Subsequently, the sections were incubated with horseradish peroxi

Subsequently, the sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Vector Laboratories Inc., Burlingame, CA) diluted 1 : 1000 for ICG-001 30 min at room temperature. The bound antibodies were visualized with 3,3′-diaminobenzidine tetrahydrochloride. The numbers of α-smooth muscle actin-positive cells were counted in three high-power (× 400) fields of each section and averaged. Fibroblastic cell line Rat-1 cells (RIKEN BioResource Center, Ibaraki, Japan) were grown at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Tokyo, Japan) supplemented with

10% fetal bovine serum (Biowest, Nuaillé, France) and antibiotics (100 U mL−1 penicillin, 100 μg mL−1 streptomycin; Nacalai Tesque). The cells were seeded in 12-well plates at 4 × 104 cells

per well. When the cells became subconfluent, a medium containing 1, 10, 50 and 100 μM 3-oxo-C12-HSL or 0.1% DMSO was added. After 24 h of treatment, the Bafilomycin A1 concentration cells were fixed with 4% paraformaldehyde in phosphate buffer for 20 min at room temperature, washed three times in PBS containing 0.05% Tween 20 (T-PBS) for 5 min and incubated for 30 min at room temperature with the same anti-α-smooth muscle actin primary antibody as that used for the tissue histological examination. After washing in T-PBS, the cells were incubated with a biotinylated anti-mouse immunoglobulin G secondary antibody (Vector Laboratories Inc.) diluted 1 : 1000 in PBS for 30 min at room temperature. The cells were then washed in T-PBS and incubated with Texas-red-conjugated avidin (Vector Laboratories Inc.) for 30 min at room temperature in the dark. The nuclei were stained with Hoechst 33258. tetracosactide The stained cells were observed using a DMI 4000 B fluorescence microscope (Leica, Wetzlar, Germany). The percentages of α-smooth muscle actin-positive cells relative to the total cell count were calculated to evaluate the effects of 3-oxo-C12-HSL on fibroblast differentiation. RNA samples were collected from

cultured cells treated with 10 μM 3-oxo-C12-HSL using Nucleospin® RNA II (Macherey-Nagel GmbH and Co., Duren, Germany) according to the manufacturer’s instructions. RT-PCR amplifications were performed for Cox-2, transforming growth factor (TGF)-β1, and interleukin-6 (IL-6). cDNA was generated using a High Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For quantitative PCR, the amplification of the target-specific region of cDNA was performed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min after preheating at 95 °C for 10 min, and monitored using a real-time PCR system (ABI prism 7700, Applied Biosystems). The relative expression level of the target genes of the AHL-treated cells to the value of the DMSO control was calculated by the Ct method using β-actin gene as an internal control.

© 2010 Wiley-Liss, Inc Microsurgery, 2011 “
“The use of th

© 2010 Wiley-Liss, Inc. Microsurgery, 2011. “
“The use of the bone flap transfer has been reported to be successful in

treatment of patients with early to medium stage (Ficat and Arlet stage I-III) osteonecrosis of the femoral head (ONFH). We examined the vascular anatomy and blood supply of the greater trochanter area and evaluated the feasibility of revascularization of the femoral head by using the bone flap pedicled with transverse and gluteus medius branches of the lateral circumflex femoral artery. Based on the anatomy study, from January 2002 to May 2004, 32 ONFH patients were treated with the greater trochanteric bone flap pedicled with double blood vessels. Fifteen femoral heads were Ficat and Arlet stage II ABT-263 molecular weight and 17 were stage III. The mean follow-up was 99.5 months. Two of the 32 patients required a total hip replacement

due to severe hip pain after surgery. The overall Harris hip score improved from a mean of 55.2 points to 85 points. CYC202 chemical structure Our data suggest the procedure is relatively easy to perform, less donor-site morbidity and useful for young patients with stages II to III disease with or without mild collapse of the femoral head. © 2013 Wiley Periodicals, Inc. Microsurgery 33:593–599, 2013. “
“Background: Superior gluteal artery perforator (SGAP) flaps are a useful adjunct for autologous microvascular breast reconstruction. However, limitations of short pedicle length, complex anatomy, and donor site deformity make it an unpopular choice. Our goals were to define the anatomic

characteristics of SGAPs Tangeritin in cadavers, and report preliminary clinical and radiographic results of using the lateral septocutaneous perforating branches of the superior gluteal artery (LSGAP) as the basis for a modified gluteal flap. Methods: We performed 12 cadaveric dissections and retrospectively reviewed 12 consecutive breast reconstruction patients with gluteal flaps (19 flaps: 9 LSGAP, 10 traditional SGAP) over a 12-month period. The LSGAP flap was converted to traditional SGAP in 53% of flaps because of dominance of a traditional intramuscular perforator. Preoperative 3D computed tomography angiography (CTA) and cadaveric dissections were used to define anatomy. Anatomic, demographic, radiographic, perioperative, and outcomes data were analyzed. Mean follow-up was 4 ± 3.4 months (range 4 weeks to 10 months). Results: Compared with the pedicle in the SGAP flap, the mean pedicle length in the LSGAP flap was 1.54 times longer by CTA, 2.05 times longer by cadaver dissection, and 2.36 times longer by intraoperative bilateral measurement. These differences were statistically significant (P < 0.001). Clinically, 100% of the flaps survived.

LI YANHONG1, LI MENGXIA1, LI XIAOZHONG1, FENG XING2 1Department o

LI YANHONG1, LI MENGXIA1, LI XIAOZHONG1, FENG XING2 1Department of nephrology, Children’s Hospital of Soochow University; 2Department of neonatology, Children’s Hospital of Soochow University Introduction: Acute kidney injury (AKI) is an independent risk factor for mortality. Since multiple Ivacaftor ic50 factors that influence kidney function and predispose to the development of AKI occur in combination in the neonates, critically ill neonates are at a high risk of having AKI. Cystatin-C is normally filtered freely and completely reabsorbed and catabolized in the proximal tubule. The appearance of increased concentrations of CysC in urine reflects renal tubular injury. In neonates, urinary cystatin-C is demonstrated to be a biomarker

for predicting AKI. This study evaluated the value of urinary cystatin-C level during the first day of life and determined whether urinary cystatin-C can predict mortality in the critically ill neonatal population. Methods: We enrolled 98 critically ill neonates who were admitted to a neonatal intensive care unit GDC-941 during the first day of life between July 2010 and April 2011. Urinary samples were collected in the first 24 hours after admission, and the level of cystatin-C was determined. The score for neonatal acute physiology (SNAP) were calculated during the first

24 hours after admission. Major outcome measure was 30-days mortality. Results: Of the 98 neonates, 7 (7.1%) died during the first 30 days of life. The median (min-max range) urinary cystatin-C level in critically ill children was 0.6 mg/g urinary Creatinine (0.0–65.5). The urinary cystatin-C level during the first day of life was significantly associated with 30-days mortality (odds ratio [OR] = 1.28; 95% confidence interval [CI], 1.02–1.60; p = 0.030), even after adjustment for gestational age, gender, and the severity of illness assessed by the SNAP score. Multivariate regression analysis showed

that high urinary cystatin-C level, high SNAP score (OR = 1.24; 95% CI, 1.01–1.53; p = 0.041), and mechanical ventilation (OR = 15.79; 95% CI, 1.69–153.10; p = 0.017) were independent risk factors for 30-days mortality in generally critically ill neonates. Urinary cystatin-C achieved an area under-the-receiver-operating-characteristic curve (AUC) of 0.85 (95% CI, 0.74–0.96; p = 0.005) for predicting 30-days mortality, which was C59 clinical trial similar to SNAP (AUC = 0.83; 95% CI, 0.71–0.95; p = 0.008). Urinary cystatin-C displayed a sensitivity of 83% and a specificity of 80% to predict 30-days mortality at an optimal cut-off value. Conclusion: A high urinary level of cystatin-C during the first day of life is independently associated with and predictive of 30-days mortality in the general population of critically ill neonates. JEYAKUMAR YOGARANI1, AGUIAR SANDRA2 1Monash Health; 2Monash Health Introduction: Focal Segmental Glomerulosclerosis(FSGS) is a glomerular disease that can affect both children and adults.

State differences in the willingness to consider home dialysis, t

State differences in the willingness to consider home dialysis, the degree of choice in dialysis location, the desire to change current dialysis type and/or location, and

the provision of information about dialysis were identified. Conclusion:  MG-132 nmr The delivery of pre-dialysis education is variable, and does not support all options of dialysis for all individuals. State variances indicate that local policy and health professional teams significantly influence the operation of dialysis programs. “
“Chronic kidney disease (CKD) is a major public health issue and early detection may prevent morbidity and mortality. Screening for CKD is simply assessed using the Kidney Health Check (KHC), a compilation of blood pressure (BP), estimated glomerular filtration rate (eGFR) and urinalysis (UA). KHC screening p38 MAPK inhibitor of high risk hospital inpatients is recommended, but its implementation and cost-effectiveness is unknown. We aimed to determine the proportion of patients currently tested for all components of the KHC during an acute hospital admission, and to compare the estimated costs of screening

and subsequent follow-up with other screening programs. A retrospective audit was conducted of consecutively admitted adult patients, and the frequency of BP, eGFR and UA testing recorded. Using published data, the likely costs and benefits of components of the KHC were estimated. Two hundred patients (median age 75 years, range 20–98) were assessed. All had a documented BP and eGFR, and 55% had a UA, representing a complete KHC. Of the total, 141 (71%) had one or more abnormalities detected, and of 71 with an eGFR <60 mL/min per 1.73 m2, only 22 (31%) had a recorded diagnosis of CKD. Estimated

costs of opportunistic in-hospital KHC screening are below those of current Australian screening programs. Hospital in-patients frequently have a full KHC and most have abnormalities detected. Opportunistic inpatient KHC screening would have little impact on hospital costs, but may result in significant health benefits. The KHC should be included in routine discharge documentation. “
“KAMIJO-IKEMORI ATSUKO1,2, SUGAYA TAKESHI1, KIMURA KENJIRO1 Dichloromethane dehalogenase 1Department of Nephrology and Hypertension, Internal Medicine, St. Marianna University School of Medicine, Japan; 2Department of Anatomy, St. Marianna University School of Medicine, Japan Deterioration of diabetic nephropathy (DN) is largely determined by the degree of tubulointerstitial changes rather than the extent of histological changes in the glomeruli. Therefore, a tubular marker that accurately reflects tubulointerstitial damage may be an excellent biomarker for early detection or prediction of DN. Liver-type fatty-acid binding protein (L-FABP) is a 14 kDa small molecule that is expressed in the cytoplasm of human proximal tubules.

Moreover, it seems that caspase-11 also

Moreover, it seems that caspase-11 also HM781-36B mw regulates the cell death mechanism known as pyroptosis, a crucial defense mechanism against certain pathogens

escaping phagosome–lysosome fusion [4]. In this review, we will discuss the latest studies that highlight the emerging importance of caspase-11 driving the noncanonical inflammasome pathway and consider the implications of their conclusions. Murine caspase-11, also known as Ich-3 or caspase-4, is a member of the caspase-1 subfamily of proteases [5], sharing 46% identity with murine caspase-1. In humans, the ortholog of mouse caspase-11 may be either caspase-4 or caspase-5, based on amino acid sequence homology; however, only caspase-5 seems to be regulated in a similar way to murine caspase-11 in response to extracellular stimuli, such as lipopolysaccharide (LPS) and interferons [6]. Caspase-11 is synthesized as 43-kDa and 38-kDa precursors, but in contrast to other caspases, procaspase-11 expression requires inflammatory stimulation. Administration of LPS to mice induces rapid protein expression of procaspase-11

in thymus, spleen, liver, lung [5], and, in particular, in splenic macrophages and B cells [7]. As well as the purified form of LPS, whole Gram-negative bacteria (Vibrio cholerae, flagellin-deficient Salmonella enterica serovar Typhimurium (ΔFlag Salmonella), Escherichia coli, enterohemorrhagic E. coli (EHEC), Legionella pneumophila, Citrobacter rodentium), all of whose outer membranes contain LPS, can induce procaspase-11 expression in macrophages [3, 8-10], while Gram-positive

bacteria cannot [9]. Some of these pathogens activate primarily caspase-1 by the canonical PF-02341066 clinical trial pathway via NLRC4 (wild-type Salmonella and Legionella) or NLRP3 (V. cholerae) [11-13]. As LPS is specifically detected by Toll-like Adenosine triphosphate receptor (TLR) 4, researchers began to interrogate this pathway. It was shown that induction of procaspase-11 expression was delayed in Myd88−/− macrophages infected with ΔFlag Salmonella, although procaspase-11 processing itself remained intact [8]. TRIF is required for the processing of procaspase-11 into the cleaved caspase-11 forms (∼26–30 KDa) (Table 1) [8, 9]. However, the role of TRIF in procaspase-11 expression remains controversial. In two independent studies, it was shown that procaspase-11 upregulation was reduced in Trif−/− macrophages infected with C. rodentium [14], E. coli [14], and EHEC [9, 14]. In two other studies, although procaspase-11 induction was delayed in macrophages after ΔFlag Salmonella infection, the protein levels were maintained [8, 10]. These observations indicate that the role of TRIF in procaspase-11 induction may be context dependent. So how does stimulation of the TRIF pathway by LPS from Gram-negative bacteria mechanistically link to capase-11 production? A series of observations suggest that IFN-mediated pathways downstream of TRIF are key drivers of noncanonical inflammasome activation.

epidermidis biofilms and the reduction in coverage was significan

epidermidis biofilms and the reduction in coverage was significant (P<0.001) for strains PAO1,

6750, 14:2, 23:1 and 27:1, but not for 15159. As for the dual-species biofilms shown in Fig. 3, a pronounced effect was seen for Ceritinib mw strain 14:2. Similar effects were seen with the P. aeruginosa supernatants for the other S. epidermidis strains (Mia and C103), although the effects were less pronounced (data not shown). To determine whether the dispersal effect on S. epidermidis biofilms was due to cell lysis, S. epidermidis cells remaining in the biofilms after exposure to the P. aeruginosa biofilm supernatants were examined with the BacLight LIVE/DEAD stain. For all the S. epidermidis strains (Mia, C103 and C121), over 90% of the cells were viable after treatment

with each of the P. aeruginosa supernatants (data not shown). this website Similarly, the level of viability of the dispersed cells was over 90% as shown by staining or growth on 110 agar. In order to investigate what might be responsible for the variable effect of the P. aeruginosa strains (PAO1, NCTC 6750, 14:2, 23:1, 27:1 and 15159), biofilm supernatants were investigated for the release of a number of known virulence factors. The type strain PAO1 and the clinical isolate 15159 were found to be positive for the production of the quorum-sensing signal C4-HSL, while all the other strains were negative (Table 1). All the P. aeruginosa strains were positive for pyocyanin production, except 14:2 and 27:1, which were negative in this assay (Table 1). These results indicate that the repertoire of extracellular

products released from the CYTH4 cells varies according to the strain. The secretion of extracellular proteases from P. aeruginosa cells growing in biofilms was investigated with zymography of culture supernatants (Fig. 5a). This showed differences between the strains in their degree of gelatinase activity. The supernatants from the two laboratory strains: PAO1 and NCTC 6750 as well as the clinical isolate 15159 contained at least three major bands of proteolytic activity at >150, 70 and 50 kDa. The >150 kDa enzyme has been identified previously by immuno-blotting and N-terminal sequencing as a multimeric form of P. aeruginosa elastase (Schmidtchen et al., 2003). In the same study, P. aeruginosa alkaline protease was demonstrated to band at around 50 kDa. This 50 kDa band, but not the higher molecular weight fractions, was also present in supernatants from strains 23:1 and 27:1 while the culture supernatant from biofilms of strain 14:2 appeared to lack any proteolytic activity. SDS-PAGE of the same material under reducing conditions confirmed differences in the extracellular protein profiles between the strains (Fig. 5b). Two different protein banding patterns could be identified, with strains PAO1, NCTC 6750 and 15159 showing a similar pattern and 14:2, 23:1 and 27:1 strains sharing many common bands.

Data confirm that, under our experimental conditions, inhibition

Data confirm that, under our experimental conditions, inhibition of MPO by 4-ABAH inhibits the formation of NETs. Therefore the product of MPO, HOCl was supplemented directly to neutrophils and was observed to elicit NET release LDK378 in vitro (Fig. 4a,b). This effect was found to be specific to the product of MPO as another chlorine-based acid (hydrochloric acid) evoked no detectable NET release (Fig. 4c). To confirm the physiological relevance of our hypothesis, we then exposed neutrophils obtained from patients with CGD to HOCl

to ascertain whether NET release could be evoked, despite the absence of a functional NADPH oxidase system (confirmed by chemiluminescent assay, data not shown). Neutrophils from CGD patients did not release NETs when stimulated with PMA, but were able to release NETs upon exposure to exogenous HOCl (Fig. 4d). Taurine is found abundantly within the cytoplasm of neutrophils (at ∼50 mM [28]) and is known to neutralize HOCl by forming taurine chloramine and essentially removing H2O2 and HOCl to promote cell survival [29]. Indeed, taurine chloramine activates

Nrf2 and a battery of downstream cytoprotective anti-oxidant enzymes (including haem-oxygenase-1; glutathione-transferase; peroxiredoxin; thioredoxin), thus promoting cell survival [29]. Therefore the role of taurine was examined by its addition prior to stimulation of NET release using both PMA (to stimulate endogenous HOCl generation) and also following direct addition of HOCl (0·75 mM). FK506 solubility dmso Taurine treatment reduced NET release significantly in response to to PMA at 100 mM and in response to HOCl at only 10 mM (Fig. 4e). This difference is likely to be due to both taurine and HOCl being added exogenously, and therefore the HOCl was likely to have been neutralized prior to entering the cell, unlike PMA which stimulates HOCl generation by direct intracellular activation of PKC. Direct neutrophil exposure to 0·75 mM HOCl resulted in the release of NET structures between 30 and 70 min (Fig. 5a), whereas stimulation with PBS did not

result in release of nuclear DNA (Fig. 5b) and treatment with 1% Triton X-100 killed neutrophils almost instantly to release non-NET DNA (Fig. 5c). The recent discovery of NET release [2] led to a plethora of studies describing their potential physiological role as a vitally important anti-microbial strategy in humans. However, their apparent complete dependence upon ROS activity suggests that the physiological heterogeneity surrounding ROS generation probably also pertains to NET release. Thus neutrophil hyperactivity and hyper-reactivity [19] with respect to ROS release may lead to disproportionate, physiologically discordant and/or displaced NET release with potentially pathogenic sequelae, such as autoimmune disease [8–10].

However, the precise role of LFA-1 in the pathogenesis of EAE has

However, the precise role of LFA-1 in the pathogenesis of EAE has so far remained unclear. We describe here the disease development in LFA-1−/− mice compared with WT controls. Ablation of LFA-1 resulted in more severe EAE with increased demyelination and increased numbers of myelin oligodendrocyte glycoprotein-reactive CD4+ T cells in the CNS. However,

the production of the Pexidartinib cost pro-inflammatory cytokines IL-17 and IFN-γ was unchanged on the level of antigen-specific T cells. Interestingly, LFA-1-deficient mice showed a clearly reduced frequency of Treg in the inflamed CNS. Moreover, Treg counts in spleens and thymi of unimmunized LFA-1−/− mice were lower in comparison to the WT controls, indicating an impairment of Treg generation. In combination,

these results suggest a substantial role of LFA-1 in Treg generation and subsequent expansion of effector T cells and highlight the importance of Treg in limiting EAE. EAE is a T-cell-mediated inflammatory disease of the CNS and serves as an animal model for multiple sclerosis. The autoimmune phenotype can be induced in rodents sensitized to proteins such as myelin basic protein or myelin oligodendrocyte glycoprotein (MOG). The disease is initiated by infiltration of peripheral lymphocytes and macrophages into the CNS and is characterized by local selleck products inflammation and demyelination. The migration of leukocytes into the CNS is facilitated by interactions of cell-surface adhesion molecules and their endothelial ligands 1. The family of β2-integrins is involved in leukocyte–vascular cell interactions as well as in the communication between T cells and antigen-presenting cells. The αLβ2-integrin LFA-1 (CD11a/CD18) is widely expressed by leukocytes including peripheral blood lymphocytes, monocytes, and NK cells 2. Among the members of CYTH4 the β2 family of integrins, only LFA-1 is expressed by CD4+ T cells and CD4+ CD25+ Treg 3. Interestingly, CD18-deficient mice, which do not express

β2-integrins, showed an impaired development of thymic and peripheral Treg, but it remained unclear which of the β2-integrins is responsible for this phenotype 3. The function of LFA-1 in EAE has been extensively studied. However, in part controversial and conflicting results have been obtained. For example, treatment with anti-LFA-1 Ab led to either protection against EAE 4 or more severe disease development 5. More recently, a deficiency for LFA-1 was suggested to dampen EAE upon active induction of an autoimmune response 6. On the other hand, adoptive transfer of WT encephalitogenic T cells into LFA-1−/− mice profoundly exacerbated the EAE course in comparison to WT mice, indicating an anti-inflammatory role of LFA-1, which would limit disease progression 7. It remained, however, elusive how LFA-1 exerts its immunosuppressive effects.

Lgals3−/− mice developed more pronounced footpad swelling startin

Lgals3−/− mice developed more pronounced footpad swelling starting from 35 days postinfection and exhibited an increased parasite burden (at day 35) compared with WT mice (Fig. 1A). To examine the possible mechanisms underlying the increased susceptibility to L. MLN0128 major infection, we examined the impact of galectin-3 deficiency in different immune cell types. We found no significant differences in the frequency of F4/80+ macrophages, CD11c+

dendritic cells (DCs), and CD4+ and CD8+ T cells in draining LNs from Lgals3−/−- and WT-infected mice at day 35 postinfection (Fig. 1B). However, we found a higher percentage of CD4+CD25+ TREG cells in L. major infected Lgals3−/− versus WT mice (Fig. 1C). To further characterize this CD4+CD25+ T cell population, we isolated CD4+ T cells from Lgals3−/−- or WT-infected mice and analyzed the frequency of Foxp3+ cells within the CD4+CD25+ gate. The

percentage of CD4+CD25+Foxp3+ T cells was higher in draining LNs from Lgals3−/− compared with WT mice (Fig. 1D). To determine whether the number of TREG cells was increased at sites of infection in Lgals3−/− mice, footpad lesions were assessed for Foxp3 by immunohistochemistry. The frequency of Foxp3+ cells in the footpad tissue from Lgals3−/−mice was considerably higher when compared with WT mice (Fig. 2A and B). In addition, real-time RT-PCR analysis showed check details increased Foxp3 mRNA expression in footpad tissue from Lgals3−/−-infected animals as compared with their WT counterpart (Fig. 2C). else Of note, galectin-3 protein was detected at high levels in footpad tissue from WT mice (Fig. 2A; panel

a). As CD103 facilitates the homing and retention of TREG cells at sites of L. major infection [17], we examined whether expression of this molecule was altered in the absence of galectin-3. CD4+CD25+ T cells from L. major infected Lgals3−/− mice displayed higher CD103 expression compared with their WT counterpart. However, we found similar CD62L expression in CD4+CD25+ T cells from Lgals3−/− and WT mice (Fig. 2D), showing selectivity in galectin-3-mediated control of TREG cell specific markers. Taken together, these data suggest that endogenous galectin-3 controls the frequency of Foxp3+ TREG cells and modulates CD103 expression on these cells during the course of L. major infection. Because TREG cells were found at higher numbers both in draining LNs and footpad lesions of L. major infected Lgals3−/− mice, we investigated the contribution of endogenous galectin-3 to the suppressive function of these cells. CD4+CD25− T cells (TEFF) were purified from LNs of WT-infected mice (Fig. 3A) and were restimulated in vitro with L. major antigen in the presence of CD4+CD25+ TREG cells from either Lgals3−/– or WT mice at various TEFF:TREG ratios (Fig. 3B). Analysis of T-cell proliferation in co-cultures of TEFF:TREG cells (ratios of 1:1 and 1:0.

To remove SDS, gels were washed with renaturing buffer for 30 min

To remove SDS, gels were washed with renaturing buffer for 30 min at room temperature and incubation was then performed overnight at 37 °C on a shaking platform in developing GSK458 concentration buffer. Gels were stained with Coomassie blue G-250 in 20% ethanol for 3 h and destained in 25% ethanol. Protease-containing fractions were visualized as clear bands against a dark background. The total repertoire of extracellular proteins was also investigated by mixing biofilm culture supernatants with NuPAGE sample buffer

(Invitrogen) and subjecting them to electrophoresis on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels under reducing conditions for 1 h at 180 V. Gels were then stained with Coomassie blue according to the manufacturer’s instructions. For the detection of P. aeruginosa elastase, proteins from the gels were electroblotted onto PVDF membranes (Immobilon-P, Millipore) at 50 V for 2 h at 4 °C. After blocking with 5% skim milk in Tris-buffered saline with 0.05% Tween-20,

membranes were incubated first with a rabbit anti α-elastase antibody [a generous gift from Dr J. Fukushima; see also Schmidtchen et al. (2003)] diluted 1 : 750 and then an HRP-conjugated goat anti-rabbit Ig antibody diluted 1 : 2500. Antibody binding was visualized using the ECL Western blotting reagent (Pierce). The production of extracellular polysaccharides by P. aeruginosa strains was studied using the lectins Hippeastrum hybrid agglutinin (HHA) and Marasmium oreades agglutinin (MOA) (recognizing galactose and mannose residues, respectively) MLN0128 cell line (Ma et al., 2007). Twenty four-hour biofilms prepared as described below were washed twice in 100 μL PBS and then incubated with MOA or HHA [0.1 mg mL−1 in PBS (7 mM K2HPO4, 2.5 mM KH2PO4, pH 7.3, containing 0.1 M Erastin concentration NaCl)] for 2 h at room temperature. Biofilms were washed four times (100 μL) with PBS before examination

using CLSM. Statistical analysis was performed using a one-way anova with a Bonferroni post-test to compare different strains. Investigation of the different P. aeruginosa strains showed that they varied in their ability to form biofilms over 6 h in the flow cells. The clinical isolates (14:2, 23:1, 27:1 and 15159) and PAO1 showed a low degree of biofilm formation (1.5–5% surface coverage), while the type strain NCTC 6750 was a relatively good biofilm former (22% surface coverage) (Fig. 1a). Because we were interested in studying the effect of different P. aeruginosa strains on biofilm formation by S. epidermidis, the ability of a number of different, freshly isolated, S. epidermidis strains to form mono-species biofilms was also investigated. After 6 h of growth in flow cells, the clinical isolates of S. epidermidis showed substantial differences in biofilm-forming ability, with the surface coverage ranging from 0.4–0.2 mm2 for strains Mia, C103, C121 and C164, to 0.009 mm2 for strains C116 and C191 (Fig. 1b).