Macrophage Stimulating Factor Research Articles

A first-in-human phase I clinical study with MVX-ONCO-1, a personalized active immunotherapy, in patients with advanced solid tumors.

Purpose Over two decades, most cancer vaccines failed clinical development. Key factors may be the lack of efficient priming with tumor-specific antigens and strong immunostimulatory signals. MVX-ONCO-1, a personalized cell-based cancer immunotherapy, addresses these critical steps utilizing clinical-grade material to replicate a successful combination seen in experimental models: inactivated patient's own tumor cells, providing the widest cancer-specific antigens repertoire and a standardized, sustained, local delivery over days of a potent adjuvant achieved by encapsulated cell technology. Experimental Design We conducted an open-label, single-arm, first-in-human, phase I study with MVX-ONCO-1 in advanced refractory solid cancer patients. MVX-ONCO-1 comprises irradiated autologous tumor cells co-implanted with two macrocapsules containing genetically engineered cells producing granulocyte macrophage stimulating factor (GM-CSF). Patients received six immunizations over nine weeks without maintenance therapy. Primary objectives were safety, tolerability, and feasibility, while secondary objectives focused on efficacy and immune monitoring. Results Data from 34 patients demonstrated safety and feasibility with minor issues. Adverse events included one serious event (SAE) possibly related to investigational medicinal product (IMP), and two moderate-related adverse events (AE). Over 50% of patients with advanced and mainly non-immunogenic tumors showed clinical benefits, including partial responses (PR), stable diseases (SD), and prolonged survival. In recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC), one patient achieved a PR, while another survived for more than 7 years without anticancer therapy for over 5 years. Conclusion/Discussion MVX-ONCO-1 is safe, well-tolerated, and beneficial across several tumor types. Ongoing phase IIa trials target advanced R/M HNSCC patients post initial systemic therapy.

Keratinocyte integrin α3β1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism.

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3β1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3β1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3β1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3β1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3β1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3β1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3β1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3β1 as a potential target of wound therapy.

Macrophage immunomodulation accelerates skeletal muscle functional recovery in aged mice following disuse atrophy.

Poor recovery of muscle size and strength with aging coincides with a dysregulated macrophage response during the early stages of regrowth. Immunomodulation in the form of ex vivo cytokine (macrophage-colony stimulating factor) or polarized macrophage delivery has been demonstrated to improve skeletal muscle regeneration. However, it is unclear if these macrophage-promoting approaches would be effective to improve skeletal muscle recovery following disuse in aged animals. Here, we isolated bone marrow-derived macrophages from donor mice of different ages under various experimental conditions and polarized them into proinflammatory macrophages. Macrophages were delivered intramuscularly into young adult or aged recipient mice during the early recovery period following a period of hindlimb unloading (HU). Delivery of proinflammatory macrophages from donor young adults or aged mice was sufficient to increase muscle function of aged mice during the recovery period. Moreover, proinflammatory macrophages derived from aged donor mice collected during recovery were similarly able to increase muscle function of aged mice following disuse. In addition to the delivery of macrophages, we showed that the intramuscular injection of the cytokine, macrophage-colony stimulating factor, to the muscle of aged mice following HU was able to increase muscle macrophage content and muscle force production during recovery. Together, these results suggest that macrophage immunomodulation approaches in the form of ex vivo proinflammatory macrophage or macrophage-colony stimulating factor delivery during the early recovery phase following disuse atrophy were sufficient to restore the loss of aged skeletal muscle function.NEW & NOTEWORTHY A single intramuscular administration of polarized macrophages into muscles of aged mice following a bout of disuse atrophy was sufficient to improve functional recover similarly to young adults after disuse atrophy regardless of the age or experimental condition of the donor mice. Additionally, intramuscular delivery of macrophage-colony stimulating factor into aged mice was similarly effective. Targeting macrophage function early during the regrowth phase may be a novel tool to bolster muscle recovery in aging.

Soluble CD163 is produced by monocyte-derived and alveolar macrophages, and is not associated with the severity of idiopathic pulmonary fibrosis.

The soluble form of the membrane hemoglobin scavenger receptor CD163 (sCD163), released by shedding, is a strong marker for macrophage activation. Serum sCD163 levels rise in several acute inflammatory states and some fibrosing diseases. Monocyte-derived macrophages (MoDM) differentiated by macrophage colony-stimulating factor (M-MoDM) contribute to the pathophysiology of idiopathic pulmonary fibrosis (IPF), an irreversible and rapidly fatal interstitial lung disease. Since M-MoDM express high membrane CD163 levels, we thus postulated that sCD163 could be a relevant biomarker for macrophage activation in IPF. We found that M-MoDM constitutively released higher amounts of sCD163 (49.5 ± 24.5 ng/ml) than monocytes (0.45 ± 0.32 ng/ml) or MoDM differentiated with granulocyte macrophage-stimulating factor (2.24 ± 0.98 ng/ml). The basal production of sCD163 by M-MoDM was increased following stimulation with lipopolysaccharide (123.4 ± 54.9 ng/ml) or ATP (168.9 ± 41.8 ng/ml). The sCD163 release was controlled by metalloproteases but not through ADAM17 activation. Moreover, CD163-positive macrophages and sCD163 were detected in pulmonary tissues and alveolar fluids of Caucasian patients with IPF, respectively. IPF alveolar macrophages constitutively secreted sCD163 amounts (67.6 ± 44.6 ng/µg RNA) which were significantly higher than those released by alveolar macrophages isolated from controls (19.2 ± 7.6 ng/µg RNA) or patients with other interstitial lung disease (31.5 ± 16.6 ng/µg RNA). However, the concentrations of sCD163 in blood serum collected from 155 patients with IPF did not correlate with the severity of their disease. In conclusion, our results show that M-MoDM constituted a pertinent model to study the regulation of sCD163 production. Yet, serum sCD163 values could not provide a prognostic biomarker for IPF in our cohort.

Comparison of blood tonic efficacy and chemical constituents of Kadsura interior A.C. Smith and its closely related species

BackgroundThe stems of Kadsura interior A. C. Smith are used as traditional Chinese medicine (TCM) Kadsurae Caulis, with the traditional efficacy of tonifying and invigorating the blood, therefore being favored to treat blood deficiency (BD) widely. However, the stems of K. interior and its closely related species are morphologically similar and they may readily be misused as Kadsurae Caulis, thus likely to exert negative effects on clinical efficacy and clinical medication safety.MethodsFirstly, blood tonic efficacies of the stems of K. interior (KIS) and its closely related species were compared using BD mouse model induced by 1-acetyl-2-phenylhydrazine (APH) and cyclophosphamide (CTX). Secondly, the chemical constituents from the stems of K. interior and its closely related species were evaluated and compared using a plant metabolomics approach. Plant metabolomics in this study aims at discovering differential metabolites and comprehensively assessing the chemical constituents by combining state-of-the-art high-resolution UPLC-Q/TOF–MS/MS technique and multivariate data analysis. Finally, based on the pharmacological data and the chemical constituents in UPLC-Q/TOF–MS fingerprints, the potential blood tonic active markers were screened by the spectrum-effect relationship analysis and quantified by UPLC-UV-DAD.ResultsThe ethanol extract of the stems of K. interior significantly increased the levels of hematocrit (HCT), hemoglobin (HGB), and red blood cells (RBC) in BD mice. In addition, it significantly increased the serum levels of interleukin 3 (IL-3), granulocyte–macrophage colony-stimulating factor (GM-CSF), and macrophage-stimulating factor (M-CSF) in BD mice (P < 0.01). The blood tonic efficacy of the stems of K. interior was superior to those of its closely related species, especially at the dose of 200 mg/kg. Six differential compounds in the stems of K. interior were screened out to distinguish it from its closely related species. In combination with the results of the spectrum-effect relationship analysis, heteroclitin D, interiorin C, and heteroclitin G were identified as potential bioactive markers. The contents of heteroclitin D and heteroclitin G in the freeze-dried powder of KIS were 15.90 and 3.74 μg/mg.ConclusionsThis study illustrated the differences in the blood tonic efficacies and the chemical constituents of the stems of K. interior and its closely related species, and pinpointed the potential bioactive markers of K. interior.

Global Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Lung Transplant Recipients.

Lung transplant recipients (LTxRs) with acute rejection (AR) and chronic rejection (bronchiolitis obliterans syndrome [BOS]) induce circulating exosomes known to contain donor human leukocyte antigens and lung-associated self-antigens. Here, we sought to identify proteomic signatures in circulating extracellular vesicles (EVs) that differentiate LTxRs in 4 groups: stable, AR, BOS, or respiratory viral infection (RVI). EVs were isolated from plasma from patients in each group via ultracentrifugation. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography–tandem mass spectrometry. We identified 2 unique proteins for AR, 4 for RVI, 24 for BOS, and 8 for stable LTxRs. Differential analysis of AR, BOS, RVI, and stable proteins identified significantly deregulated proteins (p < 0.05, log2(fold change) > ±1) in each condition (31, 2, and 2, respectively). EVs from LTxRs with AR contained proteins involved in immunoglobulin, complement regulation, coagulation, and innate and adaptive immune response pathways. EVs from LTxRs with BOS revealed enriched immunoglobulin receptors and a carboxypeptidase N catalytic chain. EVs from LTxRs with RVI had an enriched macrophage-stimulating factor. We found unique signatures in LTxRs with AR, BOS, and RVI, highlighting complex immune mechanisms underlying lung allograft rejection. Proteomic signatures in LTxRs’ circulating EVs provided insights into immunological mechanisms of graft rejection and RVI.

Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Plasma of Human Lung Transplant Recipients

We demonstrated induction of circulating exosomes in lung transplant recipients (LTxRs) with acute/chronic rejection (bronchiolitis obliterans syndrome [BOS]) containing donor human leukocyte antigen and lung self-antigens (SAgs) (Kα-1 Tubulin, Collagen V), costimulatory molecules (CD80, CD86), transcription factors (NFkB, 20S proteasome) (Am J Transplant 17(2):474-484, 2017). In the current study, we identified unique proteomic signatures in circulating extracellular vesicles (EVs) that can differentiate human LTxRs with acute rejection (AR), BOS, respiratory viral infection (RVI), and stable. EVs were isolated using ultracentrifugation from LTxRs plasma (stable, AR, BOS or RVI) following transplant. EVs were characterized for the presence of lung SAgs (Kα-1 Tubulin, Collagen V) and size (50-200nm) was determined using NanoSight. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified unique proteins (2 AR, 24 BOS, 4 RVI, 8 stable cohort) using LC-MS/MS. Differential analysis of the proteins from AR, BOS and RVI to stable yielded significant unique proteins (p-value <.05, fold-change >2) in each clinical cohort (27 AR, 2 BOS, 2 RVI). EVs from AR contained proteins involved in immunoglobulin, complement regulation, innate and adaptive immune response pathways. EVs from BOS revealed enrichment of immunoglobulin receptors and carboxypeptidase N catalytic chain. EVs from RVI were enriched for macrophage stimulating factor. Our results demonstrate unique signatures in acute/chronic rejection and RVI highlighting complex immune mechanisms underlying lung allograft rejection. Presence of complement proteins and immunoglobulins in circulating EVs from AR can be helpful in differentiating cellular rejection with a mixed phenotype (cellular and humoral). We identified immunoglobulins and complement proteins in BOS in support of significance of Abs in the pathogenesis of BOS. Macrophage stimulating factor contained in EVs specific to RVI can be useful for the differential diagnosis of RVI from AR. To conclude, proteomic signatures of circulating EVs from LTxRs provided insights into immunological mechanisms at play in graft rejection and RVI leading to increased risk for chronic lung allograft dysfunction.

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains.

Background and purpose:Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3).Experimental approach:The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method.Findings / Results:A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/μg was obtained.Conclusion and implications:The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).

Cloning, Optimization of Periplasmic Expression and Purification of Recombinant Granulocyte Macrophage-Stimulating Factor in Escherichia coli BL21 (DE3).

Background:Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by Escherichia coli. However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of E. coli BL21 (DE3).Materials and Methods:The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the pelB secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 104 IU/μg.Conclusion:The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by E. coli.

Mechanisms involved in enhancement of osteoclast formation by activin-A.

Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-β (TGF-β) family is known to be present in bone tissues and an important regulator in osteoclastogenesis with SMAD-mediated signaling being crucial for inducing osteoclast differentiation. In the present study, we examined the effect and underlying mechanisms of activin-A on osteoclast formation in vitro culture systems. Activin-A enhanced osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW 264.7 cells induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and/or macrophage stimulating factor (M-CSF). We also found that activin-A stimulated bone resorption and actin ring formation induced by RANKL and/or M-CSF. Furthermore, activin-A enhanced RANKL-induced expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis, thereby increasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, and cathepsin K. Blockage of receptor binding by follistatin, an activing-binding protein suppressed the activin-A-mediated stimulation of NFATc1. In addition, activin-A increased RANKL-induced c-fos expression without significantly affecting the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment of the cells with a specific inhibitor of SMAD2/3 attenuated the activin-A-induced expression of NFATc1 and co-immunoprecipitation assay revealed that treatment with activin-A increased physical interaction of phosphorylated-c-fos and phosphorylated-SMAD2 protein induced by RANKL. These results suggest that activin-A enhances RANKL-induced osteoclast formation mediated by interaction of c-fos and smad2/3.

Elevated inflammatory cytokines in aqueous cytokine profile in HIV-1 infected patients with cataracts in Uganda

BackgroundCataracts occur earlier among HIV-infected adults and this is attributed to various intraocular inflammatory processes that result in early degeneration. In this study we purposed to investigate whether HIV infected individuals with cataracts develop heightened intraocular inflammatory processes compared to their HIV negative counterparts by determining the concentration of 8 cytokines in the aqueous humour of HIV-positive adults with cataracts and their HIV-negative counterparts.MethodsA cross-sectional study was conducted among consecutive adults with cataracts that were operated in an ophthalmology surgical camp in western Uganda. We determined levels of Granulocyte macrophage stimulating factor (GM-CSF), interleukin 6 (IL-6), interleukin 8 (IL-8), tumour necrotic factor alpha (TNF-a), interferon gamma (IFN-g), interleukin 4 (IL-4), interleukin 2 (IL-2), and interleukin (IL-10) in the aqueous fluid using a multiplexed cytokine analysis.Data was entered in the SPSS version 10 and analyzed using STATA statistical software version 7.0. Categorical and continuous variables were compared using the χ2 test, Fisher’s exact test and the Student’s t-test. Bonferroni correction was used to cater for multiple comparison of p values for the various cytokines.ResultsThe 50 adults that underwent cataract surgery were outdoor peasants with similar exposure hours to UV radiation. The HIV-positive patients were younger {median age 43 years (SD 11.741)} compared to the HIV -negative patients {median age 66.5 years (SD 21.4)}. The mean CD4+ T cell count of the HIV-positive patients was 161 cells /mm3, and 12(48%) had started anti-retroviral therapy (ART). Pro inflammatory cytokines, GM-CSF, IL-8 and IL-10 were significantly higher among HIV-positive individuals (p = 0.001, 0.030, < 0.001 respectively). HIV-positive individuals on ART also showed significantly higher levels of GM-CSF, IL-8 and IL − 10 (p = 0.002, 0.021, < 0.001 respectively). TNF-a and IL-4 were significantly higher among those with a CD4+ T cell count greater than 200cells/mm3 compared to those with CD4+ T cell count less than 200 cells/mm3 (p = 0.022, 0.032 respectively).ConclusionCataracts among HIV-positive adults were associated with higher intraocular inflammation relative to the healthy elderly individuals with cataracts. There is need to explore the potential role of intra-ocular anti-inflammatory agents in the management of cataracts among HIV positive patients.

IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression.

Virtual screening and pharmacophore design for a novel theoretical inhibitor of macrophage stimulating factor as a metastatic agent.

Metastasis is a crucial aspect of cancer. Macrophage stimulating protein (MSP) is a single chain protein and can be cleaved by serum proteases. MSP has several roles in metastasis. In this in silico study, MSP as a metastatic agent was considered as a drug target. Crystallographic structure of MSP was retrieved from protein data bank. To find a chemical inhibitor of MSP, a library of KEGG compounds was screened and 1000 shape complemented ligands were retrieved with FindSite algorithm. Molegro Virtual Docker (MVD) software was used for docking simulation of shape complemented ligands against MSP. Moldock score was used as scoring function for virtual screening and potential inhibitors with more negative binding energy were obtained. PLANS scoring function was used for revaluation of virtual screening data. The top found chemical had binding affinity of -183.55 based on MolDock score and equal to -66.733 PLANTs score to MSP structure. Based on pharmacophore model of potential inhibitor, this study suggests that the chemical which was found in this research and its derivate can be used for subsequent laboratory studies.

Cytokine Profiles in Asthma Families Depend on Age and Phenotype

BackgroundCirculating cytokine patterns may be relevant for the diagnosis of asthma, for the discrimination of certain phenotypes, and prognostic factors for exacerbation of disease.Methodology/Principal FindingsIn this study we investigated serum samples from 944 individuals of 218 asthma-affected families by a multiplex, microsphere based system detecting at high sensitivity eleven asthma associated mediators: eotaxin (CCL11), granulocyte macrophage stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-4 (IL-4), IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17 and tumor necrosis factor alpha (TNFα). Single cytokine levels were largely similar between asthmatic and healthy individuals when analysing asthma as single disease entity. Regulatory differences between parental and pediatric asthma were reflected by six of the eleven mediators analyzed (eotaxin, IL-4, IL-5, IL-10, IL-12, TNFα). IL-12 (p40) and IL-5 were the best predictor for extrinsic asthma in children with an increased odds ratio of 2.85 and 1.96 per log pg/ml increase (IL-12 (p40): 1.2–6.8, p = 0.019, and IL-5: 1.2–2.5, p = 0.025). Frequent asthma attacks in children are associated with elevated IL-5 serum levels (p = 0.013). Cytokine patterns seem to be individually balanced in both, healthy and diseased adults and children, with various cytokines correlating among each other (IL-17 and IFNγ (rs = 0.67), IL-4 and IL-5 (rs = 0.55), IFNγ and GM-CSF (rs = 0.54)).Conclusion/SignificanceOur data support mainly an age- but also an asthma phenotype-dependent systemic immune regulation.

Effects of nanosized titanium dioxide on innate immune system of fathead minnow ( Pimephales promelas Rafinesque, 1820)

Effects of nanosized (<100 nm) titanium dioxide (TiO 2) particles on fish neutrophils and immune gene expression was investigated using the fathead minnow ( Pimpehales promelas). Expanded use of TiO 2 in the cosmetic industry has increased the potential exposure risk to aquatic ecosystems and human health. Effects of nano-TiO 2 on neutrophil function of the fathead minnow was investigated using oxidative burst, neutrophil extracellular traps (NETs) release and degranulation of primary granules. The innate immune gene expression was determined with quantitative PCR (qPCR). Application of 0.1 μg mL −1 of nano-TiO 2 in vitro stimulated oxidative burst and NET release. Intraperitoneal injection of 10 μg g −1 of nano-TiO 2 caused a significant decrease in oxidative burst, NETs release and degranulation (21%; 11%; and 30%, decrease, respectively). Fish exposed to nano-TiO 2 for 48 h in vivo had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2 (4; 2.5; and 2 fold increase, respectively). Nano-TiO 2 has potential to interfere with the evolutionary conserved innate immune system responses, as evidenced with observed changes in gene expression and neutrophil function. This finding encourages the use of fish models in the studies of nanoparticle immunotoxicity. The lowest significant response concentration studied in vitro is four times greater than the estimated environmental concentration for TiO 2 (0.025 μg mL −1) causing concern about potential impact of nano-TiO 2 on aquatic animals and ecosystems.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach

BackgroundDespite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV).ResultsTwo-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36).ConclusionsThe genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

Hepatocyte Growth Factor Family Negatively Regulates Hepatic Gluconeogenesis via Induction of Orphan Nuclear Receptor Small Heterodimer Partner in Primary Hepatocytes

Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4alpha-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.

Bone Marrow Stromal Cells Attenuate Lung Injury in a Murine Model of Neonatal Chronic Lung Disease

Neonatal chronic lung disease, known as bronchopulmonary dysplasia (BPD), remains a serious complication of prematurity despite advances in the treatment of extremely low birth weight infants. Given the reported protective actions of bone marrow stromal cells (BMSCs; mesenchymal stem cells) in models of lung and cardiovascular injury, we tested their therapeutic potential in a murine model of BPD. Neonatal mice exposed to hyperoxia (75% O(2)) were injected intravenously on Day 4 with either BMSCs or BMSC-conditioned media (CM) and assessed on Day 14 for lung morphometry, vascular changes associated with pulmonary hypertension, and lung cytokine profile. Injection of BMSCs but not pulmonary artery smooth muscle cells (PASMCs) reduced alveolar loss and lung inflammation, and prevented pulmonary hypertension. Although more donor BMSCs engrafted in hyperoxic lungs compared with normoxic controls, the overall low numbers suggest protective mechanisms other than direct tissue repair. Injection of BMSC-CM had a more pronounced effect than BMSCs, preventing both vessel remodeling and alveolar injury. Treated animals had normal alveolar numbers at Day 14 of hyperoxia and a drastically reduced lung neutrophil and macrophage accumulation compared with PASMC-CM-treated controls. Macrophage stimulating factor 1 and osteopontin, both present at high levels in BMSC-CM, may be involved in this immunomodulation. BMSCs act in a paracrine manner via the release of immunomodulatory factors to ameliorate the parenchymal and vascular injury of BPD in vivo. Our study suggests that BMSCs and factor(s) they secrete offer new therapeutic approaches for lung diseases currently lacking effective treatment.

Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice.

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.

Phosphotidylserine modulates the phenotype of mouse dentritic cells

Abstract Introduction: Dentritic cells (DCs) are the most potent professional antigen presenters and are required to initiate adaptive immunity. Immature DCs (iDC) capture antigen efficiently, but express low levels of MHC II and costimulatory molecules. DCs mature in response to danger signals, migrate into secondary lymphoid organs and activate T cells. Maturation is characterized by increase in MHC II and costimulatory molecule expression. Apoptotic cells inhibit DCs maturation. Phosphotidylserine (PS) is an anionic phospholipid that is limited to the inner leaflet of plasma membranes in live cells and externalized upon apoptosis. We examined the effects of PS on DCs maturation. Methods: Bone marrow derived mouse DCs were generated in the presence of granulocyte macrophage stimulating factor (GM-CSF), exposed to PS or phosphotidylcholine (PC) containing liposomes on day 5, and treated with lipopolisaccharide (LPS) on day 6. On day 7, DCs were analyzed by flow cytometry. Results: DCs exposed to media or PC and treated with LPS to induce maturation expressed significantly high levels of MHCII, CD80, CD86 and CD40 compared to iDCs. DCs exposed to PS expressed significantly less CD40 in response to LPS than control DCs. ∗MHC IICD80CD86CD40iDC17 (± 5.33)43 (± 24.3)14 (± 4.8)8 (± 7.4)mDC54 (± 28.2)100 (± 44.1)70 (± 28.6)∗57 (± 16.5)∗PC-DC55 (± 27.0)105 (± 46.2)73 (± 24.4)63 (± 17.5)∗PS-DC49 (± 29.5)102.6 (± 49.3)56 (± 8.6) † 41 (± 14.2)∗∗Statistically significant (p Conclusions: We demonstrated that PS modulates mouse DC maturation, specifically reducing CD40 expression. Diminished CD40 signaling is likely to lead to reduced IL-12 production and inhibition of Th1 responses. These findings suggest that PS, externalized by apoptotic cells and some tumor cells, could prevent autoimmunity and contribute to immune evasion by tumor.