compared with the glycoside hydrolase family amylomaltases from o

compared with the glycoside hydrolase family amylomaltases from other bacteria, plants, and archaea. Because MalQ and maltose transport proteins have been implicated in expression of virulence factors in V. cholerae and streptococci, respectively (Lång et al., 1994; Shelburne et al., 2006), presumably to relay information about the environment,

we assayed whether malQ has a similar role in B. burgdorferi. Neither the malQ mutation nor varying carbohydrates available affected the expression of outer surface lipoprotein C (data not shown), which is essential for MK-2206 supplier transmission or mammalian infection (Grimm et al., 2004; Pal et al., 2004). While our data suggest that MalQ does not have an essential role in disaccharide utilization in vitro, we hypothesized that MalQ

may be important in the enzootic cycle for metabolism or gene regulation in vivo. Therefore, we assayed the malQ::aadA Small molecule library ic50 mutant strain in the experimental tick–mouse model. Wild-type, malQ::aadA, and complemented strains were needle-inoculated into mice; ear biopsies were collected 3 weeks after injection, cultured in BSK II, and examined for spirochetes by dark-field microscopy. In addition, ear, ankle, and bladder tissues were dissected and cultured for B. burgdorferi at 5 weeks postinoculation. The malQ mutant was infectious by needle inoculation and successfully disseminated to the ear, ankle, and bladder of the mice (Table 2). To examine the role of MalQ in B. burgdorferi acquisition, naive I. scapularis larvae were allowed to feed to repletion on mice infected with wild-type 297, malQ::aadA, or complemented strains. Five to 10 days after feeding to repletion, PCR analysis revealed that larvae acquired B. burgdorferi from infected mice independent of the presence of malQ (seven of seven ticks were infected with each strain). Larvae Isotretinoin that had

fed to repletion on infected mice were allowed to molt into nymphs to examine whether MalQ functions in tick persistence. After 3 to 4 weeks, five nymphs infected with each strain were then fed to repletion on naive mice. About 7 days after feeding to repletion, the midguts were dissected and processed for immunofluorescence microscopy using anti-Borrelia antibodies (green) and wheat germ agglutinin-AlexaFluor® 594 that stains tick cells (red). All midguts examined contained B. burgdorferi at similar densities by immunofluorescence microscopy (Fig. 4), suggesting that survival during molting and persistence in nymphs following the blood meal does not require MalQ. Although mouse infection by needle inoculation was malQ independent, the natural route of transmission is by tick bite. Nymphs infected with wild-type, malQ::aadA, or complemented strains were allowed to feed to repletion on naive mice to test whether transmission of B. burgdorferi by tick bite requires malQ. Five nymphs infected with each strain were fed on three separate mice. Three weeks after tick feeding, ear biopsies were taken, cultured and screened for B.

Results: Fifty two AKI patients, who collectively underwent 248 d

Results: Fifty two AKI patients, who collectively underwent 248 dialysis treatments, were studied prospectively. Mean (±SD) age was 69.4 ± 16.9; 50% were male. At dialysis initiation, APACHE buy Etoposide II score was 20.6 ± 6.2 and SOFA score 8.2 ± 3.1. The frequency of HD treatments averaged 2.0 ± 0.5/patient/week. Mean session length was 3.54 ± 0.81 h, and 78.9% used a femoral venous catheter. The mean delivered Kt/V of each session was 1.20 ± 0.58

while 64.1% of treatments delivered a Kt/V less than 1.3. The results showed that the mean weekly delivered Kt/V at first, second, and third week was 2.49 ± 1.14, 2.55 ± 1.31 and 2.36 ± .076 respectively. Minority of patients (15.8%) achieved the recommended weekly Kt/V of 3.9. Mortality rate was lower in patients who achieved adequacy target (weekly Kt/V ≥ 3.9) but the different was not statistically significant (33.3% vs 40.6%, P = 0.73). Conclusion: Majority of our AKI patients received a lower dose of dialysis than recommendation. Survival benefit of delivering higher dose of dialysis was not shown in this study due to a small number of patients. YAMAGUCHI JUNNA, TANAKA TETSUHIRO, ETO NOBUAKI, NANGAKU MASAOMI Division of Nephrology and Endocrinology, the University of Tokyo Graduate School of Medicine Introduction: Tubulointerstitial this website hypoxia is a critical mediator in the pathogenesis of kidney

disease. In light of accumulating knowledge on protective roles of HIF-1, we aimed to identify novel HIF-1 regulators in kidney. Methods: An shRNA library was created against hypoxia-inducible genes screened from a microarray analysis of rat renal artery stenosis model. The impact of candidate genes on HIF-1 was evaluated in vitro by HREluc, HIF-1α immunoblot, and VEGF protein levels, leading to identification of a novel upregulator of HIF-1. Its regulation of HIF-1 and the underlying mechanisms were investigated in human proximal tubular cells (HK-2). Furthermore, we attempted to characterize the inflammatory nature of this gene and link inflammation to the HIF response. Results: An

shRNA library experiment identified CEBPD, a transcription Etomidate factor, as a novel HIF-1 regulator in kidney. CEBPD was induced in kidneys subjected to systemic hypoxia, as well as in models of acute and chronic hypoxic kidney injuries, with predominant expression in the nuclei of proximal tubular cells, the most susceptible portion of kidney to hypoxia. In vitro, CEBPD siRNA knockdown and overexpression mediated down- and upregulation of HIF-1α as well as its target genes. Mechanistically, promoter and chromatin immunoprecipitation (ChIP) assay confirmed that CEBPD directly promoted the transcription of HIF-1α. Notably, CEBPD was rapidly inducible by inflammatory cytokines, such as interleukin-1β, in an NF-κB-dependent manner, and was indispensable for the non-hypoxic induction of HIF-1α. Conclusion: These results demonstrate CEBPD as a novel HIF-1 regulator in kidney.

Polystyrene beads were used as a control with common genes induce

Polystyrene beads were used as a control with common genes induced by the beads and the bacteria, suggesting that these genes may represent a gene signature Tanespimycin related to M-cell translocation. There were, however, genes that were specifically induced by the bacteria but not by the beads. These genes included the transcription factors that mediate the immediate-early response EGR1, FOS and JUN, the negative regulator ZFP36, and the phosphatase DUSP1.

These genes have previously been linked by cluster analysis in studies examining the response of human epithelial lung cells to avian influenza, endothelial cells stimulated with IL-1 and in tumour microarray analyses.30–32 The selective activation of these genes by the bacteria but not the beads indicates that the bacteria are activating and being sensed by the M cells, whereas the beads are merely being translocated non-specifically. Escherichia

coli and B. fragilis also activated more pro-inflammatory genes than L. salivarius, which again may be related to the lower translocation efficiency of L. salivarius. These inflammatory genes included the chemokines CXCL1, CXCL2 and IL8 which are potent chemoattractants for neutrophils and other immune cell types. Neutrophil recruitment is critical for clearance of bacteria once they have translocated the epithelium.33–35CXCL2 has Dorsomorphin manufacturer also been shown to be up-regulated in vivo in Peyer’s patches and mesenteric lymph nodes coincident with the accumulation of monocytes and neutrophils in these tissues.36 It is interesting to note that the increased

translocation efficiency of E. coli and B. fragilis compared with L. salivarius, was associated with a more potent induction of pro-inflammatory genes. NFKBIZ, NFKBIA and TNFAIP3 inhibit nuclear factor-κB signalling via a negative feedback loop.37–39NFKBIZ is induced by lipopolysaccharide, which could explain why it is induced by E. coli and B. fragilis but not L. salivarius.40 The microarray data were confirmed by qRT-PCR for selected genes, including IL8 and EGR1, and the same changes Resveratrol for each of the bacteria and the beads were observed, which validates our microarray findings. The gene ANKRD37, an HIF1α-inducible gene, was also confirmed, illustrating activation by all three bacterial species,41 whereas the gene, NR4A1, which is involved with fibronectin adhesion,42 was reduced in expression in C2-M cells treated with bacteria and the beads. It is also interesting to note that we observed differential expression of PRRs in C2-M cells compared with control differentiated C2 cells. MRC1 was highly expressed in C2-M cells compared with C2 cells and has previously been observed by Hase et al.43 to be increased in the follicle-associated epithelium overlying the murine Peyer’s patch.

2A and 2B) When lymphatic vessels were not enhanced

by m

2A and 2B). When lymphatic vessels were not enhanced

by microscopic ICG lymphography, lymphatic vessels were dissected as a conventional method without intraoperative ICG lymphography guidance.[3, 4] Lymphatic vessels were anastomosed to appropriate venules in an end-to-end fashion using 11-0 or 12-0 nylon sutures.[3, 4, 12-14] Patency of the anastomosis can be confirmed by lymph fluid washout into the venule (Fig. 2C and 2D; See Video, Supporting Information Digital Content 1, which shows intraoperative microscopic ICG lymphography-guided LVA). A week after the LVA surgery, patients resumed the same compression therapy as preoperatively performed to make lymphatic pressure higher than venous pressure. Intraoperative findings and treatment efficacy were compared between LVA with and without Selleckchem BVD-523 intraoperative microscopic ICG lymphography. Edematous volume was evaluated preoperatively and 6 months after the operations using LEL index.[15] A summation of squares of circumferences C1, C2, C3, C4, and C5 (cm) divided by BMI is defined as the LEL index. C1 denotes circumference at 10 cm above the superior border of the patella, C2 circumference at the superior border of the patella, C3 circumference at 10 cm below the superior border of the patella, C4 circumference at the lateral malleolus, and C5 circumference

at the dorsum selleck chemical of the foot. Student’s t-test and Mann Whitney U test were used for statistical analysis. A statistical significance was defined as P-value < 0.05. Forty LVAs were performed on 12 lymphedematous limbs by one surgeon (T.Y.): 24 LVAs with intraoperative microscopic ICG lymphography-guidance on 7 limbs, and 16 LVAs without the guidance on 5 limbs (Tables 1 and 2). Lymphatic vessels were enhanced by intraoperative (-)-p-Bromotetramisole Oxalate microscopic ICG lymphography in 11 of 12 skin incision sites. In 1 of 12 skin incision, lymphatic vessels could not be enhanced even after additional ICG

injection. The nonenhanced site was shown diffuse pattern on preoperative ICG lymphography. All anastomoses, regardless of ICG-enhancement of lymphatic vessels, showed good anastomosis patency after completion of anastomoses. Time required for detection and dissection of lymphatic vessels in cases with intraoperative microscopic ICG lymphography-guidance was significantly shorter than that in cases without the guidance (2.3 ± 1.7 min vs. 6.5 ± 4.0 min, P = 0.010). Postoperative LEL index decreased significantly compared with preoperative LEL index (254.9 ± 35.8 vs. 238.0 ± 32.5, P < 0.001). There was no statistically significant difference in LEL index reduction between cases with and without intraoperative microscopic ICG lymphography guidance (18.3 ± 5.5 vs. 15.0 ± 5.5, P = 0.337). A representative case is shown in Figure 3. Secondary lymphedema is caused by obstruction and subsequent congestion of lymph flows.

To compare the efficacy of these female T cells, we also immunize

To compare the efficacy of these female T cells, we also immunized one female dog in vivo with PBMCs from a DLA-identical male littermate. Male-specific recognition induced by UTY-specific CTLs after in vitro immunization

was comparable to those with T cells after in vivo immunization of a female dog (Figs. 3 and 5) as male-BM was targeted with the highest efficiency, followed by DCs and PBMCs, monocytes and B cells indicating an elevated presentation of male-antigens in BM, as previously assumed by others [11, 12, PXD101 44, 45]. Nevertheless, male-BM represents the most-affected target of female T cells, indicating higher and presumably different UTY-expression/UTY-presentation [46] and confirms the high-potential of UTY in female-to-male-transplantation settings (Figs. 3 and 5). Immunization of the female dog with DLA-identical-male-PBMCs induced UTY-specific CD8+T cells, as indicated by increasing amounts of donor-PBMCs. Other mononuclear-cells, like CD4+, CD14+ and B cells, also increased within the experiment, indicating an intense immune-response (data not shown). IFN-γ-secretion was detectable against the UTY-peptides when loaded on different target cells and hT2-MHC-I-restricted cells. Thereby, immunogenicity of investigated peptides was W248 > K1234 > T368 (T2-cells).

Detailed characterization of the UTY-response of female T cells exhibited a male-specific T cell response acting in an MHC-I-restricted-fashion (Figs. 3 and 5). As these blocking-experiments did not reveal a purely CTL-directed PARP inhibitor T cell reactivity (incomplete blocking), these data could implement an additional CD4+T cell-driven reactivity [43, 47]. With these data, we can evidently exclude xenogeneic-CTL-activity as in vitro data were reproducible in vivo showing male-cell-type-specific comparable results. UTY-mRNA-expression was determined in cell-types of hematopoietic-origin (Fig. 4) and confirmed our in vitro and in vivo data: Only male cells expressed UTY-mRNA (male-BM≫DCs, PBMCs,

monocytes, Neratinib price B-cells), whereas corresponding female cells lack UTY-mRNA proofing male-restricted UTY-expression in hematopoietic-cells [48]. UTY-expression in non-hematopoietic cells was not shown in our study. This is compelling to demonstrate as UTY is ubiquitously expressed and would lead not only to GvL-reaction after adoptive immunotherapy, but also to GvHD after transfusion of CTLs. In order to show that our UTY-derived peptides are good targets for canine-/human-GVL-reactions, canine non-hematopoietic-cells like male-fibro-blasts/keratinocytes as well as gut-, liver- and epithelium-cells should be investigated in further experiments to prove this hypothesis. Three isoform-variants of the hUTY-gene are known (additional isoforms seem to exist) and splice-variants show different expression-profiles and tissue-distributions of resulting peptides [49, 50].

“Research on the influence of multimodal information on in

“Research on the influence of multimodal information on infants’ learning is inconclusive. While one line of research finds that multimodal input has a negative effect on learning, another finds positive effects. The present study aims to shed some new light on this discussion by studying the influence of multimodal information and accompanying stimulus complexity

on the learning process. We assessed the influence of multimodal input on the trial-by-trial learning of 8- and 11-month-old infants. Using an anticipatory eye movement paradigm, we measured how infants learn to anticipate the correct stimulus–location associations when Ferroptosis phosphorylation exposed to visual-only, auditory-only (unimodal), or auditory and visual (multimodal) information. Our results show that infants in both the multimodal and visual-only conditions learned the stimulus–location associations. Although infants in the visual-only condition appeared to learn in fewer trials, infants in the multimodal condition showed better anticipating behavior: as a group, they had a higher chance of anticipating correctly on more consecutive trials than infants in the visual-only condition. These findings suggest that effects of multimodal information

on infant learning operate chiefly through effects on infants’ attention. “
“Infants are attuned to emotional facial and vocal expressions, reacting most prominently when they FK228 cell line are exposed to negative expressions. However, it remains unknown if infants can detect whether

a person’s emotions are justifiable given a particular context. The focus of the current paper was to examine whether infants react the same way to unjustified (e.g., distress following a positive experience) and justified (e.g., distress following Molecular motor a negative experience) emotional reactions. Infants aged 15 and 18 months were shown an actor experiencing negative and positive experiences, with one group exposed to an actor whose emotional reactions were consistently unjustified (i.e., did not match the event), while the other saw an actor whose emotional reactions were justified (i.e., always matched the event). Infants’ looking times and empathic reactions were examined. Only 18-month-olds detected the mismatching facial expressions: Those in the unjustified group showed more hypothesis testing (i.e., checking) across events than the justified group. Older infants in the justified group also showed more concerned reactions to negative expressions than those in the unjustified group. The present findings indicate that infants implicitly understand how the emotional valence of experiences is linked to subsequent emotional expressions. “
“The ability to effectively regulate emotions is an important marker for early socio-emotional development. The uses of self-comforting behaviors and self-distraction have been empirically supported as effective regulatory strategies for infants, although research on determinants of such behaviors is scarce.


Gaithersburg, MD, USA In this study, w


Gaithersburg, MD, USA In this study, we have analyzed the in vivo dynamics of the interaction between polyclonal Foxp3+ Treg cells, effector T (Teff) cells, and DCs in order to further our understanding of the mechanisms of Treg cell-mediated Navitoclax manufacturer suppression. Cotransfer of polyclonal activated Treg cells into healthy mice attenuated the induction of EAE. Suppression of disease strongly correlated with a reduced number of Teff cells in the spinal cord, but not with Treg cell-mediated inhibition of Th1/Th17 differentiation. Cotransfer of Treg cells with TCR-Tg Teff cells followed by immunization by multiple routes resulted in an enhanced number of Teff cells in the lymph nodes draining the site of immunization without an inhibition of Teff-cell differentiation. Fewer Teff cells could be detected in the blood in the presence of Treg cells and fewer T cells could access a site of antigen exposure in a modified delayed-type hypersensitivity assay. Teff cells recovered from LNs in the presence of Treg cells expressed decreased levels of CXCR4, syndecan, and the sphingosine phosphate receptor, S1P1 (sphingosine 1-phosphate receptor 1). Thus, polyclonal Treg cells influence Teff-cell

responses by targeting trafficking pathways, thus allowing immunity to develop in lymphoid organs, but limiting the number of potentially auto-aggressive cells that are allowed to enter the tissues. Numerous mechanisms exist to both activate and dampen immune responses. A primary cell type involved in immune suppression is the selleck inhibitor thymic-derived Treg cell defined by the expression of the transcription factor Foxp3. Mutations in Foxp3 lead to severe defects of immunological homeostasis in both mouse and human 1. Treg cells have also been shown to play a pivotal role in numerous disease settings, including autoimmunity, infection, and tumor progression 2. Multiple mechanisms have been proposed for suppressor function of Treg cells including the secretion of suppressive cytokines, direct cytolysis of T effector (Teff) cells, metabolic disruption through tryptophan catabolites,

adenosine or IL-2 deprivation, and direct interference of co-stimulation via expression of CTLA-4 3. Given the obvious interest in targeting Treg cells in various disease settings through pharmacological intervention, DAPT purchase a more definitive understanding of their mechanism of action is warranted. To achieve this, the in vivo dynamics of the interaction between Treg cells, Teff cells, and DCs need to be more thoroughly evaluated. Upon immunological challenge, DCs capture antigen and migrate to draining LNs where they present the antigen to Teff cells 4. The Teff cells then become activated and undergo several rounds of division during which time they differentiate. After this has occurred, Teff cells leave the LN, enter the circulation, and ultimately enter tissues. All of these steps represent potential checkpoints where Treg cells may exert their influence.

Previous studies in our laboratory established that GTE suppresse

Previous studies in our laboratory established that GTE suppresses B cell production of IgE without inducing apoptosis, in a homogeneous U266 B-cell model [11]. In this study, cell viability in PBMC was observed (>90%) on day 10 in the presence or absence of GTE to rule out cytotoxicity as a potential mechanism of GTE’s inhibitory

potential on IgE production and is in agreement with our earlier studies that GTE suppresses in vitro IgE responses without inducing apoptosis [11]. It has been demonstrated that green tea polyphenols, including epicatechin-3-gallate (ECG) and EGCG, exhibit anti-mutagenic and anti-carcinogenic activity in microbial systems, mammalian cell systems and in vivo [20]. Studies of Nakazato, et al. [21] reported that ECG has potential as a novel therapeutic agent for patients with B-cell malignancies (e.g. multiple myeloma), Y 27632 possibly through induction of apoptosis mediated by modification of the redox system [21]. GTE has been shown to inhibit breast cancer growth by a direct anti-proliferative effect on the tumour cells as well as by indirect suppressive effects on the tumour-associated endothelial cells [22] and

can increase the inhibitory effect of tamoxifen on the proliferation of the oestrogen receptor MCF-7, ZR75, T47D human breast cancer cells in vitro [22]. Studies of Silverberg et al. [23] found that GTE inhibits hydrogen peroxide-induced GSK2126458 datasheet necrosis of human skin fibroblasts [23]. In various tumour cell stiripentol systems, green tea polyphenols have been implicated in induction of apoptosis, via a caspase 3-executed mechanism

that targeted the mitochondria [24]. In other disease states, GTE also prevented Abeta [25]-induced activation of NF-κB, ERK and P38 MAP kinase pathways in rats, suggesting that GTE may prevent the development and progression of Alzheimer’s disease [25]. Green tea extract-4 (CSI-4) has also been reported to possess anti-adhesive activity against certain pathogenic bacteria (e.g. P. acnes), with no adverse effects against beneficial bacteria (e.g. Lactobacillus acidophilus) [26]. Previous studies of Nie et al. [27] demonstrated that green tea polyphenols and their major component, EGCG at a concentration of 200 microM, exert significant protective effects against 6-OHDA-induced PC12 cell apoptosis, and EGCG was more effective than the mixture of green tea polyphenols [27]. The authors concluded that green tea polyphenols’ neuroprotective effect was because of antioxidant function [27] and has potential for the treatment of neurodegenerative diseases [27]. In this study, addition of GTE (1–100 ng/ml) resulted in suppression of IgE (up to 98%); EGCG (0.5–50 ng/ml) alone moderately suppressed IgE production (up to 28%). Addition of cat pelt antigen (1 AU/ml) and GTE (1–100 ng/ml) or EGCG (0.

To verify this possibility, the concentration dependence of infla

To verify this possibility, the concentration dependence of inflammasome activation in WT and KI cells (in the presence and absence of ATP) Epigenetics inhibitor was determined. It was found that while inflammasome activation increased in both the cell types with increasing LPS concentrations, WT cells required massive amounts of LPS (>1000 ng/mL) to activate the inflammasome in the absence of ATP, whereas KI cells required only minute amounts of LPS. It thus appears that KI cells do not require co-stimulation by ATP because the small amounts of TLR ligand that enter in the absence of ATP are sufficient to activate the altered inflammasome.

Overall, these data Selleck LDE225 are consistent with the concept previously suggested from studies of CAPS patients that NLRP3 mutations lead to changes in the conformation of the protein that, in turn, result in a reduced activation threshold and thus an inflammasome capable of responding to reduced amounts of TLR ligand or other activating factors 9, 19. However, NLRP3 may not be able to directly bind to such a wide variety of ligands including PAMP and DAMP, rather an endogenous activator induced by all these upstream stimuli may serve as the direct ligand for NLRP3 (Fig. 1). This concept has also been proposed independently by other researchers 20, 21. NLRP3 KI mice bearing an R258W

mutation raised under pathogen-free facility exhibit spontaneous clinical symptoms similar to those of the counterpart Muckle–Wells syndrome patients. These symptoms consist of poor linear growth, reduced reproductive capacity, impaired hair development and, in many animals, severe dermatitis affecting the Phosphoribosylglycinamide formyltransferase ears, top of

the head and tail base area occurring at 6–12 wk of age that is associated with a deterioration of health. The skin lesions were clinically more severe than the urticaria-like skin disease seen in human CAPS and characterized by neutrophilic infiltration of the dermis and epidermis. Spleen and draining lymph nodes were enlarged in the KI mice and showed poorly developed follicles along with a diffuse infiltrate, again containing many neutrophils. However, these KI mice were free of lung, kidney or gut inflammation and the level of circulating inflammatory cytokines was normal 9. The clinical features of mice bearing A350V and L351P mutations were qualitatively similar to those described for R258W mice, but were far more severe. These A350V/L351P KI mice had lifespan measured in days rather than weeks, and had more widespread skin inflammation and inflammatory infiltration (mainly neutrophilic) of many organs, including the joints, sinus, bone marrow and tongue. In addition, there was evidence of “necrotic degeneration” in the gut and kidney.

After washing, 20 ml 0·9% NaCl containing CaCl2 were added To de

After washing, 20 ml 0·9% NaCl containing CaCl2 were added. To determine the number of bacteria in the alginate beads the beads were dissolved to release the bacteria using 0·1 M citric acid buffer pH 5. Serial dilutions were made and cultured on a modified Conradi-Drigalski medium (SSI), selective for Gram-negative rods. After overnight incubation at 37°C PS-341 datasheet the number of colony-forming units (CFU) was determined. The concentrations of P. aeruginosa in both the small beads (SB) and large beads (LB) varied from 0·2 to 0·7 CFU/ml; in no experiment did the concentration of bacteria in the beads differ more than 19%, and the bacterial concentration was lowest in the SB in all experiments.

In the present work we made beads in two different sizes. For the SB we used the 0·250 mm nozzle, an alginate flow rate 20 ml/h and the airflow 105 mBar. For the LB the 0·500 nozzle, alginate flow rate 60 ml/h and airflow 35 mBar were used. The diameter of the beads were measured using a light microscope (Olympus, Tokyo, Japan) and a picture-analysing program (Visiopharm Image Analysis and Stereology, Alleroed, Denmark). Two diameters at right

angles were determined for each bead and presented as the mean. Female 11-week-old BALB/c mice were purchased from Taconic Europe A/S (Lille Skensved, Denmark) and allowed to acclimatize for 1 week before use. A total of 207 mice were used in the experiments. Mice had free access to chow and water, and were under the observation of trained personnel. All experiments were authorized by the National Animal Ethics Committee, Denmark. Mice were anaesthetized subcutaneously PRKD3 Aurora Kinase inhibitor (s.c.) with a 1:1 mixture of etomidate (Janssen, Birkeroed, Denmark) and midazolam (Roche, Basel, Switzerland) (10 ml/kg body weight) and tracheotomized. SB or LB seaweed alginate beads embedded with PAO579 were installed into the left lung of BALB/c mice using a bead-tipped needle. All mice received the same amount of alginate and number of P. aeruginosa (0·66 × 109 CFU/ml for the SB group versus 0·71 × 109 CFU/ml for the LB group). An additional 32 mice were challenged with

beads prepared as described but without adding P. aeruginosa to the alginate. Mice were killed using an overdose of barbiturate at days 1, 2, 3, 5 or 6 after challenge. Peripheral blood was collected by cardiac puncture and serum isolated after centrifugation of coagulated blood. Serum was kept at −70°C until analysis. Half the number of lungs were collected aseptically and transferred to 5 ml of sterile phosphate-buffered saline (PBS) and kept on ice until further analysis. The left lungs from the remaining number of mice were fixed in a 4% w/v formaldehyde solution (VWR, Copenhagen, Denmark). Evaluation of pulmonary histopathology was performed as described previously [8]. The fixed lungs were embedded in paraffin wax and cut into 5-µm-thick sections, followed by haematoxylin and eosin or Alcian blue staining.