In particular, a threshold for the minimal area Am of macrophages on the red layer (split point 3 in Fig. 1) BAY 73-4506 and thresholds for the minimal areas As and Acs of single spores and clustered spores, respectively, were used on the green layer (split points 4–6 in Fig. 1). We used different thresholds for single spores and clustered spores, Acs < As, because largely overlapping fluorescence signals in the images appear for spores that are lying close together in clusters. Furthermore, to distinguish spores from artifacts in the images, thresholds for object roundness and object asymmetry were used in addition to the area feature (split

points 5 and 6 in Fig. 1). Here, object roundness was evaluated by approximating the ROI by an outer and inner ellipse and selleck compound by computing the difference σ between the major axis of the outer ellipse and the minor axis of the inner

ellipse.[16] In contrast, the object asymmetry was computed from the ratio of the main axes rmax and rmin of an ellipse that was fitted to the ROI as α = 1 − rmin/rmax. Here, we distinguished again between thresholds for the roundness of single spores σss and clustered spores σcs, and similar for the asymmetry of single spores with threshold αss. We modified the implemented algorithm[16] to deal with the current image data by dividing the segmentation into two sub-steps. Here, we first computed for each image an intensity threshold automatically and then applied the multi-threshold segmentation algorithm. With regard to the size of the spores (see split points 4–6 in Fig. 1), we enforced only a lower but not

an upper threshold and by that enhanced the probability of detecting all spores to ensure that the number of missed spores was minimal, i.e. we were opting for a high recall. However, since this segmentation sub-step did not distinguish Calpain between ROIs that are single spores or clustered spores, a second segmentation sub-step was required where clusters of spores were split into single spores based on the features roundness and asymmetry. The ruleset distinguishes between phagocytosed and non-phagocytosed spores being adherent and non-adherent to macrophages (split point 7 and 8 in Fig. 1). The decision of the class memberships for spores was made on the blue layer, because due to the staining only adherent and non-adherent spores that were not phagocytosed appear in blue. ROIs are classified as spores or artifacts in the images depending on their average intensity I relative to the threshold value Is in the range of integer values between 0 and 255. We optimised the value of Is (see Table 1) by a validation procedure involving a manual classification on selected images. Finally, non-phagocytosed spores were classified as adherent or non-adherent to macrophages (split point 8 in Fig. 1) depending on whether or not they share a border with macrophages on the red layer.

Together, these data suggest a novel mechanism of immunosuppression by dexamethasone. To induce immune synapse formation, untransformed resting human peripheral blood (PB) T cells were incubated with superantigen (Staphylococcus aureus enterotoxin B, SEB) loaded APCs. The immune synapse formation was analyzed using multispectral imaging flow cytometry (MIFC), which combines fluorescence find more microscopy and flow cytometry. MIFC allows the spatial quantification of fluorescence signals within T cells by defining regions of interest for the measurement (Supporting Information Fig. 1). T-cell/APC couples were identified by gating on cell clusters

according to DNA content (Hoechst33342 staining) and CD3 expression (Fig. 1A, blue gate). T-cell/T-cell couples (Fig. 1A, green gate) or cell clusters that contained more than one T cell or APC (Fig 1A, black gate) were eliminated from further analysis. Then, the accumulation of the TCR/CD3 complex and LFA-1 in the T-cell/APC contact zone was used as measure for immune synapse formation. As expected, in the absence of superantigen most T cells did not show an enrichment of TCR/CD3 and LFA-1

in the contact zone (Fig. 1B, left side). selleck chemicals llc In the presence of SEB, however, T cells showed a clear formation of an immune synapse (Fig. 1B, right part). To quantify the number of T cells with an immune synapse, we acquired up to 25 000 T cells. Figure 1C shows the frequency of primary human T cells that showed an enrichment of TCR/CD3 and LFA-1 in the contact zone from 19 different donors. The mean number of

T cells with an immune synapse increased significantly in the presence of SEB. It is important to note that the variations of T cells forming immune synapses were relatively high between different donors, ranging from 0.2 to 1.5% (Fig. 1C). We therefore compared the values from experiments that were performed in triplicates to evaluate the variance in dependent samples (Fig. 1D). The mean standard deviation of the triplicates Meloxicam (intratest SD) was 7.5 per 10 000 T cells. Taken the high variations between different donors (Fig. 1C) and the low variations of the triplicates (Fig. 1D) into account, we decided to normalize the following experiments by setting the numbers of T cells of one individual with synapses in the absence of dexamethasone as 1. To analyze the effects of glucocorticoids on the formation of an immune synapse in untransformed human T cells, PB T cells were preincubated with the glucocorticoid dexamethasone (5 μM). This concentration inhibited blast formation and cell-cycle entry without having any toxic effects (Supporting Information Fig. 2). Interestingly, in dexamethasone pretreated T cells, an inhibition of TCR/CD3 and LFA-1 accumulation and thus immune synapse formation could be observed (Fig. 2A and B). The reduced maturation of the immune synapse was due to a combined failure of LFA-1 (Fig. 2C) and CD3 (Fig.

The phenothiaziniums are known to localise in the plasma membrane

of yeast.[29] Consequently, this is the cellular structure primarily damaged upon illumination and it has been proposed that the increased permeability resulting from such damage is the reason for cell death.[29] The fungicidal effect of MB has been demonstrated on various species of the Candida genus (C. albicans, C. dubliniensis, C. krusei and C. tropicalis) [30] and that of NMB on C. albicans, both in vitro and in an in vivo mouse model with infected abrasion wounds.[11] The concentration of DMMB needed to photoinactivate C. albicans (2.5–5 μmol l−1) was much lower than that for NMB (20 μmol l−1), which in turn was significantly lower than S1P Receptor inhibitor that for toluidine blue O or MB.[11] Nevertheless, our results are not completely comparable because their fluence was lower (9.75 J cm−2) than the one used in our experiments (18 and 37 J cm−2). The ROS-quenchers study revealed a different pattern of ROS contributing to the fungicidal effect of HYP and DMMB PDT. Previous studies have shown that hydrogen peroxide may be the most important ROS involved in the photoinactivation of C. albicans by HYP[31] and this agrees with the findings of this study. The involvement of hydrogen peroxide in the PDT-mediated

fungal killing could be confirmed by studies that examined the killing of Candida cells by addition of concentrations of H2O2 similar to those likely to be generated during PDT. Hydrogen peroxide generation has been reported within an hour of HYP photosensitisation followed by glutation depletion.[32] A signalling role of hydrogen CH5424802 supplier peroxide in C. albicans has been firmly established, in fact higher concentrations of hydrogen peroxide can induce programmed cell death.[33] Likewise, Price et al. [34] have demonstrated that hydrogen peroxide is a very important factor in the pro-apoptotic response to PDT, being determinant in the photokilling process. In contrast, our results point to singlet oxygen as the PJ34 HCl main cytotoxic species for DMMB, in agreement with the results found for the photobactericidal activity of the phenothiaziniums.[16]

Finally, we were unable to find significant differences in the ROS pattern among azole-resistant and susceptible C. albicans strains. This study demonstrates that aPDT is effective in eliminating in vitro C. albicans strains independent of their azole resistance pattern, even using PSs with different mechanisms of action, such as HYP and DMMB. However, there are subtle differences between them: HYP is more efficient at low yeast density whereas DMMB performs better at high density; HYP has less dark cytotoxicity than DMMB and its effect is less dependent on the type of C. albicans strain. This study was supported by grant no. PI1120/09 and Research Groups B65 and B85 from the Department of Science, Technology and University of the Government of Aragón.

Moreover, the feasibility of macrophage therapy has recently been

demonstrated in two renal transplant recipients,[124] where regulatory macrophages (IFN-γ-stimulated) were administered via central venous infusion several days prior to donor transplantation. Both patients underwent a rapid reduction in immunosuppressive therapy and maintained stable graft function during the 3-year follow-up period. These findings have now prompted The One Study, a multinational clinical trial for the use of regulatory macrophages as a potential immune-conditioning therapy in renal transplantation (see http://www.onestudy.org). As this review highlights, more needs NVP-BGJ398 to be understood in terms of macrophage phenotype and function in humans, and the processes that control their activation during the various stages of acute and chronic disease progression. A greater understanding of these different states of activation may result in the development of therapies specifically designed to capitalize

on this variation in phenotype and cellular responses. selleckchem “
“Oxidative stress plays an important role in the progression of renal interstitial fibrosis. The nicotinamide adeninedinucleotide phosphate (NADPH) oxidase (Nox) family is considered one of the major sources of reactive oxygen species (ROS). In the present study, we investigated the inhibitory effects of a novel anti-fibrotic

agent, Fluorofenidone (AKF-PD), upon Nox-mediated oxidative stress and deposition of extracellular matrix (ECM) in the development of renalinterstitial fibrosis. AKF-PD was used to treat renal fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of Nox homologues, p-Akt, collagen I and III were detected by immunoblotting or immunohistochemistry. Levels of 8-iso prostaglandin F2alpha (8-Iso PGF2a) was measured by enzyme linked immunosorbent assay. In addition, ROS and the expression of collagen I (1a), Nox subunits and p-Akt was measured in angiotensin (Ang) II-stimulated Rolziracetam rat proximal tubular epithelial (NRK-52E) cells in culture. AKF-PD treatment significantly attenuated tubulo-interstitial injury, ECM deposition and oxidative stress in fibrotic rat kidneys. In addition, AKF-PD inhibited the expression of ROS, Collagen I (1a), Nox2, p-Akt in Ang II-stimulated NRK-52E cells. AKF-PD attenuates the progression of renal interstitial fibrosis partly by suppressing NADPH oxidase and ECM deposition via the PI3K/Akt signalling pathway, suggesting AKF-PD is a potential novel therapeutic agent against renal fibrosis. “
“Renal transplant recipients are at risk of developing Pneumocystis pneumonia (PcP), especially in the first 2 years after transplantation, with a mortality rate of up to 50%.

Pra1 is an important multifunctional fungal immune evasion protein [[15]]. The pro-inflammatory cytokine response to Candida

is complement- and cell-mediated and is distinct from the previously defined TLR-induced cytokine response to fungi defined by Netea et al. [[16]]. Cheng et al. [[1]] confirm the importance of complement in this process by using heat-inactivated serum, which lacks an active complement system, and also by blocking specific complement activation pathways, that is, the alternative, the classical, or the lectin pathways. In each scenario, release of pro-inflammatory cytokines, that is, IL-1β, TNF-α and IL-6 by PBMCs was significantly reduced. In addition, in the study by Netea et al. [16], the complement-induced inflammatory cytokine response via C5a–C5a receptor signaling was shown to cooperate and interact PF-6463922 mw MAPK Inhibitor Library synergistically with TLR2 and TLR4 signaling induced by the ligands Pam3Cys and lipopolysaccharide (LPS), respectively. In order to confirm that the inflammatory response is indeed complement mediated and induced by the inflammatory activation fragment C5a, Cheng et al. [[1]] use recombinant C5a in competition assays to block C5a

receptors on human PBMCs. Recombinant C5a alone has no effect on the inflammatory response, but C5a added together with Candida augments IL-6 and IL-1β production, but does not affect TNF-α release. Furthermore blocking experiments with antibodies against complement components clearly defines that C5a and C5a-receptor functions mediate this cytokine response. Cheng et al. [[1]] also identify host genetic susceptibility factors by analyzing the immune response of serum Methamphetamine derived from patients with defined genetic deficiencies. Previously, two authors (Schejbel and Garred) of Cheng et al. [1], were also involved in the identification of patients with inherited complement defects, that is, patients with C5-, C6-, and C7 deficiencies

[[17]]. C5-deficient serum, when activated, forms a C3 convertase and generates C3a and C3b; however complement progression is blocked at the C5 stage. When cultivated in C5-deficient serum, the cytokine response to Candida is abrogated, thus underlining the relevance of C5 for cytokine production. This C5-deficient serum forms neither C5a nor C5b. In order to conclude whether the block in the complement-mediated cytokine response is mediated by C5a or C5b-triggered TCCs, Cheng et al. [[1]] also used serum from patients who were deficient for single components of the terminal pathway, that is, C6 or C7. Both sera, when activated by Candida, form C3- as well as C5-activation products, that is, C5a and C5b. However, progression of the terminal pathway and TCC pore formation does not occur.

Influenza was included in the viral antigen mix, as it is known to initiate the adaptive immune response, provoking a multi-step process with a sudden ‘cytokine storm’ at 48 h [25]. In general, the immune defence of the human body is a multi-step process triggered and executed by different cell Luminespib chemical structure defence lines. The major sources of cell-mediated immune response are leucocytes, whereby B and T cells and their release of cytokines play the most important role. The test presented in this study reflects reactions of both cell types and also of other defence lines as represented, e.g. by macrophages. As the human immune system is a complex organ,

the in-vitro test in this STI571 cost study is testing for the overall response. The two important mechanisms are the B cells and their

capacity to produce antibodies, and more importantly the T cell activation followed by the T cell-dependent and -independent B cell activation [26]. Cytokines play a key role in these activation processes. Recent investigations found that the cytokine release is not only limited to T cells but that B cells also have the potency and capacity to produce cytokines [27]. For this reason, the test introduced in this study uses the cytokine responses as a read-out parameter, reflecting both cell lines. Testing for the most suitable and representative read-out parameters to mirror a DTH-like immune response, we focused on three representative cytokines which are involved in T cell-mediated immune responses: IL-2, IFN-γ and TNF-α. IL-2 is one of the key cytokines involved Carbohydrate in T cell activation and proliferation [28]. After incubation with the different antigens of either a bacterial, viral or fungal nature, the concentrations of IL-2 in the culture supernatants increased significantly at 24 h and even more significantly at 48 h after onset of incubation, reflecting a strong and time-dependent Th1 response. Moreover, IL-2 is known to be a potent inductor of IFN-γ during Th1 and Th2 differentiation [29]. In addition, IFN-γ has been also identified

previously as one of the important cytokines involved in mediating skin DTH reactions [30]. Accordingly, the time kinetic of IFN-γ followed mainly the IL-2 slope, and showed high concentrations at 24 and 48 h. TNF-α secreted by macrophages as well as by T cells is a potent initiator, enhancer and primer of T cell signalling and activation [31] in the inflammatory cascade. It is known to be released very early in the inflammation process [32]. This was confirmed by our findings showing peak levels of TNF-α for bacterial, viral and fungal antigen stimulation as early as after 12 h after onset of incubation. This is in further accordance with previous results from virus-induced TNF-α secretion, which also occurs very early in the inflammatory process [33, 34].

TRAMP PCa cells retrovirally transduced to express human PSMA (TRAMP-PSMA+HHD−) and/or HHD (TRAMP-PSMA+HHD+) or (TRAMP-PSMA−HHD+) were used as targets. The PSMA27, PSMA663, and PSMA711-specific CTLs demonstrated high levels of cytolytic activity (over 75% specific lysis) against target cells loaded with the respective PSMA peptide (Fig. 3A–C). The PSMA27 and PSMA663-specific CTLs were also able to specifically and effectively kill the target cells endogenously

expressing human PSMA and HHD (approximately 60 and 75% specific lysis, respectively, Fig. 3A and B). This confirms the processing and presentation of both PSMA27 and PSMA663 peptides from the protein backbone. However, despite displaying

high cytotoxic capacity against peptide-loaded cells, the PSMA711-specific CTLs were unable to kill the human PSMA and HHD-expressing cells find more (Fig. 3C). These observations were confirmed in multiple experiments and indicate that the PSMA711 peptide may be poorly processed and presented. As strong ex vivo CD8+ T-cell responses were generated against p.DOM-PSMA27 and p.DOM-PSMA663 constructs (Fig. 1B), and these CTLs were able to specifically lyse target cells which expressed PSMA endogenously (Fig. 3A and B), the in vivo Raf inhibitor cytotoxicity of these CTLs was investigated. Testing of the ability of the CTLs induced in HLA-A*0201 transgenic mice to kill tumor cells in vivo is hampered by the fact these mice lack expression of endogenous mouse MHC class I (H-2b) antigens. This means that the H-2b antigens expressed by the TRAMP PCa cell line will be immunogenic, preventing their long-term survival in standard tumor challenge experiments. We therefore used two approaches: the first was to demonstrate that the induced CD8+ T cells could kill peptide-loaded autologous target cells (splenocytes; Fig. 4A–C). For this, mice were immunized with p.DOM-PSMA27, p.DOM-PSMA663, or p.DOM control vaccine and boosted 28 days later with electroporation. Eight days after boosting, CFSE-labeled HHD splenocytes loaded with PSMA27, PSMA663, or control peptide

were injected as target cells. Representative flow cytometry plots are shown in Fig. 4A. Mice vaccinated with p.DOM-PSMA27 had a significantly reduced ratio of surviving CFSEhi PSMA27-loaded Thiamet G cells in respect to CFSElo control cells, with ∼33% fewer cells persisting compared with those in control mice (p=0.0011, Fig. 4B). The level of lysis of target cells observed in individual mice correlated with IFN-γ ELISpot responses detected in vitro (p=0.0049, Fig. 4B). After p.DOM-PSMA663 vaccination, the effect was even greater, with an approximately 50% reduction in the survival of PSMA663-positive cells in the vaccinated group compared with controls (p=0.0076, Fig. 4C). Again the level of lysis of target cells correlated with IFN-γ ELISpot responses (p<0.0001, Fig. 4C).

Cells were analyzed on an FACSCanto (BD Biosciences), followed by analysis with FlowJo software (Tristar). Expression of γc in T cells was analyzed by Western blotting using a rabbit anti γc as first antibody (1:500; Santa Cruz) and a peroxidase-conjugated secondary antibody (1:6000; Amersham). Nuclear proteins were extracted from spleen CD3+ T cells and the amounts of activated NF-κB p65 subunit and NFATc1 were measured with commercial kits (Nuclear Extract Kit and TransAM™, Active Motif), according to the manufacturer’s instructions. Allograft-survival comparisons between

groups were analyzed using the log rank method. RT-PCR data were analyzed by the non-parametric Kruskal–Wallis and Mann–Whithney test. Other statistical analyses were performed selleck products using bilateral Student t test or ANOVA followed by protected least significance difference Fisher test when multiple groups were compared

(Statview). Results with p<0.05 were considered statistically significant. All values are means±SEM. This work was supported by the INSERM and by the Faculté de Médecine Pierre et Marie Curie. Additional support was provided by grants from the Association pour la Recherche sur le Cancer (No. 9946), the Ligue Nationale contre le Cancer (Comité de Paris), the Baxter Extramural Grant Program, and the Agence de la Biomédecine. E. L. was supported by grants from INSERM and Fondation pour la signaling pathway until Recherche Médicale. C. D. C. was supported by the Else-Kröner-Fresenius Foundation. We thank Philippe Fontanges and Romain Morichon for confocal microscopy experiments, Olivier Lantz (Laboratoire d’Immunologie, INSERM U932, Institut Curie, Paris, France) for valuable discussions and all participating centers of the

European Renal cDNA Bank-Kroener-Fresenius biopsy bank (ERCB-KFB) and their patients for their cooperation. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Although many case–control studies have investigated the association between P2X7 gene polymorphisms and tuberculosis susceptibility, the interpretation of these data has been difficult due to limited power. As a means of better understanding the link between P2X7 and tuberculosis, a systematic review of the literature was conducted using metaanalysis. This approach provided a quantitative summary estimate on the association between P2X7 and tuberculosis. We searched databases (MEDLINE, PUBMED, and OVID) between January 1998 and July 2010 using the search words ‘gene’ or ‘P2X7’ in combination with ‘tuberculosis,’ performed manual citation searches from relevant original studies and review articles and corresponded with researchers in the field of study. The pooled odds ratios (ORs) for studies examining variations in the P2X7 gene 1513 C and −762 C loci were 1.44 [95% confidence interval (CI) 1.23–1.68; P<0.

Our results revealed that CML-specific CTL crucially contribute to disease control and are characterized by high IL-7Rα expression. Interestingly, CML cells produced IL-7 that was crucial for the

maintenance of specific CTL. Therefore, CML maintains Wnt inhibitor leukemia-specific CD8+ T-cell-mediated immunosurveillance by IL-7 signaling. Bone marrow was cotransduced with retroviral particles encoding for BCR/ABL and NUP98/HOXA9 and injected into irradiated syngeneic recipient mice. As shown previously, coexpression of BCR/ABL and NUP98/HOXA9 led to the development of CML and progression to blast crisis within several weeks 17. Granulocyte counts rose up to 9×107/mL (C57BL/6 control mice:<2×106 granulocytes/mL blood). Phenotypically, the leukemic cells consist of a population Y-27632 cost of immature myeloid blasts (MAC-1+,

GR-1+ and c-kit+) of up to 10% and a majority of mature granulocytes 17. This cotransduction was chosen to model the transition from chronic phase to blast crisis. To study antigen-specific immune responses, H8 transgenic mice were chosen as bone marrow donors. In this experimental setup, all leukemia cells expressed the immunodominant CTL epitope gp33 of lymphocytic choriomeningitis virus (LCMV) on MHC class I molecules as a model leukemia antigen (H8-CML mice). To analyze the impact of CD8+ T cells on disease progression, H8-CML mice with high granulocyte counts (>5×107 granulocytes/mL blood) were depleted of CD8+ TCL T cells by monoclonal antibody or were left untreated. Depletion of CD8+ T cells

led to disease progression and death of 83% of the animals within 4–19 days (Fig. 1A). CML progression was significantly slower in untreated control mice and 50% of the mice survived up to 75 days. On the contrary, treatment with IgG from rat serum (as control for αCD8 antibody YTS169.4) did not prolong survival when compared with untreated mice (Supporting Information Fig. 1). These results suggest that CD8+ T cells are crucially involved in the control of CML disease progression. The fact that a minority of the CD8+ T-cell-depleted animals still could control CML suggests that other effector mechanisms may contribute to the immunosurveillance of CML. In agreement with our earlier results, in CML mice no gp33-specific CTL response was detectable in the blood by tetramer staining 17. Naïve C57BL/6 mice and LCMV-immune mice which had been infected 8 wk previously with 200 pfu LCMV-WE were used as controls (Fig. 1B and D). The absence of specific CD8+ T cells in blood of CML mice by tetramer staining was in contrast to the rapid leukemia progression in CD8+ T-cell-depleted animals. Using a dextramer enrichment approach, we could detect gp33-specific CTL in pooled spleens and lymph nodes of H8-CML animals above the background of naïve C57BL/6 mice (Fig. 1C and D and Supporting Information Fig. 2A and B) 18.

In the immunostimulation setting after transplant, rapamycin

decreases lymphocyte proliferation and reduces rejection [39]. Nevertheless, in the setting of renal injury, where organ repair depends on tubular cell proliferation and well-orchestrated apoptosis, rapamycin may be harmful. Lieberthal et al. [40] have demonstrated that rapamycin inhibits proliferation and increases apoptosis of renal tubular epithelial cells in vitro and in vivo. Furthermore, there is evidence of pharmacokinetic interactions between rapamycin and CNI that augment ischaemic injury and inhibit tissue repair when used in combination [41]. Conversely, our results may demonstrate that the combination of rapamycin and tacrolimus administered to donors MAPK Inhibitor Library cost decreases apoptosis and necrosis in the graft in a syngeneic rat model. The difference

observed in our experiments, Sirolimus compared to Lieberthal et al. [40], may result from the administration setting. Once the injury is caused, rapamycin delays ATN recovery but the early administration of rapamycin, i.e. before the injury is caused, may explain the different beneficial effects observed in this exploratory study. Immunosuppressive treatment was administered in a single dose only to donor animals, 12 h before ablation. Several authors using the transplant model with rapamycin exposure after I/R injury support the hypothesis that rapamycin compromises renal function by impairing recovery rather than increasing injury severity [19,40]. In particular, Fuller et al. have demonstrated that serum creatinine in rapamycin-treated groups takes longer to recover [42]. These results show coherence regarding the specific impact of rapamycin on injured kidney. The data presented in our exploratory Cepharanthine work could provide new evidence for the use of rapamycin as a potent non-nephrotoxic immunosuppressant for its use

in donors in the DGF setting. The exact mechanism underlying the effect described for rapamycin or tacrolimus on renal I/R injury has not been explained completely. The protection by donor preconditioning has been associated with a reduction in the inflammatory response to reperfusion. Accordingly, the proinflammatory cytokines TNF-α and IL-6 were reduced by donor preconditioning with immunosuppressive treatment drugs. Other studies have also described that rapamycin suppresses IL-6 production, and that this may be associated with regulatory T cell (Treg) induction and with a decrease in the T helper 17 (Th1) population [43,44]. Regarding apoptosis, the improvement observed in the rapamycin group could be explained by in-situ up-regulation of Bcl-2, a specifically anti-apoptotic gene.