5,9,16,35,36 Once again, a range of cell surface receptors interactions play an important role at this stage. As for DC–T interactions, CD40–CD40L are also important for T–B interactions, as a lack of CD40 expression on B cell prevents activation of B cells by T cells which, in turn, results in decreased Tfh cell numbers.15 In contrast, while CD28 seems to be important at the initial stages of CD4+ T cell activation it

selleck chemicals llc does not seem to be as crucial for Tfh cell development at the later stages of T–B interactions.37,38 A recent study, however, reported that B7.2 expression on B cells was required for GC formation, suggesting the B7–CD28 interactions between T–B cells are important for the function of Tfh cells and the delivery of helper signals to the B cells.39 For the most part, however, another CD28 family member, namely ICOS, seems to be required at this later stage. Consequently, mice in which ICOS–ICOSL interactions are

disrupted, or patients with mutations in ICOS (which results in common variable immunodeficiency), have decreased Tfh cells.26,32,40,41 ICOSL is expressed widely on haematopoietic cells; however, mice that lack ICOSL expression on their B cells show decreased numbers of Tfh cells indicating that, at least in part, this ICOS–ICOSL signal is delivered by B cells.42 This requirement for ICOS signalling seems Selleckchem Cilomilast to depend on its ability to activate phosphoinositide-3-kinase (PI3K), as mice expressing a mutant ICOS molecule with defective PI3K activation41 or lacking the p110δ isoform of PI3K in T cells43 also show decreased Tfh cell generation. Several studies have demonstrated that ICOS signalling, via PI3K, is able to up-regulate Tfh cell-associated genes such as c-maf,

IL-4 and IL-21;40,41,43 however, it remains to be determined whether the primary role of ICOS signalling is to induce the differentiation of Tfh cells or simply to maintain those that have already formed. It Buspirone HCl has also become clear that the SLAM family of surface receptors play an important role in Tfh cell generation. The importance of these molecules in T–B interactions first came to light in patients suffering from the immunodeficiency X-linked lymphoproliferative disease (XLP). XLP is caused by mutations in the gene encoding SAP (i.e. SH2D1A), which is a cytoplasmic adaptor molecule that signals downstream of the SLAM family of receptors. Patients with XLP, as well as gene-targeted mice that lack SAP expression, display a deficiency in T-dependent B cell responses.44,45 Furthermore, several groups have demonstrated that loss of SAP can result in decreased numbers of Tfh cells.9,20,46,47 Members of the SLAM family including SLAM itself, CD84 and NTBA (also known as Ly108 in the mouse) are expressed highly on both activated B cells and activated CD4+ T cells, including Tfh cells.8,9,11,20,47–50 As these receptors are homotypic receptors, this expression pattern allows for SLAM family interactions between T and B cells.

Figure S2. Gating strategy for identification of B cells and monocytes. Doublets were excluded by FSC-H versus FSC-A and viable

cells were selected on the basis of exclusion of live/dead aqua. B cells were identified 3-MA solubility dmso as CD19+SSClow and monocytes as CD14+SSCmid. CD1d expression was determined by MFI of the PE channel and specificity determined by fluorescence minus one with isotype control antibody controls. Figure S3. Example of human CD1d expression on EBV-B cells after transfection. EBV-B cells were identified on the basis of size (FSC vs SSC) and dead cell excluded with the use of a viability dye, human CD1d was detected at the cell surface with a fluorescent antibody. “
“Our objective was to study the alterations of CD4+CD25+Foxp3+ Tregs in HIV-infected SPs and to examine the role of Tregs in the disease progression of HIV. The proportion of CD4+CD25+Foxp3+ Tregs in peripheral blood of 24 SPs, 30 asymptomatic HIV-infected patients, 20 AIDS patients, and 16 non-infected controls was quantified using flow cytometry. HIV Gag peptide mix-induced IFN-γ expression in CD8+ T cells in whole and CD25-depleted PBMCs was examined to evaluate the function of Tregs. The expression of CTLA-4 in Tregs was also detected to measure the suppressive effect of Tregs. HLA-DR and CD38 expression were measured to study the relationship between the frequency of Tregs

and immune activation of HIV-infected patients. The frequency of CD4+CD25+Foxp3+ regulatory T cells in SPs was lower than in asymptomatic HIV-infected patients, AIDS patients, and normal controls (P < 0.05). Tregs in SPs showed lower intracellular CTLA-4 Linsitinib in vivo expression than those of asymptomatic HIV-infected patients and AIDS patients (P < 0.05). The frequency of Tregs significantly correlated with the percentage

of CD38 expression on CD4+ and CD8+ T cells (P < 0.05). Multivariate regression analysis showed that the CD4+ T cell count was the strongest independent factor correlated with the absolute count of Tregs, while viral load had the Farnesyltransferase strongest predictive strength on the proportion of Tregs. We conclude that a lower frequency of Tregs and intracellular CTLA-4 expression of Tregs was one of the characteristics of SPs that may have important clinical impacts for the prediction of the clinical progress of HIV infection. Regulatory T cells play crucial roles in immune regulation and have been reported to suppress effector T cell responses in chronic infections, including retroviral infections (1, 2). Several studies have examined the role of Tregs in HIV pathology, although whether Tregs enhance or inhibit disease progression is still a matter of debate (3–9). Two nonexclusive roles have been attributed to Tregs: a detrimental effect mediated through the impairment of HIV-specific responses, and a beneficial effect through the suppression of chronic immune activation that has been correlated with progression of HIV to AIDS (10).

Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target-gene expression levels were measured by reverse transcription–polymerase chain reaction (RT–PCR) or real-time PCR. The JAK inhibitors CP-690,550 learn more and INCB028050 both suppressed activation of JAK-1/-2/-3 and downstream STAT-1/-3/-5, as well as the expression levels of target proinflammatory genes (MCP-I, SAA1/2) in oncostatin-M (OSM)-stimulated rheumatoid synovial fibroblasts. In contrast, the JAK-3-selective inhibitor, PF-956980, suppressed STAT-1/-5 activation but did not affect

STAT-3 activation in OSM-stimulated rheumatoid synovial fibroblasts. In addition, PF-956980 significantly suppressed MCP-1 gene expression, but did not block SAA1/2 gene expression in OSM-stimulated rheumatoid synovial fibroblasts. These data suggest that

C646 JAK-3-selective inhibition alone is insufficient to control STAT-3-dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK-1/-2, is needed to control the proinflammatory cascade in RA. The Janus kinase (JAK) family of cytoplasmic tyrosine kinases mediates signalling by association with type 1 and type II cytokine receptors [1]. JAK activation leads to activation of their downstream substrates, the signal transducer and activator of transcription Methocarbamol (STAT) proteins, followed by their nuclear translocation and subsequent activation of target genes [2]. Dysfunctional JAK/STAT signalling has been implicated in various haematological and immunological disorders [3] and other pathological inflammatory conditions, such as rheumatoid arthritis (RA) [4]. Because JAKs play an essential role in cellular signalling pathways involved in regulating the immune and inflammatory process [5, 6], targeting of the JAK family members may cause immunosuppression or anti-inflammatory effects [7]. Clarification of the

modification of downstream signalling cascades induced by JAK inhibition is thus important for elucidating the molecular mechanisms whereby JAK inhibitors might exert their beneficial effects against RA. JAK-3 is important in proinflammatory cytokine-mediated signalling [8, 9], which is involved in the pathogenesis of RA. The use of kinase inhibitors with wide-ranging effects on immune/inflammatory mediators may have a more beneficial response than biological agents that target a single cytokine [10, 11]. Small-molecule inhibitors of JAKs are emerging as promising therapies for RA [12]. However, the inhibitory activities responsible for the beneficial effects of these inhibitors against RA are unknown. The JAK-3 inhibitor CP-690,550 has demonstrated efficacy in clinical trials of RA [13-15]. Although CP-690,550 inhibits JAK-3, it also exerts overlapping activities against JAK-1 and JAK-2 [16].

Transformation efficiency was calculated as number of transformants

μL-1 of plasmid DNA (Patwardhan et al., 2008). An API assay identified 44 isolates obtained from urine samples as A. baumannii and three as Acinetobacter lwoffii, while urinary catheter samples yielded three A. baumannii isolates. CSH indices for all the 50 isolates of Acinetobacter obtained from UTI and urinary catheters were determined www.selleckchem.com/products/Staurosporine.html and they varied from 34% to 79.4%. Nine strains had an HI value between 30% and 40%; six isolates displayed HI values between 41% and 50%; for seven isolates the HI values were between 51% and 60%. For the majority of the strains (22), the HI values varied between 61% and 70%. The six strains of A. baumannii (A1, A2, A3, A4, A5 and A6) that showed the highest hydrophobicity indices are listed in Table 1. Six isolates with the lowest HI values (A45–A50) were also selected. Escherichia coli HB101 and P. aeruginosa PA01 were used as the negative and positive control cultures, respectively. The difference between Roxadustat concentration the six strains with the highest HI values and six with the lowest HI values was found to be significantly different with P<0.05. Twenty isolates displayed lectin activity while the remaining 30 did not. A1–A6 produced lectins and A45–A50 did not. Figure 1a shows the HI values of the six strains that produced lectins and displayed the highest HI values (A1, A2, A3, A4, A5 and

A6). These values were compared with those strains that had the lowest HI indices and did not produce lectins (designated as A45, A46, A47, A48, A49 and A50). Standard lectin (phytohemagglutinin) displayed hemagglutination, while normal saline and uninoculated

LB used as negative controls did not show any reaction. The biofilm formation abilities of all the 50 isolates were determined. Quantitative analysis of biofilms formed by A. baumannii on glass and polypropylene surfaces showed that shaking conditions were suitable for biofilm formation. The biofilm formation Sclareol by strains of Acinetobacter with high hydrophobicity (A1–A6) was higher and significant difference was observed compared to strains to low hydrophobicity (A45–A50) with less biofilm-forming ability with P<0.001 (Fig. 1b). Adhesion of A. baumannii on polypropylene was higher than on glass surfaces (Fig. 2). Figure 1 depicts biofilm formation by a representative A. baumannii isolate (A3). The biofilm formation by P. aeruginosa PAO1 was found to be similar to that of A. baumannii, while E. coli was ineffective in forming biofilms on these surfaces (results not shown). Biofilms of six A. baumannii isolates were formed optimally at 30 °C, at pH 7.0 and when supplemented with 5.0 g L−1 NaCl. The results of A. baumannii A3 were shown as a representative isolate. Light microscopic examination of biofilm-forming A. baumannii cells attached to the polycarbonate and glass surfaces were performed and quantified with crystal violet.

2 cells, using protein G columns according to standard protocols. Soluble TNFR1 fusion protein (sTNFR1-Ig) was a kind gift from Geoff Hale (Therapeutic Antibody Group, University of Oxford, UK). All fluorochrome-conjugated anti-mouse mAbs and secondary detection reagents used were purchased from BD Biosciences (Oxford, UK). Biotinylated anti-CD3ζ was from Upstate (Watford, UK), and purified polyclonal rabbit anti-mouse EP1, EP2, EP3 and EP4 were from Cayman Chemicals (Ann Arbor, MI). Bone marrow (BM) Mϕ were generated using a method adapted from Munder et al.21 Briefly, bone marrow cells were resuspended at 5 × 105

cells/ml www.selleckchem.com/products/ipilimumab.html in complete media supplemented with 5% v/v horse serum (Invitrogen), and 50 pg/ml macrophage colony-stimulating factor. The cell suspension was transferred to hydrophobic PTFE-coated tissue culture

bags (supplied by Dr M. Munder, University of Heidelberg, Heidelberg, Germany) and incubated for 8 days at 37° in 5% v/v CO2. Single-cell splenocyte suspensions were generated by grinding spleens through a 70-μm cell strainer (BD Biosciences) with a syringe plunger. When used as APCs, splenocytes were irradiated with 3000 Rads using a caesuim-137 source (Gravatom, Hants, UK). The OT-II CD4+ T cells were prepared by enriching CD4+ cells from single cell suspensions of C57BL/6 OT-II splenocytes, using anti-CD4 microbeads (Miltenyi Biotech, Bisley, UK) according to the manufacturer’s instructions. B cells were prepared from spleens using anti-B220 microbeads (Miltenyi Biotech). AZD1208 nmr Dendritic cells were generated from cultures of bone marrow cells as previously described.22 The 1 × 105 APCs were co-cultured with CD4+ T cells at ratio of 1 : 1 in round-bottom 96-well plates in complete media. The OVA peptide was added at the indicated concentrations. To some cultures the arginine analogue, l-NG-monomethyl arginine, the NO donor S-nitroso-N-acetyl-l,l-penicillamine,

or the cyclo-oxygenase (COX) inhibitor indomethacin (all from Sigma) was added. In some experiments, recombinant IFN-γ (Peprotech, London, UK), or PGE2 (Sigma) was added. Cells were cultured in a humidified ID-8 environment at 37°, 5% v/v CO2. Proliferation was measured by pulsing with 18·5 kBq [3H]thymidine (GE Healthcare, Bucks, UK) per well for the final 8 hr of culture and determining thymidine uptake [measured in counts per minute (c.p.m.)]. Accumulated NO production was measured after 64 hr in culture supernatants using Griess reagent (Sigma) as previously described.23 Production of IFN-γ was assessed using a murine T helper type 1 (Th1)/Th2 Flow cytomix 10plex kit (Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions. Concentration of PGE2 was measured using an enzyme immunoassay competition enzyme-linked immunosorbent assay kit (Caymen Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.

A (64%) and A/J (75%) mice. In this study, we evaluated the in situ localization of IFN-γ in the granulomatous response developed in the omentum of mice infected with P. brasiliensis. Herein,

the immunohistochemical evaluation allowed us to detect the presence of IFN-γ only in cells with lymphomononuclear morphology. Immunostained cells were located mainly at the periphery of the granulomas circumscribing macrophages, epithelioid cells, and giant cells around fungi in the center of the lesions. Other authors also demonstrated the presence of IFN-γ positive cells in cutaneous lesions of human PCM, and it was correlated to well-organized granulomas and maintenance of cellular immune response (Pagliari & Sotto, 2003). At 15 days after infection, the similar presence in both selleck compound number of positive cells and intensity of IFN-γ staining detected in susceptible and resistant mice confirms earlier data that at this

time of infection the genetic background of susceptibility and resistance is not manifested yet (Fazioli et al., 1994). On the other hand, at later phase of infection, resistant mice showed a higher IFN-γ staining in lymphomononuclear cells compared with susceptible mice, suggesting the presence of protective immune mechanisms to the control of fungal dissemination through the high activation status of phagocytes, as previously described (Calich et al., 1994). Therefore, susceptible mice although able to produce IFN-γ since the early stage of the infection, could not control the fungal dissemination, as demonstrated by the several loose granulomas Palbociclib ic50 containing viable fungal cells, indicating their incapacity to control the infection, and confirmed by the higher fungal load in omentum lesions of B10.A than in A/J mice previously observed by the same authors (Nishikaku et al., 2008). Cellular distribution of IFN-γ was similar in mice infected with the slightly virulent isolate Pb265, showing positive immunostaining localized

at the periphery of the lesions in both mouse strains. Quantitative analysis demonstrated that IFN-γ positive cells were observed in both mouse strains at the early phase of Pb265 infection, but differently from the infection with the highly virulent Pb18 in which their positivity was increased; infection with Pb265 was associated with the presence of residual 4-Aminobutyrate aminotransferase lesions with lower number of IFN-γ positive cells, suggesting the inactivation of inflammatory/immune reaction and the resolution of the infection. The presence of IFN-γ has been observed in necrotic processes (Sugawara et al., 1998), whereas TGF-β has been associated with fibrosis in the granulomatous lesions (Wynn, 2004). In previous studies using the murine model of PCM, TNF-α (Nishikaku, 2003), and TGF-β (Nishikaku & Burger, 2003a) immunostaining was detected in macrophages and multinucleated giant cells, as well as in ECM components.

3). CAPRI cell-stimulated cancer cells showed a 40% increase in mean fluorescence intensity (MFI) in HLA class I expression (MFI versus MFI) and a 60% increase

in HLA-DR class II expression (MFI versus MFI) (Fig. 3A). The enhanced MHC class II expression in cancer cells could be pivotal for the 3-MA clinical trial destructive power of CAPRI cells, as CD4 interactions augment cytotoxic T cell responses [34, 35]. Stimulated APC express high levels of MHC class I and class II molecules along with B7 and other costimulatory molecules [36]. We analysed phenotypic markers of CFSE-labelled CD14+ monocytes before activation (day 0) and 1 day (day 1) and 5 days (day 5) after activation (Fig. 4). In CAPRI cells, a considerable number of monocytes lost CD14 expression and matured, as defined by the acquisition of the dendritic cell markers CD1a and Raf inhibitor CD83 at day 1 and their marked upregulation at day 5 (Fig. 4B). Upregulation of the costimulatory molecules CD80, CD86 and CD40, and HLA-DR

class II and HLA class I molecules was also observed (Fig. 4B). In only CD3-activated PBMC, the number of CD14+ monocytes and cells expressing CD83 and CD1 remained constant. Upregulation of the costimulatory molecules CD80, CD86, CD40 and HLA class I and of HLA-DR was clearly lower than in CAPRI cell cultures (Fig. 4C). Quantitative analysis of leucocyte subpopulations in CD3-activated PBMC and CAPRI cells from five patients with cancer showed significantly more matured dendritic cells in CAPRI cultures than in CD3-activated PBMC (paired t-test, P = 0.000096) (Table 1) and

a higher percentage of monocytes in CD3-activated PBMC compared to CAPRI cells on day 5 (paired t-test, P = 0.023) (Table 1). Depletion of subpopulations Histone demethylase and the resulting effect on lysis were analysed at the following time points: 1) in unstimulated PBMC before CD3 activation; 2) in unstimulated PBMC to be added to CD3-activated PBMC; and 3) from CAPRI cells before coculture with cancer cells (Fig. 5). Depletion of CD3+CD8+ T lymphocytes at each time point prevented CAPRI cells from developing any lytic capacity (Fig. 5D), and depletion of CD3+CD4+ T cells had the same effect at each time point (Fig. 5C). Depletion of CD14+ monocytes at time point 1) or 2) completely abrogated the lytic activity of CAPRI cells (Fig. 5A), whereas depletion of monocytes at time point 3) did not significantly influence the lysis of cancer cells. Depletion of CD83+ dendritic cells reduced the development of CAPRI cell lytic efficiency by 50% (Fig. 5B). This ‘medium’ contribution to the lytic capacity of CAPRI cells may indicate a continuous supply of contact information and/or of cytokines to T effector cells during cancer cell destruction. The failure of immune responses as a consequence of rudimentary immunogenic information from cancer cells has been previously demonstrated [32, 33].

5×106 macrophages (approximate ratio 25:1). The interacting cells were incubated for 1 h at 37°C/5% CO2, in the presence or absence of 10% serum, washed, and incubated for 24 h with RPMI medium

and 10% serum. Supernatants were collected at 6 and 24 h. Interaction assays between human DC and iC3b-opsonized apoptotic cells were performed as described 8 using iC3b-opsonized apoptotic thymocytes 12. Non-opsonized interaction between human macrophages and zymosan or LPS (Sigma-Aldrich) was performed without the presence of human serum. Zymosan, at 100 μg/mL, Fluorouracil was added to macrophages for 1 h at 37°C/5% CO2. Macrophages were then washed three times with RPMI. Following the interaction, macrophages were washed three times using ice cold RPMI, followed by incubation for 24 h with RPMI medium, 10% serum. Supernatants were collected at 6 and 24 h. In mixed assays, macrophages or DC were washed three times with RPMI and then exposed for 1 h to either iC3b-opsonized

apoptotic cells or to RPMI. After 1 h, macrophages Selleck C59 wnt were washed three times with RPMI, while DC were not washed; both cell types were then exposed to zymosan 100 μg/mL for 1 h/37°C, and washed three times with RPMI. All macrophages were then incubated in RPMI with 10% human serum for up to 24 h. Supernatants were collected at 6 and 24 h. Interaction index was calculated as described previously 15. Cytokine concentrations were determined for IL-1β, Non-specific serine/threonine protein kinase IL-6, IL-10, and TGF-β using ELISA immunoassays, according to instructions provided with each kit. Data were analyzed using a log/log curve fit option from Microsoft Excel software (Microsoft Corporation, Seattle, WA, USA). In

inhibition assays, anti-IL-10 and anti-TGFβ (R&D Systems) were used. Rabbit polyclonal antibody against human phosphorylated IkB (37 Kd) (R&D Systems) was used to detect protein by immunoblotting. A total of 40×106 freshly isolated macrophages were lysed following the indicated treatments, loaded on 14% SDS-PAGE gel, transferred to a PVDF membrane (Millipore, MA, USA), and blocked with 20% skimmed milk in PBST (PBS*1, 0.05–0.1% Tween 20). The membrane was incubated with primary antibody overnight at 4°C, then washed with TBST and incubated for 30 min with 1:10  000 HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibody. Proteins were visualized with the EZ-ECL detection kit (Beit-Haemek Industries). After interaction with iC3b-opsonized apoptotic cells and or zymosan, DC were fixed with 1% PFA in PBS for 15 min at room temperature and washed twice with PBS containing 10% FBS (PBS-FBS). For microscopy, DC were layered on a microscope slide using a Shandon Cytospin centrifuge (Shandon, Pittsburgh, PA, USA) at 600 rpm for 5 min. Cells were then permeabilized for 45 min with 0.

We think that the affected part seems to be the L region because it inhibits bladder contraction and also elicits the external urethral sphincter activity. It is also possible that both storage centers may be affected. Increased Ganetespib manufacturer late latency times may also derive from suprasegmental dysfunction that can be seen in the elderly

population due to vascular lesions. However all of the patients’ neurological examination was normal and none of the brain MRI scans of the patients reveal pathology. In patients with storage LUTS, the afferent receptors and nerves of the bladder may be activated during the storage phase and, in some individuals, this may result in activation of the M region, leading to involuntary contractions and storage symptoms. However, in normal subjects, there appears to be reciprocal inhibition between the M region and the L region, facilitating either micturition or urine storage.[31] This intense

vesical afferent activity may be inhibited through activation of the L region and might not result in storage symptoms. If this inhibitory effect is delayed because of a disorder in the reticular formation, subjects may encounter storage symptoms, such as an increased response time of the orbicularis occuli muscle to the stimulus of the supraorbital nerve (increased late blink latency time). The major limitation of our study is a lack of assessment of DO Dasatinib using cystometry. Because of invasive examination, cystometry has not been performed. However, there is a close association between storage symptoms and DO in men.[9, 37] Uroflowmetry represents a noninvasive and inexpensive, but indirect, indicator of urinary performance measurements for BOO.[38] In order to eliminate BOO as a factor, patients with peak flows higher than 15 mL/sec were excluded from the storage symptom group. Storage symptoms may result secondary to BOO or changes in urothelial receptor function and to neurotransmitter release or changes in the excitability and coupling of detrusor muscle cells. Another attractive

possibility for explaining storage symptoms might be that they are related to a disorder in the pontine reticular formation, which could also lead to increases in late blink latency times. The nature of this association between the blink reflex and Casein kinase 1 storage symptoms is not clear. There may be a defect in the pontine reticular formation among patients with storage symptoms. This pathology could affect both the blink reflex and the L region, nucleus reticularis pontis oralis and lead to increased late blink latency times and storage symptoms. In order to examine the pontine reticular formation pathology in patients with storage symptoms, studies on other pontine reticular formation-regulated reflexes are needed. The authors have no actual or potential conflict of interest in relation to this article.

albicans infection in humans. WT C57BL/6 mice or mice lacking TLR7 or TLR9 were infected i.v. with a low dose (1 × 104 CFU) of C. albicans, a challenge that was found to be sublethal for WT mice

in preliminary experiments. Survival and morbidity were monitored daily. As shown in Neratinib cell line Figure 7A, most of the mice lacking either TLR7 or TLR9 succumbed to infection while all WT mice survived. To ascertain whether increased lethality was associated with a decreased ability of these mice to control in vivo infection, we measured fungal burden in the kidney, the main target of hematogenous C. albicans dissemination, at 5 days after infection with the same C. albicans dose (1 × 104 CFU) used in the lethality experiments. In these experiments, we also tested MyD88−/−, IRF1−/−, and 3d mice in addition of TLR7−/− and TLR9−/− animals. While low CFU numbers were found in kidneys of WT mice, fungal burden was significantly increased in mice lacking

either TLR7 or TLR9 (Fig. 7B). Notably, fungal burden was even higher in 3d Wnt beta-catenin pathway or IRF1−/− mice compared with TLR7−/− or TLR9−/− mice. Mice lacking MyD88 showed the most severe phenotype of all, with colony counts that were approximately 6 orders of magnitude higher than those of WT controls. Collectively, these data indicated that the TLR7/TLR9/MyD88/IRF1 pathway has a nonredundant role in defenses against C. albicans. Moreover, 3d mice (that are unable to mobilize TLR7/9 and other intracellular TLRs to phagosomes) showed a phenotype

that was similar to that of IRF1−/− mice and intermediary between MyD88−/− (highly susceptible) and TLR9−/− or TLR7−/− (moderately susceptible). Our results, showing an increased susceptibility of TLR9−/− mice to C. albicans infection, were apparently in contrast with those of previous studies showing similar [28, 38] or even decreased [14] susceptibility Dapagliflozin of TLR9−/− mice in comparison with WT animals. We hypothesized that these discrepancies could be related to the fact that the cited studies used a higher (1–2 log) challenge doses than the one we used. Therefore, to test this hypothesis, we challenged TLR7- and TLR9- defective mice with a 20-fold higher C. albicans dose than that previously used in the experiments summarized in Fig. 7. Under these conditions, no differences were found in susceptibility to infection between TLR7-or TLR9-deficient mice and WT controls, as measured by kidney colony counts (Supporting Information Fig. 5). This data indicate that the effects of TLR7 or TLR9 deficiency on the outcome of the infection are critically dependent on the challenge dose. The identification of receptors and signal transduction pathways involved in immune responses to fungi is essential to understand the mechanisms underlying the development of mycoses and to devise alternative strategies to control these difficult to treat infections.