The reason for the efficient Cldn11 induction in BMDM is unclear, although M-CSF, used to generate BMDM, and IL-4 have been shown before to co-regulate certain genes [30]. A summarized gene expression pattern of all adherence and tight junction proteins in macrophages is provided (See summary in Table 2, right columns). Although IL-4 significantly increases the mRNA levels of claudin-1, 2 and 11, this does not result in a detectable

expression of these proteins in macrophages. As a matter of fact, no reports of claudin protein expression in HTS assay macrophages exist up to now, in contrast to related cell types such as LCs and DCs. Possibly, the claudin protein expression levels in macrophages are under the detection limit of the antibodies currently used. Alternatively, we cannot exclude that post-transcriptional, such as poor

mRNA stability, and/or post-translational regulatory mechanisms preclude high claudin levels in macrophages. For example, during epithelial reorganization, claudins are ubiquitylated and undergo degradation in the lysosomes [31]. A similar mechanism might be at play in macrophages, especially if the claudins are not engaged in TJ formation. In this respect, one could imagine that claudin proteins are stabilized in vivo when intimate interactions between macrophages and epithelial cells are formed. This could help to bring macrophages in close contact with epithelial cells or with other macrophages, a phenomenon that could be relevant in several situations: (1) in tumours where Sirolimus mw fusion between macrophages and carcinoma cells might occur [32], (2) during wound healing where macrophages have to integrate in the epithelial sheet of the skin [33] and (3) during granuloma formation and the foreign body reaction where close contacts between macrophages have to be initiated to promote their fusion [29]. Interestingly, Lenzi et al. [34] reported the expression of cadherins and the tight junction–associated protein occludin during the PTK6 process of granuloma closure. Yet, the lack of claudin proteins in our assays with IL-4-treated macrophages does not preclude their use as marker genes. Indeed, the macrophage activation status in a given pathological

condition is often evaluated by the detection of M1 versus M2 signature genes [4, 25, 26, 35]. Testing different M2 activators identified TGF-β as the most potent inducer of Cldn1 gene expression in macrophages. This finding is reminiscent of TGF-β’s central role in upregulating claudin-1 expression during IL-4-/GM-CSF-treated bone marrow cultures, ultimately giving rise to Langerhans cells [18]. The association of claudin-1 mRNA with the M2 activation status was further confirmed in vivo where high levels of Cldn1 induction were observed in TAM subsets from two mammary carcinoma models and in splenic macrophages isolated from the chronic infection stage of T. congolense infections. In both models, the implication of TGF-β seems plausible.

fumigatus infection, which suggests that IFN-β is a possible adjuvant to elicit an appropriate Th reactivity to A. fumigatus. Dendritic cells were prepared as previously described.9 CD14+ monocytes were cultured with 25 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Levallois Perret, France) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN) for 5 days. On day 5, about 90% of the cells express CD1a+ and 95% express

CD14−. The DCs were starved from IL-4 and GM-CSF for 20 hr before infection or treatments. Monoclonal antibodies specific for CD1a, CD14, CD38, CD40, CD83, CD86, HLA-DR, CD3 and CD4 as well as immunoglobulin G1 (IgG1), IgG2a Caspase inhibitor and IgG2b (BD Bioscience PharMingen, San Diego, CA) were

used as direct conjugates to fluorescein isothiocyanate (FITC) or phycoerythrin. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich, St Louis, MO) was used at a concentration of 100 ng/ml to stimulate DC maturation and IFN-β expression. The IFN-β (Avonex®; Biogen Inc., Cambridge, MA) was used at 200 pm. A wild-type clinical isolate of A. fumigatus (CBS 144 89) was grown on Sabouraud–chloramphenicol agar for 3 days, at 37°, as previously described.23 Preparations of A. fumigatus were analysed for LPS contamination by the Limulus lysate assay (Biowhittaker, Verviers, Belgium) and were found to contain less than selleck compound 10 pg/ml LPS. In all experiments, DCs were infected with live A. fumigatus conidia at a 1 : 1 ratio. Amphotericin B (0·75 μg/ml; Sigma-Aldrich) was added to the cell

cultures to prevent fungal overgrowth 6 hr after infection when the internalization of A. fumigatus conidia was completed.9 For the adherence assay, A. fumigatus conidia were incubated with FITC at a final concentration of 3 mg/ml overnight at 4°, and then washed extensively with PBS. After a 6-hr incubation with FITC-labelled oxyclozanide conidia (ratio 1 : 1), DCs were washed and the adherence was measured by flow cytometric analysis. The cells were incubated with purified monoclonal antibodies at 4° for 30 min. After washing, the cells were fixed with 2% paraformaldehyde before analysis on a FACScan using the cellquest software (BD Bioscience PharMingen). A total of 5000 cells were analysed per sample. RNA extraction, reverse transcription (RT) and real-time RT-polymerase chain reaction (PCR) assays were performed as previously described.24 Sequences of the primer pairs used for glyceraldehyde 3-phosphate dehydrogenase (GaPDH), IFN-β, IL-12p35, IL-23p19 and IL-27p28 quantification were previously described.24 Cytokine concentration in filtered supernatants was evaluated with the human inflammation cytometric bead array (CBA) [for IL-12p70, IL-10, tumour necrosis factor-α (TNF-α) and IL-6: BD Bioscience PharMingen] and enzyme-linked immunsorbent assay (ELISA; for IFN-β: PBL Biomedical Laboratories, Piscataway, NJ; for IL-23: eBioscience, San Diego, CA).

Future directions in this field will also be discussed. MiRNAs were first found in the nematode Caenorhabditis elegans in 1993.1 Since then they have also been described widely in plants and mammals.2 MiRNAs are first transcribed in the nucleus as stem-loop primary miRNA, which are then cleaved into shorter precursor miRNA by Drosha, an RNase III, and its essential Selleck Rapamycin cofactor called DGCR8 (DiGeorge syndrome critical region 8), a double-stranded RNA-binding protein (Fig. 1).3–6 The precursor miRNAs are transported out of the nucleus via Exportin-5 and once in the cytosol are cleaved into their mature form of 20–22 nucleotides by Dicer, another

RNase III.7,8 After cleavage, the miRNA duplex is unwound and the functional strand is loaded onto the RNA-induced silencing complex (RISC) and functions as its guide.9 The mature miRNA guides the RISC complex to a (near) complementary sequence, usually in the 3′ untranslated region (UTR), of a target messenger RNA (mRNA).9 Upon binding, the RISC causes post-transcriptional gene silencing by

either cleaving the target mRNA or by inhibiting its translation, XAV 939 so that miRNAs are usually negative regulators of gene expression.10 In addition to their role in such post-transcriptional repression, miRNAs have now been implicated in transcriptional gene silencing by targeting the promoter region but have also been reported to have a positive effect on transcription.11–13 Each miRNA can potentially regulate the translation

of a large number of different mRNA and each mRNA can check details possess multiple binding sites for a single or for many different miRNA because the specificity of miRNA is mainly determined by Watson-Crick base pairing at the 5′ region of the miRNA. Estimates have suggested that the total number of different miRNA sequences in humans may exceed 1000.14 Computational analysis also predicts that over 60% of human genes are potential targets of miRNAs and that there are a large number of other non-coding RNAs of greater nucleotide length than microRNA, which are also likely to have important functions.15 However, direct experimental evidence defining mRNA targets of miRNA regulation has been reported for only a small number of miRNAs and target mRNAs. Assaying the levels of specific microRNA sequences was initially cumbersome; however, advances in technology now allow detection with a sensitivity and specificity that can enable monitoring in a clinical setting. Originally, RNA blot analyses provided both quantitative and qualitative information about the various forms of a miRNA within a total RNA sample.1,16 As the number of miRNAs in the miRBase registry17 has increased, microarray technology has been adapted to enable the parallel screening of thousands of miRNAs in one sample.18 More recently, real time reverse transcription-polymerase chain reaction has been adapted to enable relative quantification and quantitative analysis of miRNA levels.

To assess the localization of the cytoskeletal protein paxillin, we applied DqDF to DiO-stained neutrophils

of mice expressing an mCherry–paxillin fusion protein. Results:  The footprint topographies obtained from DiO and DiI in the plasma membrane were identical. The z-coordinates of the microvilli tips obtained with the two fluorochromes in the footprint were also identical. Paxillin was found to be localized to some, but not all ridges in the neutrophil footprint. Conclusions:  Our data suggest that the spectral properties of the fluorochrome do not affect the results. DqDF will be useful for simultaneous visualization of two fluorochromes in the footprint of rolling cells. “
“Please cite this paper as: Tajbakhsh N, Sokoya EM. Regulation of cerebral vascular function by sirtuin 1. Microcirculation 19: 336–342, 2012. Objective:  Endothelial dysfunction, associated with reduced nitric oxide bioavailability selleck screening library and oxidative stress, is a common feature of vascular-related diseases. Sirtuin 1 (SIRT1)

is a protein deacetylase that has been shown to target endothelial nitric oxide synthase in large arteries and is protective during oxidative stress. However, within resistance-sized vessels, the expression and functional effects of SIRT1 remain unknown. Methods:  Immunoblotting and immunohistochemistry were used to determine SIRT1 expression and localization in cultured brain endothelial cells and intact rat middle cerebral artery. The influence of SIRT1 on vascular

function Rapamycin in vitro was then studied in intact middle selleck kinase inhibitor cerebral arteries using pressure myography. Results:  We report for the first time that SIRT1 is expressed in the resistance-sized vessels in the brain and is present in both the endothelium and smooth muscle. Pharmacological inhibition of SIRT1 demonstrated reduced endothelium-dependent dilation mediated by nitric oxide. However, endothelium-independent dilations were comparable in the presence and absence of SIRT1 block. Conclusions:  Our results support a role for SIRT1 in endothelium-dependent relaxation in the cerebral vasculature and reveal a potential for SIRT1 as a therapeutic target in vascular-related diseases by restoring endothelial function. “
“Please cite this paper as: Markiewicz, Nakerakanti, Kapanadze, Ghatnekar and Trojanowska (2011). Connective Tissue Growth Factor (CTGF/CCN2) Mediates Angiogenic Effect of S1P in Human Dermal Microvascular Endothelial Cells. Microcirculation18(1), 1–11. Objective:  The primary objective of this study was to examine the potential interaction between S1P, a pleiotropic lipid mediator, and CTGF/CCN2, a secreted multimodular protein, in the process of endothelial cell migration. The secondary objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have a specific function in cell migration.

There were no significant MI-503 research buy differences among 0–24-hr hypoxia in control groups (n= 20) for all the measured cytokines. As shown in Table 1, 6-hr hypoxia evoked an obvious elevation of IL-17A (mean 7.10 pg/mL, n= 20), IL-1β (mean 37.00 pg/mL, n= 20) and IL-23 (mean 377.49 pg/mL, n= 20) from PBMC in

chronic stage SCI patient groups, while 24-hr hypoxia induced a slightly decreased release of IL-17A (mean 5.74 pg/mL, n= 20). On the contrary, no obvious elevation of IFN-γ (mean 11.81 pg/mL, n= 20) was detected in SCI patients’ PBMC culture supernatants under hypoxia exposure (Table 2). This study provides evidence that hypoxia might induce immunological response by upregulating Th17 ratio and IL-17A expression in severe RXDX-106 purchase cerebral infarction patients during the chronic stage. Previous studies have found increased peripheral blood IL-17A mRNA levels in acute cerebral infarction patients (7, 19). However, it was difficult to demonstrate in vivo whether the IL-17A upregulation was induced by hypoxia but not by

other potential stimuli. It has been demonstrated that hypoxia could upregulate the expression and function of pro-inflammatory cytokines and inhibitors of these cytokines might prevent related neurotoxicity in ischemic stroke rodent models (20–23). But to our knowledge, the hypoxia induced Th17 participating pathogenesis of brain ischemic injury has not been reported. The results of this study indicate that the primary event following hypoxia treatment of cultured patients’ PBMC involves Th17 upregulation, accompanied by increased IL-17A expression and release. Previous studies have demonstrated Th17 and IL-17A are essential for the expression of pro-inflammatory cytokines triggered by transcription factor nuclear factor-κB in multiple those sclerosis (24). Our data revealed that only the

patients but not healthy volunteers’ PBMC responded significantly higher to hypoxia exposure for IL-17A expression as well as Th17 upregulation in vitro, suggesting that local ischemic brain lesions might already contribute to PBMC differentiation toward Th17 direction during the acute stage in vivo and the activated PBMC obtained during the chronic stage of ischemic stroke might be more allergic to hypoxia stimulation compared to normal control groups. How do ischemic neural cells in the central nervous system (CNS) affect Th17 upregulation in vivo? Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, TGF-β and IL-23 produced in the CNS may enter the peripheral blood and upregulate Th17 in PBMC. Alternatively, peripheral blood T cells and monocytes/macrophages may enter the CNS by means of chemokines induced in the ischemic brain and be activated, and then return to the peripheral blood. Previous studies have revealed that activated monocytes/macrophages played an important pathogenic role in hypoxic and ischemic brains (25–27).

SAs bind the complex from the exterior in an unspecific manner, as compared to conventional specific TCR antigen binding. As a result, https://www.selleckchem.com/products/azd5363.html SAs produce undifferentiated, exaggerated activation of T lymphocytes, which generates increased production of cytokines. If SAs escape into the blood, the serum concentrations of TNF-α, IL-2 and IFN-γ produced by circulating lymphocytes rapidly reach toxic levels, which can cause death by toxic shock (9). SAs activity is evaluated by measuring P50 (h),

the concentration which activates half of the human T cells. SEA has the lowest P50 (h) (0.1 pg/ml) of all SEs (10). SEs are coded by plasmids, transposomes, prophages, and pathogenicity islands. They have a complex structure, with two important domains: one responsible for digestive toxicity and another for superantigenic activity (11). So far, it is not clear whether these two functions can be separated (12). Apart from its effects in food-borne toxic shock, the impact of SEA on the function of the enteric immune system is connected with the immunological characteristics of the digestive tract. The intestine has an estimated mucous surface of 300 square meters and processes annually 30 kg of proteins. Daily absorption

of 130–190 g of peptides occurs; these have not only a nutritive role, JAK inhibitor but also an antigenic function (13). There are approximately 1000 billion bacteria which stimulate local immunity per gram of

feces, and as many lymphocytes per meter of intestine (14). Thus, there is more lymphoid tissue in the whole digestive tract than in the whole of the rest of the human body (15). This lymphoid tissue is distributed between the intestinal epithelium and the lamina propria, the sub-epithelial connective tissue of the mucosa. In the epithelial layer, lymphocytes are located in the spaces between the latero-basal sides of normal enterocytes. It is estimated that there are 20 intraepithelial lymphocytes for every 100 enterocytes (13). In the lamina propria, the lymphoid tissue is organized in the form of solitary lymph nodes or Pazopanib order classical Peyer’s patches, which are veritable secondary lymphoid organs. IELs are relatively difficult to classify according to the classical criteria used for T cells. The majority of IELs express αEβ7-integrin (which binds the E-cadherin expressed on enterocytes) and belong to the CD8+ type; however the CD8 molecule is heterodimeric, as is true in the general circulation, in only 50% of cases (16). Some of the homodimeric CD8+ IELs are autoreactive, and these are functionally more similar to γδTCR T cells than to αβTCR T cells (17). Likewise, some of the CD8+ IELs with αα-homodimeric CD8 are MHC-II restricted, and not MHC-I restricted (18). IELs are the result of intestinal migration of lymphocytes, which begins in the neonatal period, sometimes after antigenic stimulation in secondary lymphoid organs.

This “outside-in” signaling pathway requires ITAM signals from DAP12 and FcRγ, and also involves early effectors such as the Src family kinases and Syk in neutrophils and macrophages [14, 15]. Because β2 integrins signal through

ITAM adapters in myeloid cells, we hypothesized that β2 integrin signaling may also inhibit TLR responses. There have been conflicting reports in the literature regarding the influence of β2 integrin signaling on TLRs, with some studies demonstrating that β2 integrins can promote TLR-induced inflammation [16-18], whereas others have reported negative roles for these integrins in TLR responses [19, 20]. Therefore, the nature in which β2 integrins interface with TLR activation and cytokine secretion is complex selleck compound and unclear.

To better define the contribution of β2 integrins to regulation of TLR signaling, we have examined inflammatory responses in the absence of all β2 integrins. Here we demonstrate that deletion of all β2 integrins rendered myeloid cells hypersensitive to TLR stimulation in vitro and in vivo, showing an inhibitory role for β2 integrins in TLR responses. Furthermore, https://www.selleckchem.com/products/ink128.html we examined potential direct and indirect mechanisms by which β2 integrins caused this inhibition, and found that β2 integrins have a direct effect on IκBα degradation that was pronounced in β2 integrin-deficient cells through both early and late phases of TLR stimulation, thus implicating β2 integrin signals in inhibiting NF-κB pathway activation to calibrate inflammatory responses. The four β2 integrins, LFA-1 (lymphocyte function-associated antigen 1, αLβ2), Mac-1 (macrophage-1 antigen, αMβ2), CR4 (αXβ2), and CD11d-CD18 (αDβ2) are heterodimers that consist of distinct CD11 alpha subunits in association with the common

beta chain, CD18 (β2), which is encoded by the Itgb2 gene [21]. To examine whether β2 integrin signaling regulates TLR responses, we compared the cytokine secretion profiles of bone marrow-derived (BM-derived) macrophages from wild-type Erastin cell line (WT) and Itgb2−/− mice, which are deficient in CD18 and thus are unable to express any of the β2 integrins on the cell surface (Supporting Information Fig. 1A) [22]. Despite the inability of Itgb2−/− BM-derived macrophages to express Mac-1, these cells exhibited surface F4/80 expression and upregulated MHC II in response to IFN-γ treatment (Supporting Information Fig. 1A and B), demonstrating that they were bona fide macrophages. Furthermore, β2 integrin-deficient macrophages exhibited similar or slightly lower levels of cell surface TLR2, TLR4, and Dectin-1 protein and TLR9 mRNA (Supporting Information Fig. 1C and D). To determine how β2 integrin signals influence TLR activity, we stimulated Itgb2−/− BM-derived macrophages with a panel of TLR agonists, including LPS (TLR4), CpG B DNA (TLR9), and zymosan (TLR2).

Target cells were labeled with Na251CrO4 (Hartmann,

Analytik, Braunschweig, Germany) for 1.5 h at 37°C, washed, and added at a concentration of 1×105 cells/well resulting in the indicated effector/target ratios. To study the underlying mechanisms of NK cell induced tumor cell death, neutralizing anti-FasL (BD Pharmingen), anti-TRAIL (BioVender), or isotype control antibody was added to the co-culture system. To inhibit perforin-mediated cytolysis, CMA (Sigma-Aldrich, Taufkirchen, Germany) was added to the NK cells 2 h prior to co-culture with target cells. The radioactive content of the supernatant was measured in a gamma counter (Berthold, Wildbad, Germany). Specific lysis was determined according to the following formula: specific lysis (%)=100×(Exp−Spo)/(Max−Spo), where Exp is the experimental release, Spo is the spontaneous release, and Max is the maximum release. Assays were Nivolumab in vivo performed as triplicates/quadruplicates, and data are depicted as means±standard deviation (SD). The experimental design of the Treg cell-NK co-culture experiments is illustrated in the Supporting Information Fig. S1. Student’s t-test for means (two-tailed, paired samples) from at least three individual experiments was used to calculate significance, and p-values equal or below 0.05 were considered as significant. We thank Kirsten Bruderek for her excellent

technical assistance. We also thank Johannes Schulte for his help with the chromium release assays. Antibodies directed against ULBP1, ULBP2, ULBP3, MICA, and MICB were a kind gift from Annette Tyrosine Kinase Inhibitor Library mouse Paschen (UK Essen). Research described in this article was supported in part by the IFORES program

of the Medical Faculty, University Duisburg-Essen (to S. B.) and the Deutsche Forschungsgemeinschaft (DFG 4190/1-1 to C. B.). Conflict of interest: The authors have declared no conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. Celecoxib They are made available as submitted by the authors. “
“Cyclooxygenase-2 is a promising target for cancer immunotherapy. Here, we designed the analogues p321-9L and p321-1Y9L (YLIGETIKL) from cyclooxygenase-2-derived native peptide p321. Then, we tested the binding affinity and stability of the analogues and their ability to elicit specific immune response both in vitro (from PBMCs of HLA-A*02+ healthy donors) and in vivo (from HLA-A2.1/Kb transgenic mice). Our results indicated that the activity of cytotoxic T lymphocytes induced by p321-9L and p321-1Y9L was more potent than that of p321. In conclusion, the epitope analogue, especially p321-1Y9L, may be a good candidate which could be used to the immunotherapy of patients with tumours expressing cyclooxygenase-2. Cytotoxic T lymphocytes (CTLs) specific for various tumour antigens play an important role in elimination of tumour cells [1, 2].

[12], namely the HLA-DQB1*02:02 subtype, an eventual allele for ABPA–CF susceptibility and HLA-DQB1*02:01, a possible allele of ABPA–CF protection. The difference between DQB1*02:01 and DQB1*02:02 is in exon 3 (amino acid 135). The DQB1*02:01 allele is genetically linked to DQA1*05:01 and has classically been associated with celiac disease, Type 1 diabetes and other autoimmune diseases. However, DQB1*02:02 is linked to several DQA1 alleles, namely DQA1*02:01 and DQA1*03:03. Thus, in future studies we will investigate other HLA genes to clarify other possible associations. In addition, because ABPA is an uncommon complication of CF, it will also be important to further investigate and corroborate

these interesting findings with a larger number Selleck GDC973 of patients in the future. We found no differences between the groups used as comparison controls, which consolidates our findings. Our findings allow us to both corroborate and rule out partnerships with primary genetic pathology in patients with CF. With regard to patients with asthma, they allow us to discard possible associations with other allergic pulmonary pathology and, by making comparisons with healthy subjects, to determine general population frequencies. NVP-LDE225 In this context, several reports have shown that a strong Th2 response to A. fumigatus antigens, as indicated by prominent eosinophil infiltration, could be responsible for development of ABPA [21, 22].

Thus, it is possible that particular HLA class II alleles play critical roles in the outcome of T-cell responses (Th1 vs Th2) to A. fumigatus antigens. Thus, patients with CF but without ABPA who Astemizole lack permissive alleles possibly have Th1 type responses against the fungus A. fumigates, which would prevent colonization of the lung and development of ABPA. The opposite situation would occur in patients with ABPA–CF and susceptibility alleles; they mount a Th2 type response [11, 15]. In this context, other authors have also demonstrated that altered T cell receptor-mediated signals can lead to altered T lymphocyte phenotypes [23]. This

does not mean that a susceptibility allele alone can cause ABPA; however, these alleles could influence the outcome of exposure to A. fumigatus. In conclusion, these data corroborate previous studies showing correlations between HLA-DRB1*15:01, –DRB1*11:01, –DRB1*11:04, –DRB1*07:01, –DRB1*04 alleles, and ABPA–CF susceptibility. Indeed, our data show that HLA-DQB1*02:01 is a possible ABPA–CF resistance allele. This work was possible in part thank to technical support from projects from Fondo de Investigación Sanitaria (FIS) (PI11/02686) (CIBERehd) funded by the Instituto de Salud Carlos III, Spain and Seneca Foundation No. 04487/GERM/O6 y CajaMurcia. None of the authors has a conflict of interest to disclose. We confirm that we have read the journal’s position on issues involved in ethical publication and we affirm that this report is consistent with those guidelines.

On the other hand, when IL-1β is highly produced by host cells after Borrelia recognition, high levels of Th17 cells may be produced. Borrelia-primed Th17 cells might facilitate development of a chronic stage of Lyme disease, as already described in other diseases,

such as RA 41. At this moment, it is still unknown which specific T-cell population is responsible for the induction of IL-17 (CD4+,γδT cells, NK T cells, CD4−/CD8). One of our future plans is to detect which specific T-cell population is responsible for the induction of selleck chemicals IL-17 by Borrelia spp. In summary, Borrelia is a strong inducer of inflammasome activation and caspase-1-mediated IL-1β induction amplifies the production of IL-17 after Borrelia exposure. The Borrelia-induced IL-17 production is modulated by the IL-18-driven IFN-γ. These data indicate that caspase-1-dependent cytokines IL-1β AZD6244 cost and IL-18 determine the development and clinical outcome of Lyme disease, which was also demonstrated by our in vivo data. These findings give more insight into the pathogenesis of Lyme disease

and may provide useful information for the development of new therapeutic strategies targeting the inflammasome. B. burgdorferi pKo strain and B. afzelii, patient isolate were cultured at 33°C in Barbour-Stoenner-Kelley -H medium (Sigma-Aldrich) supplemented with 6% rabbit serum. Spirochetes were grown to late-logarithmic phase and examined for motility by dark-field microscopy. Organisms were quantitated by fluorescence microscopy after mixing 10 μL aliquots of the culture material with 10 μL of an acridine orange solution to concentrations. Bacteria were harvested by centrifugation of the culture at 7000×g for 15 min, washed twice with sterile PBS (pH 7.4), and diluted in the specified medium to required concentrations of 1–3×106 spirochetes per mL. Heat-killed B. burgdorferi and B. afzelii were prepared by heating at 52°C for 30 min before dilution. Heat-inactivated bacteria

were used according to Wang et al. 6. C57BL/6 and Balb/c mice were obtained from Charles River Wiga (Sulzfeld, Germany). IL-1β gene-deficient mice were kindly Thiamet G provided by J. Mudgett, Merck (Rahway, NJ, USA). Caspase-1-deficient mice were originally obtained from R. A. Flavell, New Haven, CT, USA and generation of these mice was previously described 49, 50. The generation of IL-18 knockout mice was previously described 51. Male WT and knockout mice between 6 and 8 wk of age were used. The mice were fed with sterilized laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. The experiments were approved by the Ethics Committee on Animal Experiments of the Radboud University, Nijmegen. Bone marrow from mice (age between 8 and 20 wks) was flushed out after dissecting mouse legs.