Presence Of Autologous Serum Research Articles

Evaluation of the In Vitro Capacity of Anti-Human Cytomegalovirus Antibodies to Initiate Antibody-Dependent Cell Cytotoxicity.

In the setting of infectious diseases, antibodies show different functions beyond neutralizing activity. In this study, we investigated the activation of NK cells in vitro in the presence of human cytomegalovirus (HCMV)-specific antibodies and their potential role in the control of HCMV infection through antibody-dependent cell cytotoxicity (ADCC). Retinal pigmented epithelial cells (ARPE-19) infected with the HCMV strain VR1814 were co-cultured with cytokine-activated peripheral blood mononuclear cells (PBMCs) in the presence of sera collected from 23 HCMV-seropositive and 9 HCMV-seronegative donors. Moreover, 13 pregnant women sampled 3 and 6 months after HCMV primary infection and 13 pregnant women with pre-conception immunity were tested and compared. We determined the percentage of activated NK cells via the analysis of CD107a expression as a marker of degranulation. Significantly higher levels of NK-cell activation were observed using 1/100 and 1/10 dilutions of sera from HCMV-seropositive individuals, and when cells were infected for 96 and 120 h, suggesting that NK cells are activated by antibodies directed against late antigens. In the absence of serum NK cells, activation was negligible. In seropositive subjects, the median percentages of CD107a-positive NK cells in the presence of autologous serum and pooled HCMV-positive serum were similar (14.03% [range 0.00-33.56] and 12.42% [range 1.01-46.00], respectively), while NK-cell activation was negligible using an HCMV-negative serum pool. In HCMV-seronegative subjects, the median percentage of activated NK cells was 0.90% [range 0.00-3.92] with autologous serum and 2.07% [0.00-5.76] in the presence of the HCMV-negative serum pool, while it was 8.97% [0.00-26.49] with the pool of HCMV-positive sera. NK-cell activation using hyperimmune globulin is comparable to what is obtained using autologous serum. Sera from subjects at 3 and 6 months post primary infection showed a lower capacity of NK-cell activation than sera from subjects with past infection (p < 0.001). NK activation against HCMV-infected epithelial cells is dependent on the presence of HCMV-specific antibodies. This serum activity increases with time after the onset of HCMV infection. The protective role of NK-cell activation by HCMV-specific serum antibodies should be verified in clinical settings.

The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

CD4+ T cells are sensitive to peripheral changes of cytokine levels and metabolic substrates such as glucose and lactate. This study aimed to analyze whether factors released after exercise alter parameters of human T cell metabolism, specifically glycolysis and oxidative phosphorylation. We used primary human CD4+ T cells activated in the presence of autologous serum, which was collected before (CO) and after a 30-min exercise intervention (EX). In the course of activation, cells and supernatants were analyzed for cell viability and diameter, real-time oxygen consumption by using PreSens Technology, mRNA expression of glycolytic enzymes and complexes of the electron transport chain by real-time PCR, glucose, and lactate levels in supernatants, and in vitro differentiation by flow cytometry. EX did not alter T cell phenotype, viability, or on-blast formation. Similarly, no difference between CO and EX were found for CD4+ T cell activation and cellular oxygen consumption. In contrast, higher levels of glucose were found after 48 h activation in EX conditions. T cells activated in autologous exercise serum expressed lower HK1 mRNA and higher IFN-γ receptor 1. We suggest that the exercise protocol used was not sufficient to destabilize the immune metabolism of T cells. Therefore, more intense and prolonged exercise should be used in future studies.

A novel 4-dimensional live-cell imaging system to study leukocyte-endothelial dynamics in ANCA-associated vasculitis

Neutrophils, monocytes and the endothelium are critical to ANCA-associated vasculitis (AAV) pathogenesis. This study aimed to develop a 4-dimensional (4D) live-cell imaging system that would enable investigation of spatial and temporal dynamics of these cells in health and disease. We further aimed to validate this system using autologous donor serum from AAV patients and polyclonal ANCA IgG, as well as exploring its potential in the pre-clinical testing of putative therapeutic compounds. Neutrophils and monocytes were isolated from peripheral venous blood of AAV patients or healthy controls and co-incubated on an endothelial monolayer in the presence of autologous serum. Alternatively, polyclonal ANCA IgG was used, following TNF-α priming, and imaged in 4-dimensions for 3 h using a spinning disc confocal microscope. Volocity 6.3® analysis software was used for quantification of leukocyte dynamics. The use of autologous serum resulted in increased neutrophil degranulation (p = .002), transmigration (p = .0096) and monocyte transcellular transmigration (p = .0013) in AAV patients. Polyclonal MPO-ANCA IgG induced neutrophil degranulation (p < .001) in this system. C5aR1 antagonism reduced neutrophil degranulation (p < .0002). We have developed a novel 4D in vitro system that allows accurate quantification of multiple neutrophil- and monocyte-endothelial interactions in AAV in a single assay. This system has the potential to highlight dynamics key to pathophysiology of disease, as well investigating the impact of potential therapeutics on these functions.

Altered monocyte response to the dengue virus in those with varying severity of past dengue infection

ObjectiveWe sought to investigate the differences in monocyte immune responses to the dengue virus (DENV) in those who previously had either severe disease (past SD) or non-severe dengue (past NSD) following a secondary dengue infection. MethodMonocytes from healthy individuals who had either past SD (n = 6) or past NSD (n = 6) were infected at MOI one with all four DENV serotypes following incubation with autologous serum. 36-hours post infection, levels of inflammatory cytokines and viral loads were measured in the supernatant and expression of genes involved in viral sensing and interferon signaling was determined. ResultsMonocytes of individuals with past SD produced significantly higher viral loads (p = 0.0426 and cytokines (IL-10 p = 0.008, IL-1β p = 0.008 and IL-6 p = 0.0411) when infected with DENV serotypes they were not immune to, compared to those who has past NSD. Monocytes of individuals with past SD also produced significantly higher viral loads (p = 0.022) and cytokines (IL-10 p < 0.0001, IL-1β < 0.0001 and IL-6 p < 0.0001) when infected with DENV serotypes they were previously exposed to, despite the monocytes being infected in the presence of autologous serum. A significant upregulation of NLRP3 (p = 0.005), RIG-I (0.0004) and IFNB-1 (0.01) genes were observed in those who had past SD compared to past NSD when infected with non-immune DENV serotypes. ConclusionMonocytes from those with past SD appear to show marked differences in viral loads, viral sensing and production of inflammatory mediators in response to the DENV, when compared to those who experienced past NSD, suggesting that initial innate immune responses may influence the disease outcome.

Natural killer cell cytotoxicity is enhanced in the presence of 1 h post-exercise serum

Natural Killer-cell cytotoxicity (NKCA) increases during recovery from exercise. The mechanism(s) underlying enhanced NKCA remain unknown. We therefore investigated the role of exercise-induced changes in cortisol and NK-cell subsets on NKCA. Methods Thirteen adults (7 women; 32 ± 7 years) cycled 30 min 15% above lactate threshold. NK-cells from pre-, post-, and 1 h post-exercise were incubated with U266 tumor-target cells in the presence of autologous serum. NKCA was assessed after 4 h in a flow cytometry based assay. To determine the effect of cortisol released during exercise, pre-exercise NK-cells and targets were incubated with pre-, post- and 1 h post-exercise serum. To determine the effect of shifts in NK-cell subsets, pre-, post-, and 1 h post-exercise NK-cells and targets were incubated with pre- exercise serum. Serum cortisol was assessed by ELISA. Results were analyzed using linear mixed models. Results The NKCA of pre-exercise NK-cells was enhanced in the presence of 1 h post-exercise serum (p Conclusions Serum obtained 1 h post-exercise enhanced resting NKCA and decreased the proportion of inhibitory NK-cells. This suggests that factors present in serum after exercise can alter effector cell function. Future work will analyze cytokines present in 1 h post-exercise serum.

Exploring the Mechanisms Underlying Exercise-Induced Changes in Natural Killer Cell Cytotoxicity

Natural Killer (NK) cells are cytotoxic effectors of the innate immune system that eliminate tumor cells. We have previously shown that NK cell cytotoxicity (NKCA) is enhanced by acute bouts of exercise. However, the mechanisms underlying exercise-induced changes in NKCA are not yet fully understood. Exercise-induced mobilization of highly cytotoxic NK cell subsets has been proposed as one mechanism. Alternatively, exercise increases glucocorticoid and cytokines levels, which have also been shown to alter NKCA. PURPOSE: To examine the role of shifts in proportions of NK cell subsets, cytokines, and hormones on exercise induced changes in NKCA. METHODS: Adults (n=13) cycled 30 min at 115% of their lactate threshold power. Blood was collected pre, post, and 1h post exercise. Effector cells isolated from blood were incubated with K562 or U266 tumor target cells in the presence of autologous serum. NKCA was assessed after 4h by measuring lysed target cells in a flow cytometry based assay. To investigate the effects of factors (hormones and cytokines) released during exercise, pre exercise effectors were incubated with targets in the presence of pre, post, and 1h post exercise serum. The effect of shifts in NK cell subsets was determined by incubating pre, post and 1H post exercise cells with targets in presence of pre exercise serum. We also tested the cytotoxicity of pre, post, and 1h post effectors incubated with serum from the corresponding time point. RESULTS: Autologous pre, post, and 1h post exercise serum did not cause tumor cell lysis in the absence of effectors. The cytotoxicity of pre exercise effector cells was significantly increased against U266 target cells when incubated in 1h post exercise serum (Pre vs. post vs.1H post =0.318±.039 vs. 0.334±.039 vs. 0.438±.039, p<0.05). There was no difference in cytotoxicity of pre, post, 1h post effectors incubated with pre-exercise serum (Pre vs. post vs.1H post =0.321±.046 vs. 0.282±.047 vs. 0.323±.047, p<0.05). CONCLUSION: 1h post exercise serum enhanced resting NK cell cytotoxicity, indicating that factors present in serum after acute exercise can prime effector cell function. Future work will identify levels of glucocorticoids and cytokines present in 1h post exercise serum.

Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

ObjectivesTo examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing...

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24–40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1–37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant β-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1β, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

High levels of anti-tuberculin (IgG) antibodies correlate with the blocking of T-cell proliferation in individuals with high exposure to Mycobacterium tuberculosis.

To determine the effect of anti-tuberculin antibodies in the T-cell proliferation in response to tuberculin and Candida antigens in individuals with different levels of tuberculosis (TB) risk. Sixteen high-risk TB individuals, 30 with an intermediate TB risk (group A), and 45 with a low TB risk (group B), as well as 49 control individuals, were studied. Tuberculin skin test (TST) results were analyzed and serum levels of antibodies (IgG and IgM) against purified protein derivative (PPD) were measured by ELISA. Tuberculin and Candida antigens were used to stimulate T-cell proliferation in the presence of human AB serum or autologous serum. High levels of anti-tuberculin IgG antibodies were found to be significantly associated with the blocking of T-cell proliferation responses in cultures stimulated with tuberculin but not with Candida antigens in the presence of autologous serum. This phenomenon was particularly frequent in high-risk individuals with high levels of anti-tuberculin IgG antibodies in the autologous serum when compared to the other risk groups, which exhibited lower levels of anti-tuberculin antibodies. Although cellular immunity plays a central role in the protection against TB, humoral immunity is critical in the control of Mycobacterium tuberculosis infection in high-risk individuals with latent TB infection.

Toll‐like receptor‐mediated immune responses are attenuated in the presence of high levels of hepatitis B virus surface antigen

It has been recently shown that Toll-like receptor (TLR) signalling in murine nonparenchymal liver cells (NPCs) is suppressed in the presence of Hepatitis B virus surface antigen (HBsAg). It is not clear, however, whether this is also relevant for the adaptive immune responses and how this effect is mediated. Peripheral blood mononuclear cells (PBMCs) from Hepatitis B virus (HBV) patients and controls were stimulated by TLR ligands in the absence or presence of autologous serum. Interestingly, TLR-mediated cytokine expression (Interleukin-6 and -10) as well as TLR3-induced interferon (IFN) expression in PBMCs of HBV patients was significantly higher than in the healthy volunteers, showing a negative correlation with the levels of HBsAg. In addition, TLR3-mediated IFN-γ production was inhibited in the presence of HBV-containing serum. To mechanistically analyse this observation, murine Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) were stimulated with TLR3 ligands in the presence or absence of HBsAg. Mixed lymphocyte reactions were performed to study T-cell activation induced by TLR-stimulated NPCs. Gene expression of cytokines and TLR3 was analysed by quantitative rt-PCR, and activation of transcription factors was assessed by Western blot or reporter gene assays. TLR-induced expression of interferon γ, interferon sensitive genes and proinflammatory cytokines in murine KCs and LSECs was efficiently suppressed in the presence of HBsAg, whereas the expression of anti-inflammatory cytokines was enhanced. Activation of NFκB, IRF-3 and MAPKs in these liver cells was potently suppressed by HBsAg. T-cell activation mediated through TLR3-stimulated KCs or LSECs was suppressed by HBsAg which could be reverted by anti-IL-10 antibodies. These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to maintain a persistent infection.

Interferon gamma autoantibodies associated with susceptibility to disseminated mycobacterial infections

Disseminated non-tuberculous mycobacterial (NTM) infections typically occur in the context of iatrogenic immunosuppression or inherited genetic mutations. Rarely, cytokine-specific autoantibodies have also been described in patients with disseminated infections. Here, we present two cases of disseminated NTM infection in unrelated healthy adult females associated with autoantibodies to interferon-gamma (IFN-γ). The two HIV-negative patients had Mycobacterium avium and Mycobacterium abscessus infections, respectively, that presented as subacute multifocal bone, lung and lymph node involvement. Routine studies revealed normal immunoglobulin concentrations, T-cell proliferation to mitogens and granulocyte oxidative burst. Blood mononuclear cell stimulation analyses with cytokine bead array revealed normal IL-12p40 production and IFN-γ production in response to exogenous IL-12. Both patients had normal tumour necrosis factor-α (TNF-α) production in response to IFN-γ that was remarkably abrogated in the presence of autologous serum. By ELISA, both patients had high titre (>1:5000) anti-IFN-γ IgG in the serum. The patients' IgG inhibited IFN-γ-induced MHC class II protein upregulation on CD14 + monocytes. Targeted B cell depletion with rituximab has started for one patient. Autoantibodies against cytokines may disrupt normal immunity and predispose to more severe and resistant NTM disease. In the latter circumstances, screening for anti-IFN-γ autoantibodies may clarify the pathophysiology and allow targeted therapy.

Reduced Mycobacterium tuberculosis association with monocytes from diabetes patients that have poor glucose control

The re-emerging importance of type 2 diabetes mellitus (DM) to tuberculosis (TB) control is of growing concern, but the basis for this relationship is poorly understood. Given the importance of mononuclear phagocytes for TB control and the reported alterations in monocytes of DM patients, we evaluated whether the initial interaction between both was affected in diabetics. Mycobacterium tuberculosis-naïve individuals with and without DM were group matched by age and gender and the efficiency of M.tuberculosis association (attachment and ingestion) with their monocytes was assessed in the presence of autologous serum. The association of M.tuberculosis with monocytes was significantly lower in diabetics (19.2±6.1) than non-diabetics (27.5±7.9; p=0.02). Multivariate analysis controlling for host socio demographics, DM characteristics and serum lipids indicated that male gender (p=0.04) and poorly-controlled DM (high HbA1c and hyperglycemia; p=0.01) were significantly associated with the lower interaction of M.tuberculosis with monocytes. Serum heat-inactivation reduced the association of M.tuberculosis to similar levels in both study groups (p=0.69) suggesting alterations in the complement pathway of DM patients. These findings suggest an altered route of entry of the pathogen in DM patients that may influence the downstream activation of signaling pathways in the monocyte and the survival of mycobacteria.

Type of serum influences the rituximab dependent cytotoxicity and apoptosis of chronic lymphocytic leukemia cells in vitro

The aim of the study was to compare the influence of types of serum on the in vitro viability and on either spontaneous or rituximab (RIT)-induced apoptosis of chronic lymphocytic leukemia (CLL) cells. The influence of fetal calf serum (FCS), patients' autologous serum (AS) and human AB-serum (ABS), used alone and in combinations consisting of two of them (v/v-1:1), on RIT-dependent cytotoxicity, apoptosis, detection of active forms of caspases-3,-9,-8 and disruption of mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. RIT was used at the concentration of 10 µg/ml. The spontaneous apoptosis was assessed in culture without RIT. AS revealed the protective action on CLL cells, however this serum added in vitro to the culture either alone or in combination with FCS was the only one to allow RIT to exert its cytotoxic action against CLL cells. RIT-induced apoptosis involved changes in ΔΨm and activation of caspases-3,-8,-9 when AS+FCS was applicated. Drug induced apoptosis (DIA) was 6.02 and 0.34, when FCS+AS and FCS alone were used, respectively (p<0.01). The RIT-dependent cytotoxic effect decreased when FCS+AS or FCS+ABS were used, as compared to effect of AS used separately. The cytotoxic effect of RIT did not depend on drug concentration, but on the type of serum added to the culture. The strongest cytotoxic effect of RIT in the presence of AS suggests that this drug activity towards CLL cells is enhanced by known cytotoxic mechanisms, caspase-dependent apoptotic pathway and possible influence of other extracellular factors present in the patients' sera.

Bevacizumab-induced tumor calcifications can be elicited in glioblastoma microspheroid culture and represent massive calcium accumulation death (MCAD) of tumor endothelial cells

AbstractBähr and colleagues reported that 22 of 36 glioblastoma patients treated with bevacizumab showed tumor calcifications on 8 week post therapy follow up with MRI. Early tumor calcification strongly predicted for response, time to progression, and overall survival. The authors didn’t understand the mechanism, but speculated that it was vascular in nature. At the 13th International Anti-Angiogenic Symposium (2011), we presented our discovery of the phenomenon of massive calcium accumulation death, wherein MCAD occurred in endothelial cells (tumor, circulating, and HUVEC), in response to VEGF depletion by bevacizumab and other putative anti-angiogenic agents, but not in response to non-specific cytotoxins. In subsequent work, we have documented marked MCAD to occur in primary microcluster cultures from 6 fresh human glioblastoma biopsies, following 96 hours of VEGF depletion in vitro by bevacizumab. The presence and degree of MCAD is strikingly dependent on the type of serum in the culture medium (RPMI-1640 + 25% serum) -- typically most striking in (very low VEGF) fetal calf serum, but inhibited (often) or enhanced (rarely) by 25% human serum from different patients or normal donors containing variable quantities of VEGF. There was not a linear relationship between VEGF concentration and MCAD inhibition (or enhancement), suggesting that other pro-angiogenic (or anti-angiogenic) serum factors may play a role. In epithelial metastatic tumors, circulating peripheral blood endothelial cells may be easily tested, using our methods, and the serum inhibition (or, rarely, enhancement) is faithfully reproduced on circulating endothelial cells, in comparison with the tumor cluster-associated endothelial cells. We propose MCAD as the mechanism of glioblastoma calcification following bevacizumab and further propose that testing tumor microclusters and/or circulating endothelial cells, in the presence of autologous serum, could be a useful predictive biomarker and research tool.

Contribution of Decreased Migration of Monocytes to Impaired Inflammation in Gaucher Disease

Abstract 2162 Background:Gaucher disease (GD) is an autosomal recessively inherited lysosomal storage disorder caused by an inborn glucocerebrosidase (GBA) deficiency. This leads to the accumulation of glycolipids in macrophages, mainly in the bone marrow, spleen and liver, resulting in organ damage. However, recent studies suggest that clinical presentation of GD is primarily attributed to the impaired inflammatory process induced by “loaded macrophages”, rather than to their physical presence. This impairment could be explained by dysfunction of circulating monocytes, normally capable of migrating into sites of vascular injury and inflammation. The current in vitro study investigated potential changes in migratory capacity of GD-derived monocytes that would contribute to the inflammatory response. Methods: CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of untreated GD patients and healthy volunteers, using immunomagnetic bead separation (Miltenyi Biotec). Monocyte migration capacity was assessed with 5μm Polycarbonate Membrane Transwell inserts which contained either stromal cell-derived factor 1 (SDF1; CXCL12) (500 and 1000 ng/ml) or serum (0.25%) at the bottom chamber. Monocytes were allowed to migrate from the upper chamber through the membrane, over 4 hours. Migrating monocytes were collected from the bottom chamber and quantified by FACS. Blockage of the CXCR4 receptor was examined using AMD3100 (10μg/ml) added to the migration assay. Serum SDF-1 levels were evaluated by ELISA (Quntikine® human CXCL12/SDF-1α R&D systems). Results: Upon exposure to SDF-1, migration capacity of GD monocytes (n=7) was significantly lower than that observed in monocytes obtained from healthy volunteers (n=6), approaching 5.7% vs. 14%, p=0.02, for SDF1of 500 ng/ml, and 8.7% vs 21.8%, p=0.013, for SDF1of 1000 ng/ml). Similar results were observed in the presence of autologous serum: migration capacity of GD monocytes (n=7) was lower than demonstrated by healthy monocytes (n=5), (18.6% vs. 45%, p=0.05). Notably, substitution of “GD serum” (n=7) with “healthy serum” (n=7) resulted in significant improvement in migration capacity of GD-derived monocytes (from 18.6% to 33.6%, p=0.038), while a reciprocal substitution of the serum caused a marked reduction in migration of monocytes derived from healthy volunteers (34% instead of 45%). Blockage of the CXCR4 receptor led to a decreased migration capacity of healthy monocytes cultured in autologous serum (from 45.2% to 39.6%) (n=4). Remarkably, this blockage had no impact on migration of both healthy and GD-derived monocytes cultured with GD serum (n=6). However, GD monocytes treated with AMD3100 in the presence of healthy serum, exhibited a reduced migration rate compared to that observed in untreated cells (22.6% vs. 33.6%; p=0.06, n=7).CXCR4 expression on GD-derived monocytes appeared to be lower than that measured on cells derived from healthy donors [64.5% (n=6) vs. 83.6% (n=3)]. Of note, serum levels of SDF-1 were significantly increased in GD subjects compared to healthy ones (2603pg/ml vs 2039 pg/ml; p=0.004, n=10). Conclusions: GD patients exhibit an impaired SDF1-dependent migration of monocytes, despite expressing a significantly increased SDF-1serum level. The impaired migration capacity is partly explained by low levels of CXCR4 on monocyte surface. Additionally, the reduced migration appears to be dependent on as yet undetermined serum-derived factors, as demonstrated by the reversibility of this impairment with substitution of the GD serum for “healthy serum”. The observed reduction in migratory capacity may significantly contribute to the abnormal inflammation accounting for GD-associated vascular and bone complications. Further studies exploring the precise mechanisms involved in this process and their direct impact on GD-related organ damage are warranted. Disclosures:No relevant conflicts of interest to declare.

Differentiation and Glucocorticoid Regulated Apopto-Phagocytic Gene Expression Patterns in Human Macrophages. Role of Mertk in Enhanced Phagocytosis

The daily clearance of physiologically dying cells is performed safely mainly by cells in the mononuclear phagocyte system. They can recognize and engulf dying cells utilizing several cooperative mechanisms. In our study we show that the expression of a broad range of apopto-phagocytic genes is strongly up-regulated during differentiation of human monocytes to macrophages with different donor variability. The glucocorticoid dexamethasone has a profound effect on this process by selectively up-regulating six genes and down-regulating several others. The key role of the up-regulated mer tyrosine kinase (Mertk) in dexamethasone induced enhancement of phagocytosis could be demonstrated in human monocyte derived macrophages by gene silencing as well as blocking antibodies, and also in a monocyte-macrophage like cell line. However, the additional role of other glucocorticoid induced elements must be also considered since the presence of autologous serum during phagocytosis could almost completely compensate for the blocked function of Mertk.

Granulocyte‐colony stimulating factor drives the in vitro differentiation of human dendritic cells that induce anergy in naïve T cells

G-CSF is a modulator of T-cell and DC functions. Previous reports show that monocytes from G-CSF-treated (post-G) healthy donors differentiate into tolerogenic DC in vitro in the presence of autologous serum, containing high levels of IL-10 and IFN-α, and in turn induce type 1 Treg (Tr1) cells. However, the direct effect of G-CSF on DC differentiation was not investigated. Here, we show that monocytes differentiated in the presence of exogenous G-CSF (G-DC) remain CD14+CD1a−, but acquire a DC-like morphology, express CD83 and CD86 and low levels of the tolerogenic markers Ig-like transcript (ILT)4 and HLA-G. G-DC spontaneously produce IL-10 and, upon stimulation, low levels of IL-12. G-DC display low stimulatory capacity and induce anergy in naïve T cells, but do not confer suppressive function. Therefore, in vitro differentiation of monocyte-derived DC in the presence of G-CSF can replicate some but not all features of post-G DC. These findings indicate that the tolerogenic properties of G-CSF do not exclusively reside in its direct effect on DC, which in turn induce T-cell anergy, but also in its ability to generate a tolerogenic milieu in vivo, which is necessary for Tr1 cell induction and cannot be replicated in vitro.

In Vitro Cytokine Responses to Periodontal Pathogens: Generalized Aggressive Periodontitis is Associated with Increased IL-6 Response to Porphyromonas gingivalis

Generalized aggressive periodontitis (GAgP) is an inflammatory condition resulting in destruction of tooth-supporting tissues. We examined the production of IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in cultures of peripheral mononuclear cells (MNC) from 10 patients with GAgP and 10 controls stimulated with periodontal pathogens or a control antigen, tetanus toxoid (TT) in the presence of autologous serum. The pathogens used were Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, either as type strains or bacteria isolated from the participants' inherent oral flora. The P. gingivalis -induced production of IL-6 was approximately 2.5-fold higher in patients with GAgP than in healthy controls (P < 0.05), while the corresponding TNF-alpha production was non-significantly elevated. IL-1beta production induced by P. gingivalis, as all cytokine responses induced by Pr. intermedia, F. nucleatum and TT was similar in the two groups. A reduced IL-12p70 response to Pr. intermedia and F. nucleatum was observed in smokers compared to non-smoking patients (P < 0.02). To assess the role of serum factors in the elevated IL-6 response to P. gingivalis, MNC from two donors free of disease were stimulated with this bacterium in the presence of the various patient and control sera. An elevated IL-6 and TNF-alpha response was observed in the presence of patient sera (P < 0.01 and P < 0.04, respectively). The data suggest that an exaggerated production of IL-6 occurs in GAgP, and that pro-inflammatory serum factors play an essential role in the response.

CD44 Deficiency Is Associated with EnhancedEscherichia coli-Induced Proinflammatory Cytokine and Chemokine Release by Peritoneal Macrophages

CD44 is involved in several immune responses, such as cellular adhesion, migration, proliferation, and activation. Peritonitis is an important cause of sepsis, and Escherichia coli is one of the major pathogens involved therein. We sought to determine the role of CD44 in the host response to E. coli-induced abdominal sepsis and to assess the function of CD44 in the activation of primary peritoneal macrophages by E. coli or lipopolysaccharide (LPS) purified from this bacterium by using wild-type (WT) and CD44 knockout (KO) mice. CD44 KO mice already demonstrated enhanced CXC chemokine levels in peritoneal lavage fluid at 6 h after infection, whereas tumor necrosis factor alpha (TNF-alpha) and interleukin-6 levels were elevated at 20 h postinfection. In line with this, CD44 KO mouse peritoneal macrophages released more TNF-alpha and macrophage inflammatory protein 2 (MIP-2) than did WT cells upon stimulation with E. coli or LPS in the presence of autologous serum. In contrast, plasma TNF-alpha levels were lower in CD44 KO mice and CD44 KO blood leukocytes secreted similar amounts of TNF-alpha and MIP-2 upon ex vivo incubation with E. coli or LPS. The proinflammatory phenotype of CD44 KO macrophages was not associated with an altered expression of inhibitors of Toll-like receptor signaling, whereas it could be partially reversed by addition of WT serum. CD44 deficiency did not impact on leukocyte recruitment into the peritoneal cavity or organ failure. These data suggest that CD44 differentially influences cytokine and chemokine release by different leukocyte subsets.