As an additional staining, Gallyas-Braak was performed for selected sections. For immunohistochemistry, the following primary antibodies were used: mouse monoclonal anti-phosphorylated neurofilament protein (p-NFP) (Clone SMI31; diluted 1:5000; Covanse, Princeton, NJ, USA), rabbit polyclonal anti-ubiquitin (diluted 1:400; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-Cu/Zn SOD (SOD1) (diluted 1:2000; Stressgen Bioreagents, www.selleckchem.com/products/PD-0325901.html Victoria,

Canada), mouse monoclonal anti-phosphorylated tau protein (p-tau) (clone AT8; diluted 1:1000; Innogenetics, Ghent, Belgium), mouse monoclonal anti-tau protein 3-repeat isoform RD3 (Clone 8E6/C 11-05-803; diluted 1:2000; Millipore, Billerica, MA, USA), mouse monoclonal anti-tau protein 4-repeat

isoform RD4 (Clone 1E1A6-05-804; diluted 1:100; Millipore, Billerica, MA, USA), mouse monoclonal anti-transactivation response DNA-binding protein of 43 kDa (TDP-43) (Clone 60019-2-Ig; Epitope amino acids 203–209; diluted 1:4000; Proteintech Group, Chicago, IL, USA), mouse monoclonal anti-TDP-43 (Clone K1B8; Epitope amino acids 1–260; diluted 1:3000; LifeSpan Biosciences, Seattle, WA, USA), mouse monoclonal anti-TDP-43 phosphorylated at 403/404 codons (p-TDP43) (Clone 11-9; diluted 1:3000; Cosmo Bio, Tokyo, Japan), and rabbit polyclonal anti-fusion, TLS, translocated in liposarcoma protein, pigpen, POMp75 (FUS) (Clone polyclonal; Epitope amino acids 1–50; diluted 1:200; Sigma-Aldrich, St. Louis, MO, USA). Prior to staining for SOD1, RD3, RD4, TDP-43, BMS-354825 datasheet Etofibrate p-TDP43 and FUS, sections were pretreated by microwaving in 10 mmol/L citrate buffer, pH 6.0 (800 W, 95°C, 5 min). These primary antibodies were diluted with phosphate-buffered saline (PBS), pH 7.5 containing 5% bovine serum albumin. All sections were incubated at 4°C overnight. Following secondary antibody administration, the sections were washed and incubated with the avidin-biotinylated enzyme complex using the respective Vectastain Elite ABC kits (Vector Laboratories, Peterborough, UK), and immunoreactive product deposits were finally visualized with 0.5 mg/mL 3,3′-diaminobenzidine tetrahydrochloride as the chromogen (Sigma-Aldrich, Dorset,

UK) mixed with 0.05% hydrogen peroxidase in PBS. After taking microphotographs of HE-stained abnormal structures, the sections were decolored in 70% ethanol containing 1% hydrogen chloride, washed in distilled water, quenched with hydrogen peroxide, rinsed in PBS, and incubated with the antibodies as described above to identify immunohistochemical localization of the antigens. Genomic DNA was extracted from frozen brain tissue by standard methods. The entire coding region of the SOD1 gene (MIM 147450) was amplified by performing PCR, and sequenced with an Applied Biosystems 3130 DNA sequencer (Life Technologies, Carlsbad, CA, USA). The research procedure was approved by the ethics committees of Hiroshima University and Kansai Medical University.

model. According to the RPA Guidelines, it is reasonable to withhold dialysis treatment if the patient is over 75 years of age with two or more of the following risk factors: A response of ‘No, I would not be surprised if my patient died within the next 12 months’ to the Surprise Question. Patients with high comorbidity scores (e.g. MCS ≥ 8). Marked check details functional impairment (e.g. Karnofsky performance status score < 40). Severe chronic malnutrition (serum albumin < 25 g/L using the bromcresol green method). At present we suggest using the following predictive

models and risk calculators for decision-making: For CKD stage 3 to 5 patients: The JAMA KFRE in patients with CKD stages 3 to 5.[1] For patients being considered for a non-dialysis pathway (particularly the elderly): The clinical score by Couchoud et al.[18] involving a mortality risk score obtained from nine risk factors. The Surprise Question (despite lack of validation in this population).[16] For dialysis patients being considered for transition to a non-dialysis pathway (particularly the elderly with comorbidities):

Inclusion of the Surprise Question into regular clinical practice for all dialysis patients, for example monthly patient review.[16] The MCS.[3, 5, 8] The clinical Hydroxychloroquine supplier score by Cohen et al.[9] involving a mortality score obtained from combining the answer to the Surprise Question with four routine Immune system variables – age, serum albumin, presence of dementia and peripheral vascular disease.[9] Predictive modelling and risk calculators can provide a prognostic perspective and highlight the likely outcomes in this largely elderly population with multiple comorbidities and limited functional

status. However, a predictive model that comprehensively incorporates variables relevant to the prognostic outcome of the non-dialysis population has yet to be developed. As such, we have made recommendations taking into consideration the strengths and weaknesses of pre-existing predictive tools. It is important to also recognize the weaknesses that currently exist with the development and use of multivariable risk prediction models.[7] Elizabeth Josland Patients with end-stage kidney disease (ESKD) are known to have a worse quality of life (QOL) than age-matched general population What constitutes a poor QOL of life varies from person to person and the potential impact of dialysis on an individual will be unique for each person Patients need good information in order to allow them to assess the potential impact of renal replacement therapy on their lives The Short Form 36 Health Survey (SF-36) QOL questionnaire is a suitable tool to be used in dialysis and non-dialysis patients to assess QOL changes The quality of life (QOL) of patients with end-stage kidney disease (ESKD) is known to be worse than that of the general population.

PET scans, demonstrating increased cellular glucose uptake, are used primarily to assess tumour metastases. selleck chemical They are also useful in detecting large vessel inflammation (Fig. 12) [61]. Computed tomography (CT) angiography demonstrates vessel involvement in Takayasu’s arteritis, but is limited by its use of ionizing radiation [62]. Angiography is the standard investigation to determine the extent of vessel involvement in polyarteritis nodosa, but imaging with magnetic resonance angiography, CT and CT angiography are alternative non-invasive techniques [63,64].

Imaging in small vessel vasculitis provides useful information on organ inflammation and damage. CT and MRI scans of the paranasal sinuses demonstrate characteristic features

in Wegener’s granulomatosis (Fig. 13) [65,66]. A high resolution CT (HRCT) scan of the lungs will provide diagnostic and prognostic information in AASV (Fig. 14) [67]. Various diseases mimic vasculitis, for example infective endocarditis, embolism from atrial myxoma 3-MA datasheet or atheroma, thrombotic disorders such as anti-phospholipid syndrome and drug-induced vasospasm [68]. The potential for confusion is compounded by the occurrence of ANCA positivity in some patients with infective endocarditis and cholesterol emboli. If suspected, these should Tolmetin be investigated with echocardiography, clotting studies, anti-phospholipid antibodies and a history of recent medication. Other diseases may cause a secondary vasculitis; these include

connective tissue diseases, rheumatoid arthritis, viral infections, malignancies or drugs. Serological tests include anti-nuclear antibody (ANA), anti-double-stranded DNA (dsDNA), complement, rheumatoid factor (RF) and anti-citrullinated peptide antibody (ACPA). Infection screens include hepatitis B and C, human immunodeficiency virus (HIV) and cryoprecipitates, particularly in cutaneous vasculitis. Vessel size is the key discriminator in the definition of primary systemic vasculitis. While not ideal, this allows the grouping of diseases which can cause significant renal disease and are associated with the highest mortality if untreated. These are the ANCA-associated vasculitides (AASV). The AASV are a group of overlapping syndromes, associated with, but not exclusively having, a positive test for P or C-ANCA and have similar clinical and histological features. They are characterized by necrotizing small to medium vessel inflammation without immune deposits. Tables 3–5 summarize the main features of these conditions and are adapted from the Chapel Hill Consensus definitions [48]. Granulomatous inflammation is similar in Wegener’s granulomatosis and Churg–Strauss syndrome.

While CF patients were exclusively colonised with either S. prolificans or LY2606368 datasheet P. boydii, patients with other severe underlying diseases were colonised or infected with several (in total: six species) Scedosporium species. Remarkable is that CF patients, who were monitored over up to almost 5 years, were exclusively

colonised with a single Scedosporium species. Due to the limited amount of data, we cannot yet see species-dependent clinical prevalence or a correlation with underlying diseases. At the moment, VOR is the only licensed antifungal agent for the treatment of Scedosporium infections in Europe; all other antifungals are used off-label. MIC/MEC breakpoints for Scedosporium species have not yet been determined. Published studies of susceptibility profiles of Scedosporium species taking the latest taxonomical changes into account are lacking, since the separation

of P. apiosperma from P. boydii was published only in 2010.5 These two sibling species were found to have very similar susceptibility profiles, being most susceptible to MICA and VOR. Our results show that in general, MICA had reasonable in vitro activity against all Pseudallescheria/Scedosporium species except S. prolificans (Table 2). Monotherapy using VOR has frequently been reported to be tolerated by patients Selleck LBH589 and was successful in treatment of S. apiospermum infections.25–28 MICA exerts antifungal activity via inhibition of (1,3)-β-d-glucan synthase,29 and therefore may enhance in combination therapy the fungicidal effect of other antifungal compounds targeting different cellular elements. Flavopiridol (Alvocidib) In vitro synergistic effects of azoles combined with echinocandins were reported by Cuenca–Estrellas et al.1 Other studies demonstrated a profound synergistic effect of azole-terbinafin combinations.3–32 Therefore, in addition to terbinafin, MICA should be also taken into consideration as possible combination therapy option for Scedosporium infections, preferably in combination

with VOR. In contrast to other fungi, such as Aspergillus fumigatus and Candida albicans, the molecular epidemiology of Scedosporium/Pseudallescheria has received far less attention in medical literature. The few studies having addressed this are reviewed by Harun et al.33 More importantly, since the latest taxonomical change in 2010, no studies have addressed the molecular epidemiology of these fungi in patients; so, the true value of several previous studies cannot be ascertained. In 2003, AFLP was shown to be a powerful method for identifying closely related Canidida species, including differentiation between the sibling species C. albicans and C. dubliniensis.34 A similar observation was made for filamentous fungi exemplified on A. fumigatus and its sibling species Neosartorya fisheri, by Klaassen and Osherov.35 In addition, AFLP analysis can provide high resolution fingerprints for intraspecific discrimination.

1). BMDCs lacking both DAP12 and FcRγ had no staining for TREM-2 similar to those grown from TREM-2-deficient BM, suggesting that FcRγ may minimally contribute to cell surface expression of TREM-2 in these cells. To address whether TREM-2 regulates TLR responses in DCs, we generated BMDCs from WT and TREM-2-deficient mice. We first investigated whether TREM-2 deficiency affected DC development from BM cells cultured in the presence of GM-CSF.

Total cell number was decreased in TREM-2-deficient BM cell culture after 5 days of culture (Supporting Nutlin-3 molecular weight Information Fig. 1A), however the percentage of total cells that were CD11c+ DCs was not changed between WT and TREM-2-deficient cultures (Supporting Information Fig. 1B). We next stimulated these BMDCs using various TLR ligands (LPS, CpG DNA and Zymosan) for 16 h and performed ELISA to evaluate secretion of IL-12 p70 and TNF. Though Zymosan is a complex particle selleck kinase inhibitor that interacts with multiple pattern recognition receptors, such as dectin-1, it also signals through a TLR2/TLR6 heterodimer 18, 19. TREM-2-deficient DCs produced significantly more IL-12 p70 than WT DCs after stimulation with a range of doses of LPS, CpG DNA and Zymosan (Fig. 2A). TNF secretion from TREM-2-deficient DCs was

modestly increased over WT DCs (Fig. 2B). In addition to IL-12 p70 and TNF, IL-6 and IL-10 secretion was also higher in TREM-2-deficient DCs than WT DCs after stimulation with these TLR ligands (Fig. 2C and D). Interestingly, we did not see any cytokine production from unstimulated TREM-2-deficient DCs (Ito and Hamerman, unpublished observation). We next compared pro-inflammatory cytokine secretion between WT, DAP12/FcRγ-deficient

and TREM-2-deficient DCs (Fig. 3A). DAP12/FcRγ-deficient and TREM-2-deficient DCs showed higher IL-12 p70 production than WT DCs after 16 h stimulation with CpG DNA or Zymosan (Fig. 3A). The TLR responses in TREM-2-deficient DCs were lower than DAP12/FcRγ-deficient DCs (Fig. 3A). We also compared the pro-inflammatory cytokine production of WT, DAP12-deficient, DAP12/FcRγ-deficient and TREM-2-deficient BMDCs by intracellular cytokine staining. After both 2 and 6 h stimulation with CpG DNA, the Methocarbamol percentage of IL-12 p40+TNF+ cells was higher in TREM-2-deficient, DAP12-deficient and DAP12/FcRγ-deficient DCs than in WT DCs (Fig. 3B). Consistent with the ELISA results (Fig. 3A), DAP12/FcRγ-deficient DCs showed the highest percent of IL-12 p40+TNF+ cells after CpG DNA stimulation (Fig. 3B). Both TREM-2-deficient and DAP12-deficient DCs showed an intermediate phenotype of pro-inflammatory cytokine production in between WT and DAP12/FcRγ-deficient DCs in response to CpG DNA (Fig. 3B). Furthermore, the cytokine staining pattern of TREM-2-deficient DCs was very close to that of DAP12-deficient DCs, suggesting that TREM-2 inhibits TLR responses primarily through DAP12 in DCs.

Bioinformatic analysis revealed that sMTL-13 belongs to the ricin-type β-trefoil family of proteins containing a Sec-type signal peptide present in Mtb complex species, but not in non-tuberculous mycobacteria. Following heterologous expression of sMTL-13 and generation of an mAb (clone 276.B7/IgG1κ), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non-tuberculous

mycobacteria-derived culture filtrate proteins. In addition, sMTL-13 leads to an increased IFN-γ production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab selleck screening library against sMTL-13, a response found to be

decreased following successful anti-tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin-like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment. Tuberculosis (TB) remains a major public health problem in both developing and industrialized countries 1, 2. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, is one of the most successful human pathogens and epidemiological studies estimated that one-third of the world population is infected with the bacterium 1, 2. Although Mtb remains viable in the majority of the infected subjects, only 5–10% of individuals develop active disease later in life 1, 2. However, the mechanisms for the breakdown of latency are largely unknown 3. Evidence suggests see more that both humoral and cellular immune responses are implicated in host resistance against Mtb and cell-mediated immunity is thought to be the major component for protection 1, 4–7. While effective immune responses are critical to control Mtb growth inside macrophages, it has been demonstrated that mycobacteria-associated factors play an important role in TB immunopathogenesis 8–10. Thus, secreted molecules are amongst

the possible candidates that influence pathogen–host interactions Adenosine in vivo. Secretion of proteins is a critical process for bacterial virulence. Mtb possesses a specialized secretion system to transport virulence factors across their unique cell envelope 11, 12. Although the study of culture filtrate protein (CFP) preparations from Mtb has revealed a myriad of proteins, there remain several other molecules annotated as having “unknown function” 13, 14. For example, Malen et al. using a proteomic approach, have recently detected 257 secreted proteins in CFP fractions from the laboratory strain Mtb H37Rv 13. However, no function has yet been ascribed to 23% of those molecules. Polypeptides secreted by mycobacteria may modulate inflammatory processes and could serve as targets for immune protection.

6 ± 0.1 × 106 cells in control and immunized mice, respectively. The phenotype of the lymphocytes from NALT and NP was analysed by flow cytometry, as shown in Fig. 1. B cells

were more abundant than T cells, in both nasal tissues (NALT and NP) in control and immunized mice. In NP, the proportion of B cells was increased in the immunized group nevertheless in PD-1/PD-L1 activation NALT its proportion was not affected by immunization (Fig. 1). The proportion of CD3+ T cells recorded in NALT was higher than in NP, but their proportion did not vary because of immunization in NALT or in NP (Fig. 1). However, the proportion of both CD4+ and CD8+ T cells diminished in NALT of immunized mice in relation to control mice. Moreover, in NP, an important change was observed in the proportions of these T cell subpopulations, because there was a significant increase in CD4+ and CD8+ T cells in the immunized group with regard to the control (Fig. 1). In addition, following immunization with Cry1Ac, the amount of double negative CD4−CD8−CD3+ T cells

was increased in NALT while it was diminished in NP. The intranasal immunization with Cry1Ac induced high numbers of anti-Cry1Ac-specific IgA and IgG antibody–secreting cell (ASC) responses in NALT and NP, with the IgA responses higher with regard to IgG. In NP, the number of ASC responses recorded was greater than that induced in NALT, especially the IgA isotype, which was approximately Unoprostone three times greater. While the number of specific IgG ASC responses also was greater in NP than in NALT (Table 1). In NALT, the magnitude of the ASC IgA and IgG responses elicited with Cry1Ac was comparable to Palbociclib in vivo that induced with CT; while in NP were recorded higher IgA and IgG responses in the group immunized with Cry1Ac in comparison with the group immunized with CT. However, it is important to mention that although CT was used as a reference of a well known potent mucosal immunogen, because of its toxicity we have to use a dose five times lower to

the one used for Cry1Ac, in addition the immunization protocol used may be not the optimal scheme to achieve the maximal anti-CT responses. To determine the effect of intranasal immunization with Cry1Ac in the activation of lymphocytes residing at the nasal compartments, we analysed by flow cytometry the proportion of B220+, T CD4+ and T CD8+ lymphocytes expressing the activation markers CD25 and CD69, in cells isolated from NALT and NP, from control and immunized. The data shown in Figs. 2 and 3 indicate the frequency of either CD25+ or CD69+ cells, calculated individually for each gated lymphocyte population expressing the corresponding surface marker (CD4+, CD8+ or B220+). The proportion of B220+ cells and CD4+ and CD8+ T cells expressing CD25 was higher in NP than in NALT in control mice, and it was significantly increased in both nasal tissues after intranasal immunization with Cry1Ac.

Immunostained cells were analysed using a fluorescence activated cell sorter [(FACS)Calibur, Becton Dickinson, San Jose, CA, USA]. Analysis of the Th17 cell population was PKC412 performed

by gating on CD3+CD8– T cells. Total RNA was extracted from PBMCs or TMCs using TRIzol reagent (Invitrogen). Total RNA was isolated and reverse transcription was performed according to the manufacturer’s instructions (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed by triplicate using Bio-Rad SYBR green super mix (Bio-Rad, Hercules, CA, USA). Primer sequences were as follows: retinoic acid-related orphan receptor γt (RORγt), sense, 5′-CCTGGGCTCCTCGCCTGACC-3′, anti-sense, 5′-TCTCTCTGCCCTCAGCCTTGCC-3′; and β-actin, sense, 5′-CACGAAACTACCTTCAACTCC-3′, anti-sense, 5′-CATACTCCTGCTTGCTGATC-3′. Samples were run in triplicate, and their relative expression was determined by normalizing to the expression level of β-actin. Data were analysed using Bio-Rad CFX Manager software. In the case of TMCs, leptin, IL-17 and RORγt cDNA products were amplified by PCR with

the following primer sequences: leptin, sense, 5′-TCCTGGGCTCCACCCCATCC-3′, anti-sense, 5′-TGCAGAGACCCCTGCAGCCT-3′; and IL-17, sense, 5′-CAAGACTGAACACCGACTAAG-3′, anti-sense, 5′-TCTCCAAAGGAAGCCTGA-3′. Amplified products were electrophoresed on 2% agarose gel (Invitrogen), stained with ethidium bromide and visualized with ultraviolet transilluminator. One-way analysis of variance (anova) was performed to determine whether there was an overall statistically significant change among the groups, and the post-test comparison was carried out using Bonferroni’s test. Student’s check details buy Docetaxel unpaired t-test was performed as appropriate. Correlations between variables were determined by Spearman’s correlation coefficient. Data were analysed with GrapPad Prism version 5 software. We first compared the basal plasma leptin levels of 27 female HT patients with 22 age-, sex- and BMI-matched female healthy controls. It was found that HT patients showed an increase of leptin which was at the border of statistical significance (P = 0·06, Fig. 1a). Subsequently, we analysed the correlation

between the level of plasma leptin and BMI in HT patients and healthy controls. The results showed that plasma leptin correlated positively with BMI in healthy controls, but no correlation was observed in HT patients (Fig. 1b,c). Furthermore, the level of leptin in culture of CD4+ T cells from HT patients was higher than that from healthy controls (Fig. 1d). Flow cytometric analysis revealed that an increased proportion of Th17 cells from peripheral blood mononuclear cells (PBMCs) was observed in HT patients compared with healthy controls (Fig. 2a,b). There were no statistically significant correlations between plasma leptin concentrations and the percentage of Th17 cells or the level of RORγt in HT patients (Fig. 2c,d).

1). Excessive Treg activity is observed in persistent

infections such as murine models of Leishmaniasis, malaria and tuberculosis [39–41] and in human diseases such as upper GI persistence of Helicobacter pylori, human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections [42–45], suggesting the possibility of a link between pathogen persistence and Treg-mediated suppression. Subversion of Treg function for the generation of appropriate immune responses to effect efficient pathogen clearance may therefore be an advantage or, indeed, a necessity. Indeed, accumulating evidence supports the LBH589 mouse assertion that interactions between Tregs and an infective/inflammatory environment leads to the subversion of their suppressive function. The salient experiments demonstrate a direct effect of Toll-like receptor (TLR) ligation on Tregs to block their suppression [46,47] and modulation of dendritic cell (DC) activity by lipopolysaccharide (LPS) to induce restricted Treg activity [48] in a manner that is

independent of direct ligation of the TLR on Tregs[49,50]. Indeed, appropriately activated DC can break the ‘anergic’ state of Tregs and promote proliferation in this usually hypoproliferative population [51]. Our own (unpublished) observations and those of others suggest that proinflammatory cytokines, selleck compound in particular IL-1β, IL-6 and tumour necrosis factor (TNF)-α, released by DC following interaction with pathogens, can subvert the suppressive effects of Tregs. Both IL-1β and IL-6 can block Treg-mediated suppression of effector cell proliferation [48,52], although IL-6 may require the presence of IL-1 to overcome regulation [49]. There are some data from humans to suggest that TNF-α

can inhibit Treg function [53] with some supporting, but circumstantial, evidence showing a numerical increase in forkhead box P3 (FoxP3)+ Cyclin-dependent kinase 3 T cells and restoration of defective regulatory function in patients with rheumatoid arthritis treated with anti-TNF-α therapy [54]. The inevitable question is whether subverted Tregs remain ‘dormant’ Tregs or undergo a stable change of phenotype to an alternative lineage. IL-17 is a proinflammatory cytokine with non-redundant functions in the clearance of extracellular pathogens (see also [55] for further detail). This is seen readily in both IL-17R-deficient mice, which demonstrate great susceptibility to lethal bacterial infections [56,57], and in IL-17-deficient humans as part of the hyper-immunoglobulin E (IgE) syndrome (HIES), where recurrent infections are a feature [58,59]. The significant proinflammatory features of IL-17 have been reviewed previously, as has the compelling evidence for the role of IL-17 in inflammatory/autoimmune conditions of mice and the considerable body of evidence suggesting an important role for IL-17 in the aetiopathogenesis of inflammatory and autoimmune diseases in humans [60,61].

V. vulnificus cells (107 CFU/mL) suspended in PBS with 1% BSA were inoculated into each 5 cm segment. After 8 hr, the rabbit was killed and the intestine removed. The fluid within the loops was collected with a syringe and the viable bacterial counts in each determined by plating on 2.5% NaCl HI agar plates. Overnight cultures of V. vulnificus strains were inoculated into fresh 2.5% NaCl HI broth and grown for 2 hr. After staining with Ruthenium red, the bacterial cells were observed with a JEOL JEM 1200 EXП electron microscope (Jeol, Tokyo, Japan). Vibrio vulnificus strains Smoothened Agonist order were freshly grown on HI agar plates with 1.5% agar at 37°C. The bacteria

were inoculated onto semisolid HI agar plates containing buy PD98059 0.3% agar and incubated for at 37°C for approximately 8 hr, as previously described [31]. HeLa cells were seeded into four-well LabTec chamber slides (Nunc, Naperville, IL, USA) and bacterial adhesion assayed as previously reported [31]. Briefly, V. vulnificus cells were infected at an MOI of 250 for 30 min. HeLa cells were thoroughly washed three times with pre-warmed DMEM and stained with Giemsa solution (Merck, Darmstadt, Germany). Bacterial cells adhering to 90 HeLa cells were counted and the results reported as the average number of adhered bacteria per HeLa cell. Hemolytic and proteolytic activities in bacterial culture supernatants were assayed according to a previous report [12]. β-galactosidase activities

of PvvhA::lacZ and PvvpE::lacZ transcriptional reporters in V. vulnificus strains were assayed as previously described [12]. SPF 7-day-old CD-1 female mice were used for oral administration and 8-week-old mice for intraperitoneal injections. For each dose, five mice were given 10-fold serially diluted log Cetuximab ic50 phase bacterial suspensions. For iron-overload experiment, 8-week-old CD-1 mice were injected intraperitoneally with 900 µg of ferric ammonium citrate for 30 min before bacterial challenge. The infected mice were observed for 48 hr and LD50 values calculated by the Reed and Muench method [32]. This animal study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals

of the Korean Food and Drug Administration. The protocol was approved by the Chonnam National University Committee on the Ethics of Animal Experiments. All efforts were made to treat the mice humanely. Human cervical adenocarcinoma HeLa cells (Korean Cell Line Bank, Seoul, Korea) were maintained in high-glucose DMEM with 10% FBS (Gibco Invitrogen, Auckland, New Zealand) in a 37°C incubator with 5% CO2. HeLa cells cultured in eight-well glass chamber plates (Nalge Nunc International, Rochester, NY, USA) were infected with V. vulnificus strains at a MOI of 100 for 1 hr. F actin was visualized by Alexa Fluor 594-conjugated phalloidin and nuclei were stained with 4′,6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR, USA) as described previously [7].