Immunology and Immunotherapy P1315 P1316 P1317
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366 Abstracts Immunology and immunotherapy P1315 Kinetics of the release of nitric oxide and tumour Methods: PBMCs were isolated from human buffycoats by Hist- necrosis factor-alpha after activation of microglia via Toll-like opaque (Sigma,USA) density gradient centrifugation. Monocytes were isolated by adherence to plastic (3 h, 37C). Adherent mono- receptors-2, -4, and -9 cytes were stimulated with LPS (100 ng/mL) or STM (300 CFU/ S. Ebert, J. Gerber, S. Bader, F. Mu¨hlhauser, T. Mitchell, R. Nau mL) for 24 and 48 h. DXM and/or CFX (Sigma, USA) were used Goettingen, D; Glasgow at concentrations 1 lMa5lg/mL, respectively. CFX was added into some wells after 24 h. The supernatants were collected and Objectives: Microglial cells, the major constituents of innate immu- frozen at À70 C for quantification of IL-6, IL-10 and IL-12 by nity within the brain, express Toll-like receptors (TLRs) recognis- ELISA (Pharmingen, USA). Mph were harvested and stained for ing exogenous and endogenous ligands. The present study aimed flow-cytometric analysis using mAb against CD14 (Becton-Dickin- at quantifying the activation of microglial cells in response to son, USA), TLR2 and TLR4 (eBioscience, USA). stimulation with one or two simultaneously administered specific Results: TLR2 expression by Mph was enhanced by LPS or agonists of TLR-2, -4, and -9 at concentrations causing sub- STM Æ DXM. Unlike TLR2, DXM inhibited IL-6, IL-10, and IL-12 maximum and maximum effects. by LPS-stimulated Mph. However, DXM increased IL-10 produc- Methods: Primary mouse microglial cell cultures were stimulated tion by STM-stimulated Mph. Interestingly, Mph stimulated by with the TLR agonists Pam3Cys and heat-killed Acholeplasma LPS or STM and treated with CFX showed lower expression of laidlawii (TLR-2), endotoxin and pneumolysin (TLR-4) and oligo- TLR2 compared with those without CFX. This was associated nucleotides containing unmethylated cytosin-guanosin motifs with a decrease in IL-6 and IL-10. We did not observed any effect (CpG) (TLR-9). Nitric oxide (NO) release was quantified using the of LPS or STM on TLR4 expression by Mph. Griess reaction. TNF-a release was measured using the Quantikine Conclusion: Our results demonstrate the ability of GC to increase M Mouse TNF-a Immunoassay (R&D Systems GmbH). Cell viabi- the expression of TLR2 on human Mph. This indicates a possible lity of microglial cells was determined using the WST-1-Cell immunostimulatory effect of GC. However, involvement of TLR2 Proliferation Reagent (Roche Applied Science). induction by GC in subsequent innate and specific immune Results: Maximum stimulation of TLR-2, -4, and -9 resulted in responses requires further study. Decreased TLR2 expression approximately equal amounts of NO release. LPS was most potent associated with lower production of IL-6 and IL-10 in Mph treated in stimulating microglia (concentration causing the half-maximum by CFX indicates a role of TLR2 in the immunomodulatory effect effect [EC50]-NO: 0.00037 lg/mL; EC50-TNF-a: 0.024 lg/mL), fol- of this antibiotic. lowed by pneumolysin (0.024 lg/mL; 0.136 lg/mL), CpG Acknowledgement: The study was partly supported by the grants (0.119 lg/mL; 0.685 lg/mL) and Pam3Cys (52.3 lg/mL) (EC50- TW00915 and TW00233 NIH Fogarty International Centre. values represent means of six experiments with at least triplicate measurement, respectively). Pneumolysin was a potent activator of microglial cells at concentrations below 3 lg/mL; at higher concentrations it reduced cell viability. No cytotoxicity was noted with the other TLR agonists. Co-stimulation with maximum con- centrations of two TLR agonists did not further increase NO P1317 Exopolysaccharides from marine thermophilic release. Co-stimulation with sub-maximum concentrations had an bacilli induce a Th1 cytokine profile in human PBMC additive effect. Conclusions: Stimulation of microglial cells via different TLRs A. Arena, B. Pavone, C. Gugliandolo, T.L. Maugeri, G. Bisignano resulted in a relative uniform release of NO and TNF-a. Simulta- Messina, I neous stimulation with low concentrations of two agonists led to an additive effect. Not only microbial products, but also endog- Objectives: The exopolysaccharides (EPS) produced by a strain of enous compounds can act as agonists in the TLR system. The Geobacillus thermodenitrificans and a strain of Bacillus lichenifor- additive effect of stimulating more than one TLR does not only mis were used to evaluate their immunoregulatory and antiviral increase the sensitivity of microglia during infections, yet may effects. The two strains were isolated from a shallow marine hot also entail interactions among exogenous and endogenous agon- spring of Vulcano Island (Italy). In view of the important regula- ists of TLRs. This may be one reason for the exacerbation or tory role in antiviral defence played by cytokines modulator fac- induction of autoimmune diseases by infections. tors, we analysed both the effects of EPS of two marine bacteria on cytokine production and the antiviral activity on peripheral blood mononuclear cells (PBMC). Methods: PBMC were obtained from healthy donors, after centrif- P1316 Drug modulation of Toll-like receptors expression ugation of heparinised venous blood over a Ficoll-Hipaque gradi- by human macrophages stimulated with lipopolysaccharide and ent. Supernatants were harvested and analysed for the presence c a a bacteria of IFN , IFN , TNF , IL-12, IL-18, IL-4. In order to establish if the EPS antiviral activity was related either to the marked production O. Beran, D. Lawrence of Th1 cytokines or to a direct interaction with virus replication, Albany, USA we used WISH cell line. To evaluate the effects of the on HSV-2 replication in PBMC and WISH cell line, the amounts of virus Objectives: Human Toll-like receptors (TRL) TLR2 and TLR4 rec- production were titrated and expressed as PFU/mL. ognise the specific patterns of a variety of bacterial cell-wall com- Results: The EPS trigger mononuclear phagocytes in releasing cy- ponents and transduce signals in cells during an early phase of tokines involved in Th1 profile, such as IL-12, IFNc, IFNa, TNFa, the innate immune response. Glucocorticoids (GC) are potent im- IL-18, whereas did not induce the production of Th2 cytokine (IL- munomodulatory drugs. Although GC can supress many func- 4). Furthermore, treatment of HSV-2 infected PBMC with EPS tions of macrophages (Mph), a role of GC in enhancing host- down-regulated virus replication. This effect on HSV-2 replication immune responses, particularly through an effect on TLR expres- seems to be related to the pattern of cytokines induced in PBMC sion in Mph, remains unknown. The aim of the study was to by EPS. The addition of EPS to WISH cells, infected with HSV-2, evaluate the effect of dexamethasone (DXM) on TLR2 and TLR4 did not show any antiviral effect. expression and cytokine production by Mph upon stimulation Conclusion: We have shown that the two EPS are powerful stimu- with LPS or Salmonella typhimurium (STM). We also determined lators of Th1 cell-mediated immunity. They are immunomodulato- modulation of these parameters by ceftriaxone (CFX), a cefalospo- ry agents which could be potentially used as a therapy in the rin antibiotic used in the treatment of bacterial infections. immunocompromised host. Clinical Microbiology and Infection, Volume 10, Supplement 3, 2004 367 P1318 Differential induction of IL-8 expression by LPS (A, wild type, 0, variant) and a promoter polymorphisms (X/Y) of the MBL gene were determined by PCR and heteroduplex and LTA analysis. R. Munke, L. Hareng, S. Aulock von, T. Hartung Results: Birth weight and gestational age were both found to influ- Constance, D ence MBL levels. Neonates born at <30 weeks gestation and <1500 g were significantly lower than neonates of >30 weeks and Objectives: Lipopolysaccharide (LPS) from Gram-negative bacteria >1500 g. This difference persisted (P < 0.0005). 40% of preterm and lipoteichoic acid (LTA) from Gram-positive bacteria are key neonates had exon 1 mutations (A/0 or 0/0). 50% of these neo- inflammatory principles which differ regarding the cytokine pat- nates were diagnosed with sepsis and/or multiple organ system tern they induce: LTA is a stronger inducer of chemokines, i.e. IL- failure. In comparison, only 26% of neonates with wild-type 8 and G-CSF, than LPS, which is a stronger inducer of pro-inflam- alleles (A/A) were diagnosed with sepsis. When MBL levels were matory cytokines, such as TNFa. We have shown previously for analysed in all 50 neonates, levels of MBL were significantly less LPS stimulation that IL-8 and G-CSF production is increased by in those with sepsis when compared with those without sepsis db-cAMP, while TNFa production is decreased, and described a (mean 1 492 ng/mL (SD 1 451 ng/mL) vs. 2 342 ng/mL (SD 2 novel activating CRE in the G-CSF promotor. Thus we now inves- 070 ng/mL), P ¼ 0.037). tigated whether the IL-8 promotor also has an activating CRE ele- Conclusion: In this preliminary study, MBL deficiency appears to ment, which might explain the parallel strong induction of IL-8 be associated with increased susceptibility to severe sepsis in and G-CSF by LTA. premature neonates. Further studies are required to elucidate Methods: HL-60 cells were transfected with mutated or wild-type the true impact of this finding on neonatal susceptibility to promotor constructs with a luciferase reporter. b-galactosamine infection. was co-transfected and values were normalized to its activity. mRNA expression in whole blood was measured by real-time PCR. Protein release in whole blood supernatants was quantified by ELISA. Results: Screening of the IL-8 promoter identified a putative CRE P1320 Immunotherapy with RUTI, a new useful vaccine (TTTCgTCA).