Peripheral Blood Of Healthy Controls Research Articles

Phagocytosis by human monocytes is required for the secretion of presepsin.

Presepsin, a soluble CD14 subtype, is increasingly recognized as a useful biomarker for sepsis. However, little is known about the biological characteristics of presepsin in humans. Furthermore, there are no studies evaluating clinical validity of measuring the presepsin levels in patients after allogeneic hematopoietic cell transplantation, irrespective of the high frequency of sepsis. For in vitro assays, neutrophils and monocytes were isolated from the peripheral blood of healthy controls and treated with bacteria or inflammatory stimuli. Presepsin levels in the culture supernatants were measured by enzyme linked immunosorbent assay (ELISA). For a cohort study of patients undergoing allogeneic hematopoietic cell transplantation, serum samples were subjected to ELISA for presepsin, and the relationship of presepsin levels with the incidence of transplantation-related complications was statistically analyzed. We found that monocytes were the main source of presepsin in humans. Presepsin secretion by human monocytes was triggered by bacterial phagocytosis or sterile phagocytic stimulus, such as monosodium urate crystals, rather than soluble inflammatory stimuli. Elastase, a serine protease in human monocytes, mediated CD14 cleavage to produce presepsin. The cohort study demonstrated that high presepsin values were significantly associated with an increased incidence of hemophagocytic syndrome, as well as bacteremia. Moreover, patients with higher presepsin values revealed inferior overall survival, suggesting that presepsin can also be a prognostic marker for transplantation. In this study, we clarified the biological features of presepsin in humans. Our study may be useful for increasing the clinical application of presepsin as a biomarker.

Common Genetic Variant Association with Altered HLA Expression, Synergy with Pyrethroid Exposure, and Risk for Parkinson's Disease: An Observational and Case-Control Study.

Background:The common noncoding single-nucleotide polymorphism (SNP) rs3129882 in HLA-DRA is associated with risk for idiopathic Parkinson’s disease (PD). The location of the SNP in the major histocompatibility complex class II (MHC-II) locus implicates regulation of antigen presentation as a potential mechanism by which immune responses link genetic susceptibility to environmental factors in conferring lifetime risk for PD.Aims:The aim of this study was to determine the effect of this SNP on the MHC-II locus and its synergy with pesticide exposure.Methods:For immunophenotyping, blood cells from 81 subjects were analyzed by quantitative reverse transcription-PCR and flow cytometry. A case–control study was performed on a separate cohort of 962 subjects to determine association of pesticide exposure and the SNP with risk of PD.Results:Homozygosity for G at this SNP was associated with heightened baseline expression and inducibility of MHC class II molecules in B cells and monocytes from peripheral blood of healthy controls and PD patients. In addition, exposure to a commonly used class of insecticide, pyrethroids, synergized with the risk conferred by this SNP (odds ratio=2.48, P=0.007), thereby identifying a novel gene–environment interaction that promotes risk for PD via alterations in immune responses.Conclusions:In sum, these novel findings suggest that the MHC-II locus may increase susceptibility to PD through presentation of pathogenic, immunodominant antigens and/or a shift toward a more pro-inflammatory CD4+ T-cell response in response to specific environmental exposures, such as pyrethroid exposure through genetic or epigenetic mechanisms that modulate MHC-II gene expression.

Early Trafficking of Bone Marrow Derived-Endothelial Progenitor Cells Promotes Multiple Myeloma Progression

Background. Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. We studied the role of endothelial progenitor cells in multiple myeloma (MM) pathogenesis.Methods. EPC levels were evaluated in peripheral blood (PB) of patients with smoldering MM (SMM), and active myeloma (MM) and in PB of healthy controls, by using flow-cytometry (CD34+VEGFR2+ cells), and by performing endothelial colony forming assays. EPC levels were studied in PB from transgenic Vk*MYC mice harboring either early MM (smoldering-like stage); or late (active MM-like stage); as well as in mice injected intravenously with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Healthy syngeneic mice were used as controls.GFP-bone marrow (BM) transplantation and sub-cutaneous femur implantation were performed in recipient mice to study EPC BM mobilization (GFP+CD34+VEGFR2 cells) and EPC BM recruitment to the implanted femurs during 5TGM1 MM model progression. Transgenic ID1+/-ID3-/- mice (with a defect in EPC mobilization and differentiation ability) were injected with transplantable Vk*MYC cells and followed for survival. Wild type littermates were used as controls. Similar experiments were performed using transgenic ID1+/-ID3-/- mice transplanted with BM of wild type littermates. Therapeutic activity of DC101 anti-murine VEGFR2 Ab was evaluated in the MM1S human orthotopic xenograft model, using both early and late treatment approaches.Results.EPC levels were significantly increased in PB of MM patients, including SMM, compared to healthy controls (P<0.05). PB mononuclear cells (PBMCs) from SMM and aMM patients showed an increase ability to form both EC-CFUs (P<0.01) and ECFCs compared to those from healthy controls (P<0.05).EPC levels were significantly increased in PB of transgenic Vk*MYC mice at both early and advanced stage of MM development compared to healthy syngenic mice. In orthotopic MM models we found that levels of circulating EPCs increase early after i.v. injection of either human MM1S-GFP+/luc+ cells (1.5 and 2 times higher after 1 week and 2 weeks, respectively) or murine 5TGM1-turboRFP+ MM cells (2.5 and 3 times higher after 2 and 3 weeks, respectively) compared to naïve mice. By coupling GFP-BM transplantation and subcutaneous femur implantation, we generated a new model that allows the study of BM derived-cell trafficking from one site to another of the BM. The levels of BM-derived GFP+ EPC increased early after i.v. injection of 5TGM1-turboRFP+ cells (2 times increase after 2 weeks, P<0.01); also, the percentage of both total GFP+ cells (7.92% versus 2.635%, P<0.05) and GFP+ EPCs (0.22% versus 0.068%, P<0.05) increased in the BM of implanted femurs of tumor cell injected mice compared to controls. Survival of ID1+/-ID3-/- mice was significantly increased compared to that of wild type littermates after injection of MM Vk*MYC cells (62 versus 27 days median survival time, P=0.0005); this survival advantage was lost after wild type BM was transplanted in ID1+/-ID3-/- mice (34 versus 35 days median survival time). In human MM1S orthotopic xenograft model, treatment with anti-murine VEGFR2 DC101 Ab was able to inhibit MM progression when treatment was started early during smoldering like stages, but not when it was started after significant tumor burden (50.5, 41.5 and 40 days median survival time in DC101 early, late and untargeted IgG treated mice respectively)Conclusion. These results show that EPC mediated vasculogenesis is an important pathogenic process that occurs during the early/smoldering MM stage. Knockout mouse experiments confirm that EPCs are essential for MM tumor progression. These results indicate that anti-angiogenic agents could be effective as an early therapeutic intervention in SMM patients. DisclosuresGhobrial:Millennium/Takeda: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

Presepsin (soluble CD14 subtype) Is Secreted from Human Monocytes after Phagocytosis – in Vitro Analyses and a Retrospective Cohort Study in Patients with Allogeneic Stem Cell Transplantation

INTRODUCTIONPresepsin is a subtype of soluble CD14, which is a receptor for lipopolysaccharide (LPS)/LPS-binding protein complexes and is expressed on the myelomonocytic cell surface. Recently, it has been shown to be a useful biomarker for assessing the severity of sepsis and has come into use in the field of critical care medicine. However, the precise mechanism involved in its production in human is yet to be ascertained. Therefore, we performed in vitro analyses to determine the main sources of presepsin and the mechanism of presepsin production in humans. We also performed a retrospective cohort study to determine the clinical relevance of presepsin in the diagnosis of bacteremia or other complications after allogeneic stem cell transplantation (allo-SCT) because, so far, only one study has analyzed the utility of presepsin as a biomarker for myelosuppressed patients after chemotherapies. METHODSFor in vitro assays, we first isolated neutrophils and monocytes from the peripheral blood of healthy controls and co-cultured them (4 x 105 cells) with various stimuli, such as bacteria or cytokines, with or without inhibitors. The supernatants were subjected to ELISA for the determining the presepsin production levels, and precipitants were analyzed by immunostaining for CD14 and presepsin.For the cohort study, we enrolled patients who had undergone allo-SCT in our department during recent 5 years. Serum samples were collected at 4 different times (before the conditioning regimen, on day 0, 7, and 28 after allo-SCT). The serum specimens were analyzed using ELISA to determine the presepsin levels, and the relationship between these levels with the occurrence of SCT-related complications and overall survival rate was determined. RESULTSIn vitro co-culture assays showed that monocytes secreted significantly higher amounts of presepsin than neutrophils in response to Escherichia coli (mean ± standard error, 414.8 ± 55.9 vs. 124.7 ± 37.9 pg/mL, p = 0.01). Immunostaining showed an apparent cytosolic deposition of presepsin in monocytes, but not in neutrophils (Figure 1). The secretion of presepsin from monocytes induced by LPS, tumor necrosis factor (TNF)-ƒ¿, and Pam3CSK (Toll-like receptor 1/2 agonist) was small, and similar to the base line secretion (76.0 ± 9.2 pg/mL, p > 0.90). Monocytic secretion of presepsin in response to E. coli is partially suppressed by various kinds of phagocytic inhibitors (125.6 ± 9.5 pg/mL, p = 0.01 with wortmannin; 160.7 ± 18.3 pg/mL, p = 0.02 with cytochalasin). Moreover, other bacteria such as Staphylococcus epidermidis induced presepsin secretion (182.6 ± 37.7 p/mL, p = 0.02). These findings suggest that monocytic phagocytosis is the main factor responsible for presepsin production in humans.In order to analyze the clinical relevance of presepsin, we included 69 patients who had undergone allo-SCT (35 men and 34 women; age range, 17 – 66 y; median age, 49 y). The donors were related in 16 cases and unrelated in 53. Myeloablative conditioning was performed in 27 patients. The serum presepsin levels was 1,909 ± 540 pg/mL before allo-SCT, and no significant differences were observed between this level and those observed at the other 3 time points (p = 0.62). Bacteremia was observed in 17 and 10 patients within 5 days before serum sample collection on day 7 and day 28, respectively. These patients with bacteremia showed higher levels of presepsin compared to those without bacteremia (5,174 ± 1,693 vs 1,161 ± 151 pg/mL, p < 0.01 on day 7, and 4,877 ± 1,923 vs. 2,183 ± 537 pg/mL, p = 0.07 on day 28). No correlation was observed between presepsin levels, viral infections, and the occurrence of acute GVHD. Moreover, patients with higher presepsin levels at day 28 (≥ 2,000 pg/mL) showed a significantly worse overall survival (hazard ratio, 3.7; 95%CI, 1.7 – 7.7, p < 0.01, Figure 2) mainly due to transplant-related death. CONCLUSIONOur results showed that human presepsin is produced mainly by monocytes after phagocytosis of bacteria in vitro. This data is supported by the results of the retrospective cohort study indicating that elevation in serum presepsin levels is observed especially during bacteremia. Presepsin levels can be used as an effective diagnostic marker for the bacteremia even during the myelosuppressive period after allo-SCT. Moreover, high presepsin levels on day 28 after allo-SCT can be indicative of poor prognosis. [Display omitted] [Display omitted] DisclosuresYamashita:Mochida Pharmaceutical Co., Ltd.: Research Funding.

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.

SAT0553 Correlation of the Frequency and Differentiation Potential of Osteoclast Progenitor Cells with Disease Activity and Response to Therapy in Patients with Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is marked by the persistent inflammation and osteodestruction, causing progressive disability. The mechanisms leading to joint destruction involve differentiation and activation of osteoclasts, multinucleated cells derived from...

THU0408 Anti-Lactoferrin Autoantibodies Contribute to the Pathogenesis of Igg4-Related Disease by Inducing Neutrophil Extracellular TRAP Formation

BackgroundIgG4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition characterized by infiltration of IgG4-positive plasma cells into the pancreas, biliary tracts, or salivary glands. In addition to helper type 2...

232. Interferons Alter the Response of Neutrophils to Inflammatory Cytokines in vitro

Background: Neutrophils are central to the initiation, progression and resolution of inflammatory diseases such as RA. We have previously shown that neutrophils from RA patients have a gene expression signature indicating activation in vivo by interferons (IFNs), and higher expression of IFN-response genes is associated with a good response to TNFi therapy. The aim of this work was to investigate the functional effects of IFNs on neutrophils in vitro both (i) alone, and (ii) in combination with inflammatory cytokines. Methods: Neutrophils were isolated from peripheral blood of healthy controls and incubated with Type-I IFN (IFNa) or Type-II IFN (IFNg) at a range of concentrations in the absence or presence of GM-CSF and TNFa. Apoptosis was measured at 18h by flow cytometry using annexin V/PI; respiratory burst was measured at hourly intervals up to 5h using luminol-enhanced chemiluminescence after stimulation with fMLP or PMA; changes in protein expression and phosphorylation were measured by Western blotting; changes in gene expression were measured by RNA-Seq (Illumina). Results: Neutrophils stimulated with IFNs in vitro underwent rapid phosphorylation of STAT proteins (5–30 min), activation of IFNresponse genes (1h), and priming of the respiratory burst (3h), but only Type-II IFN delayed apoptosis, measured at 18h (unstimulated 58.6% 0.6, IFNg 41.4% 4.1, P<0.05). Addition of IFNs to neutrophil suspensions containing GM-CSF or TNFa had a profound dose-dependent effect on the function of the inflammatory cytokines. Type-I IFNs abrogated the protective effect of GM-CSF on neutrophil apoptosis at 18h (GM-CSF 25.6% 2.7, GM-CSFþ IFNa 49.5% 1.2, P<0.01), whereas Type-II IFNs enhanced the anti-apoptotic effect of TNFa (TNFa 42.3% 3.3, TNFaþ IFNg 27.4% 1.2, P< 0.05) and sustained the TNFa priming effect on the respiratory burst for up to 4h (P<0.01). Type-I and Type-II IFNs enhanced STAT3 phosphorylation by GM-CSF, and altered the activation kinetics of ERK and AKT by GM-CSF. Type-I IFN enhanced AKT phosphorylation in TNFa stimulated neutrophils. Conclusion: IFNs profoundly alter the functional effects of inflammatory cytokines on neutrophils in vitro. This may have important consequences in vivo during therapy with biologic drugs such as TNFi. The complexity and heterogeneity of inflammatory diseases such as RA, where different cytokines dominate or act synergistically to perpetuate systemic inflammation, may explain why some patients respond better to certain biologic therapies than others. We are currently investigating the consequences of TNFi (Infliximab) and JAKi (Tofacitinib) on neutrophils stimulated in vitro with IFNs, GM-CSF and TNFa. Disclosure statement: The authors have declared no conflicts of interest.

Expression of TIM‐3 on CD4+ and CD8+ T cells in the peripheral blood and synovial fluid of rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by a chronic inflammatory process that targets the synovial lining of diarthrodial joints. TIM-3 plays a key role in the negative regulation of the immune response. In this study, we investigated the expression of TIM-3 on CD4+ and CD8+ T cells from systemic (peripheral blood) and local (synovial fluid) perspectives of RA. Level of TIM-3+ cells from peripheral blood and synovial fluid of patients as well as peripheral blood of healthy controls was measured by flow cytometry. Results showed that TIM-3 expression was significantly increased in both CD4+ and CD8+ T cells in the peripheral blood of RA (p<0.001 and p<0.001, respectively). Furthermore, patients revealed even higher expression of TIM-3 in CD4+ and CD8+ T cells in synovial fluid than in peripheral blood. When comparing TIM-3 level with the severity of RA, we identified that the percentage of TIM-3 on both peripheral CD4+ and peripheral CD8+ T cells was negatively correlated with disease activity score 28 (DAS28) of the patients. Similarly, TIM-3 on synovial fluid CD4+ and CD8+ T cells also revealed inverse correlation with DAS28 of the cases. Our data demonstrate a negative correlation between TIM-3 and the disease progression of RA.

Different Subsets of Macrophages in Patients with New Onset Tuberculous Pleural Effusion

ObjectiveMacrophages are the infiltrate components of tuberculous pleural effusion (TPE). This study is aimed at examining the role of different subsets of macrophages in pleural fluid (PF) and peripheral blood (PB) from patients with new onset TPE.MethodsThe numbers of PB and PF CD163+, CD206+ and CD115+ macrophages in 25 patients with new onset TPE and 17 healthy controls (HC) were determined by flow cytometry. The concentrations of serum and PF cytokines were determined by cytometric bead array (CBA) and enzyme-linked immunosorbentassay (ELISA). The potential association between the numbers of different subsets of macrophages and the values of clinical measures in TPE patients were analyzed.ResultsThe numbers of PB CD14+CD163− M1-like and CD14+CD163− interleukin (IL)-12+ M1 macrophages were significantly higher than that in the HC, but lower than PF, and the numbers of PF CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CDll5+ M2-like, and CD14+CD163+IL-10+ M2 macrophages were less than PB in the TPE patients. The levels of serum IL-1, IL-6, IL-8, IL-12, tumor growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α in the TPE patients were significantly higher than that in the HC, but lower than that in the PF. The levels of PF IL-10 were significantly higher than that in the PB of patients and HC. In addition, the levels of serum IL-12 and TNF-α were correlated positively with the values of erythrocyte sedimentation rate (ESR) and the numbers of ESAT-6- and culture filtrate protein 10 (CFP-10)-specific IFN-γ-secreting T cells, and the levels of PF TNF-α were correlated positively with the levels of PF adenosine deaminase (ADA) and lactate dehydrogenase (LDH) in those patients.ConclusionOur data indicate that Mycobacterium tuberculosis (M. tb) infection induces M1 predominant pro-inflammatory responses, contributing to the development of TPE in humans.

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4(+)IL-17A-F(+) cells, as well as of T CD4(+) cells expressing IL-23Rp19 (T CD4(+) IL-23R(+)), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4(+)IL-17A-F(+) and T CD4(+)IL-23R(+) Th17 Teff cells in SF of clinically active joints of PsA patients.

Inhibition Of the Rho/Rho-Kinase Pathway Reduces In Vitro Adhesion Of Sickle Cell Anemia Eosinophils To Human Endothelial Cells

IntroductionSickle cell anemia (SCA) individuals present a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion. This process is a multi-step process that involves different cell types such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation. Leukocytes may have a propagating and, possibly, initiating role in SCA vaso-occlusion. In vivo and in vitro studies using animal models show that neutrophils play a key role in vaso-occlusion; however, eosinophils (EOs) may also participate in this phenomenon in humans. We have previously shown that SCA eosinophils demonstrating greater adhesion to fibronectin. A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. Y-27632 and NSC23766 are Rho-kinase inhibitors, that reduce leukocyte infiltration in several models of EOs inflammation, such as endotoxic liver and lung injury. The objective of this study was to investigate the effect of Rho kinase pathway on the in vitro adhesion of sickle EOs to activated human endothelial cell layers (umbilical vein endothelial cells, HUVEC). MethodsEosinophils were isolated from peripheral blood of healthy controls (n=5), steady-state SCA patients (SCA, n=7) and SCA patients on HU therapy (SCAHU, n=5), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion (1x106 cell/ml in Ham's F12 K) to cultured HUVEC grown to confluence was assessed using static adhesion assays. HUVEC cells were treated, or not, with 100 µM NSC23766 or Y-27632 for 1h before incubation with an inflammatory stimulus (10nM TNF-alpha – 3 h), subsequently Eos were added to the cell layer (1 hour, 37°C, 5%CO2). For adhesion under flow conditions, biochips with microchannels coated with HUVEC were pre treated or not with Y-27632 or NSC23766 per 1 h before the TNF-alphastimulus. Purified eosinophils was added to HUVEC-coated microchannels at a flow rate of 0.5 dynes/cm2. Images of adherent eosinophils were recorded using real-time video microscopy and analysed using Duco Cell software. ResultsEOs from SCA patients demonstrated a significantly greater adhesion to HUVEC than healthy controls (14.42 ± 1.8% vs 9.35 ± 1.7 %; p<0.02); no significant difference was observed between the SCAHU group, compared to the healthy control and SCA group (10.5 ± 1.6%). Pre treatment of HUVEC with TNF-alpha significantly augmented the adhesion of healthy control, SCA, and SCAHU eosinophils to HUVEC (16.1 ± 3.2%, 33.1 ± 5.1%, 17.9 ± 3.5%, P<0.01, respectively). However, this adhesion was significantly higher for SCA EOs compared to healthy control and SCAHU EOs (p<0.05). Interestingly, when the HUVEC was pre-treated with NSC23766 then stimulated with TNF-alpha, adhesion of EOs from SCA and SCAHU was significantly reduced (22.9 ± 5,6%, 17.2 ± 6.1%, respectively, p<0.05). However, when HUVEC was pre-treated with Y-27632 no significant difference was found. In addition, EOs from SCA patients demonstrated greater adhesion to HUVEC under flow conditions when compared to healthy control and SCAHU. EOs when the HUVEC was protect with Rho inhibitors (Y-27632 and NSC23766) before stimulation with TNF-alpha, EOs adhesion under physiological shear stress was diminished in all groups investigated (healthy control, SCA and SCAHU). ConclusionRho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion, in an in vitro model. Therapy with such Rho-kinase inhibitors could be used in patients with SCA, with beneficial effects on the vascular occlusion process.Financial Support: FAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

Does Oxidative Inactivation of CD45 Phosphatase in Rheumatoid Arthritis Underlie Immune Hyporesponsiveness?

The protein tyrosine phosphatase (PTP) CD45 is critical in regulating the earliest steps in T-cell-receptor signaling but, similar to all PTPs, it is susceptible to oxidative inactivation. Given the widely reported effects of oxidant damage associated with rheumatoid arthritis (RA), we examined whether CD45 phosphatase activity was altered in CD4(+) T cells from RA patients and related this to CD4(+) T-cell function and redox status. CD45 phosphatase specific activity in T cells from RA peripheral blood (PB) and synovial fluid was 56% and 59% lower than in healthy control (HC) PB, respectively. In contrast, CD45 activity in T cells from disease controls (DSC) was not significantly different from HC. Both reduced glutathione (GSH) (p<0.001) and oxidized glutathione (GSSG) (p<0.05) were significantly lower in RA PB T cells compared with HC PB T cells. Treatment of RA PB T cells with the GSH precursor N-acetyl cysteine increased CD45 phosphatase activity and proliferation, while it decreased Lck kinase phosphorylation, which is regulated by CD45. Our observations lead to the hypothesis that the largely reversible oxidative inactivation of the CD45 phosphatase may underlie the decreased signaling efficiency and functional responsiveness which are characteristic of RA PB CD4(+) T cells.

OP0072 Plasmacytoid Dendritic Cells Activated by Neutrophil Extracellular Traps Contribute to the Pathogenesis of IGG4-Related Disease

BackgroundPlasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) in response to the presence of viral or bacterial DNA. pDCs play a pivotal role in regulating immune responses by linking...

Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Abstract 387Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function. Disclosures:No relevant conflicts of interest to declare.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.

Increased frequency and clinical significance of myeloid-derived suppressor cells in human colorectal carcinoma.

To investigate the frequency and clinical significance of the myeloid-derived suppressor cells (MDSC) in human colorectal carcinoma (CRC). Samples of peripheral blood and tumor tissue from 49 CRC patients were analyzed. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and were subjected to a flow cytometry-based immunophenotypic analysis. A considerable increase in the percentage of CD33⁺HLA-DR⁻ MDSCs was observed in the peripheral blood (1.89% ± 0.75%) and tumor tissues (2.99% ± 1.29%) of CRC patients as compared with that in the peripheral blood of healthy controls (0.54% ± 0.35%). This expanded CD33⁺HLA-DR⁻ subset exhibited immature myeloid cell markers, but not lineage markers, and showed up-regulation of CD18/CD11b expression as compared with the MDSCs from healthy donors. Further studies showed that the MDSC proportion in CRC peripheral blood was correlated with nodal metastasis(P = 0.023), whereas that in tumor tissues was correlated with nodal/distant metastasis (P = 0.016/P = 0.047) and tumor stage (P = 0.028), suggesting the involvement of MDSCs in CRC tumor development. Characterization of MDSCs in CRC suggests the clinical significance of circulating and tumor-infiltrating MDSCs and may provide new insights into the CRC immunotherapy targeting MDSCs.

The pattern‐recognition receptor nucleotide‐binding oligomerization domain–containing protein 1 promotes production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts

Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-containing protein 2 (NOD-2), have been shown to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to analyze the expression, regulation, and function of the PRR NOD-1 in RA synovial fibroblasts (RASFs), and to examine its interaction with other PRRs. Expression of NOD-1 was analyzed by immunohistochemistry in synovial tissue from RA patients, psoriatic arthritis patients, gout patients, and osteoarthritis (OA) patients. RASFs and human monocyte-derived macrophages (HMDMs) were stimulated with L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid, palmitoyl-3-cysteine-serine-lysine-4, poly(I-C), lipopolysaccharide, heat-inactivated bacteria, tumor necrosis factor α (TNFα), or interleukin-1β (IL-1β). Expression levels of IL-6, CCL5, matrix metalloproteinases (MMPs), NODs, and TLRs were measured by real-time reverse transcription-polymerase chain reaction and/or enzyme-linked immunosorbent assay. NOD-1 and NOD-2 were silenced with target-specific small interfering RNA. Phosphorylation of IL-1 receptor-associated kinase 1 (IRAK-1) was measured by Western blotting. Expression of NOD-1 protein was significantly increased in RA synovium compared to OA synovium. The basal expression of NOD-1 was similar in RASFs, OASFs, healthy control peripheral blood mononuclear cells, and healthy control HMDMs. Stimulation of RASFs with TLR-3 up-regulated the expression of NOD-1. Expression of IL-6, CCL5, MMPs, TLR-2, and NOD-2 was significantly up-regulated in RASFs by stimulation with the NOD-1 ligand. A synergistic effect on IL-6 production was observed in cells stimulated with NOD-1 and TLR-2 ligands or NOD-1 and TLR-4 ligands. Silencing of NOD-1, but not NOD-2, decreased the levels of IL-6 in RASFs after stimulation with TLR-2 and IL-1β, and blocked the phosphorylation of IRAK-1. NOD-1 is strongly expressed in different cell types in the synovial tissue of patients with RA. These results indicate that NOD-1, either alone or interacting with other inflammatory mediators, can play an important role in the chronic and destructive inflammation of the joints in RA.

S47 Pseudomonas aeruginosa induces apoptosis in human dendritic cells: a potential mechanism to evade pulmonary immune responses

IntroductionAirway colonisation with Pseudomonas aeruginosa (PA) represents a hallmark of cystic fibrosis (CF). Mechanisms by which PA establishes pulmonary infection are undoubtedly complex and, in part, will reflect the capacity...

Altered Functional Properties of Eosinophils In Sickle Cell Anemia and Effects of Hydroxyurea Therapy

AbstractAbstract 2656The pathophysiology of sickle cell anemia (SCA) is now recognized to include a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion that characterize this disease. The vaso-occlusive process is a multi-step process that involves different cell types, such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. In vivo transgenic animal studies indicated that the neutrophil appears to be the most important leukocyte in vaso-occlusion in previous transgenic animal in vivo studies; however, eosinophils (EOs) may also participate in this phenomenon in humans. EOs are found in significantly elevated numbers in sickle cell disease individuals and, in static adhesion assays, demonstrate augmented adhesive properties suggesting an activated state of these cells. However, the role of EOs in the pathophysiology of SCD and the effect of hydroxyurea (HU) therapy on the functional properties of these cells are not well understood. The aim of this study was to evaluate EO adhesion, migration and degranulation in SCA patients and the effect of HU therapy on these cells. EOs were purified from the peripheral blood of healthy controls (CON) (n≥10), steady-state SCA patients (n≥12) and SCA patients on HU therapy (n≥15), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion was determined by static adhesion assays and degranulation was evaluated by measurement of eosinophil peroxidase activity. Migration of EOs was determined in 96-multiwell chemotaxis chamber assays. Plasma levels of Eotaxin-1, 2 and RANTES, important eosinophil attracting chemokines, were determined by ELISA. The absolute number of EOs in the peripheral blood of SCD patients not treated with HU was significantly higher compared to control individuals (0.504±0.09 vs 0.188±0.04; P=0.01, respectively). SCA patients taking HU presented significantly lower numbers of EOs (0.200±0.05, P = 0.01). Basal adhesion of EOs from SCA patients was significantly higher than in healthy controls (22.8±2.6 vs 13.7±1.7%, respectively, P = 0.004). Furthermore, the EOs from SCA patients on HU therapy demonstrated a significantly lower adhesion to fibronectin (12.5±2.0%, P=0.002). Spontaneous EO chemotaxis was significantly increased in SCA patients, compared to healthy controls (12.9±1.56 vs 6.97±0.8 × 105/ml, respectively, P=0.009). Interestingly, the chemotatic response of EOs isolated from SCA patients taking HU was also higher than that of control EOs (12.1± 1.9 × 105/ml). EO chemotaxis in response to RANTES, Eotaxin and IL5 was significantly augmented in all groups compared to spontaneous chemotaxis (RANTES: CON: 15.3± 2.3; SCA: 19.9±3.0; SCAHU: 25.0±4.7; Eotaxin: CON: 18.7±3.0; SCA: 24.5±4.2; SCAHU: 24.0±5.2; IL5: CON: 13.4±2.4; SCA: 18.8±3.0, SCAHU: 16.6±4.5, P<0.05). Baseline eosinophil peroxidase release was higher in SCA EOs, compared to CON EOs (0.4±0.04 vs 0.2±0.02, respectively, P=0.001), but patients on HU presented a lower EOs degranulation than SCA not on HU (0.19±0.03, P=0.003). Plasma levels of Eotaxin-1 and RANTES were significantly higher in SCA individuals, compared to CON individuals (124±7; 1057±125 vs 78±7; 540±50.2 pg/ml, P=0.0002; P=0.0009, respectively). HU therapy was not associated with any change in Eotaxin-1 and RANTES plasma levels (117.9±9.7; 1175±126.2 pg/ml, respectively). Eotaxin-2 plasma levels did not vary among groups (485±80.8, 450±34, 398±47.8 pg/ml for CON, SCA and SCAHU, respectively). EOs of patients with SCA demonstrate a higher capacity for spontaneous migration, stimulated migration and degranulation. Therapy with HU is associated with reduced adhesion and degranulation of EOs in these patients, but had no effect on the chemotactic ability of these cells or chemokine levels. The presence of relative eosinophilia along with altered functional properties in SCA patients warrants further investigation of the role of EOs in the vaso-occlusive process. In particular, complications associated with allergic reactions, such as acute thoracic syndrome and asthma in SCA children, in which EO adhesion and degranulation may be more important, should be studied. The characteristics of the effect of HU on EO also suggest that SCA events in which EO chemotaxis plays a role are likely to respond poorly to HU therapy, and should lead to the development of alternative therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.