony stimulating factor in breast cancer cells leads towards drug sensitization.

This report describes the effect of overexpressing granulocyte macrophage colony stimulating factor (GMCSF) in breast cancer cells, which otherwise is involved in proliferation and differentiation of granulocyte and macrophage lineages. The purified recombinant GMCSF cytokine is known to exert dose-dependent proliferative response on various cancer cells, but its effect during overexpression is yet to be evaluated. In our present study, we have generated MCF-7 (breast cancer) cells overexpressing GMCSF. Interestingly, cell viability studies showed pronounced sensitivity of GMCSF overexpressing MCF-7 cells towards anticancer drugs, such as, doxorubicin, 5FU and cisplatin. These findings were substantiated by cell cycle analysis of the drug-treated GMCSF overexpressing MCF-7 cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results revealed differential expressions of cyclins, and the carboxyfluorescein succinimidyl ester (CFSE)-based assay established decrease in doubling time of GMCSF overexpressed cells with respect to the control populations. Thus, overexpressing of proliferative GMCSF cytokine in breast cancer cells may increase susceptibility to anticancer drugs.