A total of 662 samples were collected

from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further Selleckchem BGJ398 characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty-five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified. “
“Despite several chemotherapeutic and preventative advances, opportunistic fungal infections

remain common unintended consequences of cancer treatment. Currently, cancer patients spend most of their time between treatments at home, where they can inadvertently selleck come across potential hazards from environmental and food sources. Therefore, infection prevention measures are of the utmost importance for these patients. Although clinicians closely observe patients throughout their treatment courses in the hospital, the focus of clinical visits is predominantly on cancer

care, and clinicians seldom provide recommendations for prevention of such infections. Herein, we provide practical recommendations for busy clinicians to help them educate patients regarding potential sources of fungal infections outside the hospital. “
“Although silver nanoparticles (SN) have been investigated as an alternative to conventional antifungal drugs in the control of Candida-associated denture stomatitis, the antifungal activity of SN in combination with antifungal drugs against Candida biofilms pheromone remains unknown. Therefore, the aim of this study was to evaluate the antifungal efficacy of SN in combination with nystatin (NYT) or chlorhexidine digluconate (CHG) against Candida albicans and Candida glabrata biofilms. The drugs alone or combined with SN were applied on mature Candida biofilms (48 h), and after 24 h of treatment their antibiofilm activities were assessed by total biomass quantification (by crystal violet staining) and colony forming units enumeration. The structure of Candida biofilms was analysed by scanning electron microscopy (SEM) images. The data indicated that SN combined with either NYT or CHG demonstrated synergistic antibiofilm activity, and this activity was dependent on the species and on the drug concentrations used. SEM images showed that some drug combinations were able to disrupt Candida biofilms.

Moreover, “best practices” for infant eye tracking, such as knowing which software tool enhances experimental flexibility, remain to be determined. The present investigation was designed to evaluate the temporal and spatial accuracy of data from the Tobii T60XL eye tracker through the use of visual latency and spatial accuracy tasks involving adults and infants. Systematic delays and drifts were revealed in oculomotor response times, and the system’s Panobinostat spatial accuracy was observed to deviate somewhat in excess of the manufacturer’s estimates; the experimental flexibility of the system appears dependent on the chosen software. “
“Although research

has demonstrated poor visual skills in premature infants, few studies assessed infants’ gaze behaviors across several domains of functioning in a single study. Thirty premature and 30 full-term 3-month-old infants were tested in three social and nonsocial tasks of increasing complexity

and their gaze behavior was micro-coded. In a one-trial version of the visual recognition paradigm, where novel stimuli were paired with familiar stimuli, preterm infants showed longer first looks to novel stimuli. In the behavior response paradigm, which presented infants with 17 stimuli of increasing complexity in a predetermined “on-off” sequence, premature infants tended to look away from toys more during presentation. Finally, during mother–infant face-to-face interaction, the most ICG-001 mw dynamic interpersonal context, preterm infants and their mothers displayed short, frequent episodes of gaze synchrony, and lag-sequential analysis indicated that both mother and infant broke moments of mutual gaze within 2 sec of its initiation. The propotion of look away

during the behavior response paradigm was related to lower gaze synchrony and more gaze breaks during mother–infant interactions. Results Docetaxel price are discussed in terms of the unique and adaptive gaze patterns typical of low-risk premature infants. “
“Fourteen-month-old infants were presented with static images of happy, neutral, and fearful emotional facial expressions in an eye-tracking paradigm. The emotions were expressed by the infant’s own parents as well as a male and female stranger (parents of another participating infant). Rather than measuring the duration of gaze in particular areas of interest, we measured number of fixations, distribution of fixations, and pupil diameter to evaluate global scanning patterns and reactions to emotional content. The three measures were differentially sensitive to differences in parental leave, emotional expression, and face familiarity. Infants scanned and processed differently happy, neutral, and fearful faces. In addition, infants cared for by both father and mother (divided parental leave) distributed their gaze more across faces than did infants primarily cared for by one parent (in this study, the mother).

The sequence of the complete TG2 gene obtained from the human intestinal epithelial cell line Caco-2 published by us in the National Institutes of Health (NIH) database [4], codifies for a protein of 687 amino acids long. TG2 acts as a monomer and has two closely located binding regions, one for Ca2+ and one for GTP, as TG2 also has GTPase activity. TG2 is expressed ubiquitously and has multiple physiological functions in processes such as blood clotting, wound healing, cell adhesion, cell signalling and apoptosis, among others [1–3]. TG2 has also been associated with pathological conditions, mainly inflammatory diseases find more such as encephalomyelitis and inflammatory myopathies, and neurodegenerative

disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases, as well as various types of cancer [5–7]. TG2 is involved at different molecular levels in the pathological processes of these disorders, associated mainly with protein cross-linking or deamidation, as well

as regulation of apoptosis. In particular, TG2 plays a critical role in the pathogenesis of coeliac disease (CD), because it is able to deamidate glutamine residues present in toxic proteins from wheat and related cereals. The deamidation of glutamine at selective positions leads to higher-affinity check details binding of deamidated peptides to human leucocyte antigen (HLA) proteins encoded by the CD predisposing alleles DQ2 (A1*0501, B1*0201) and DQ8 (A1*0301, B1*0302), and also to a higher gliadin-specific T cell stimulation [8–10]. The TG2 gene is regulated by the canonical nuclear factor (NF)-κB pathway in several cell lines, and it has been reported that in cancer and microglial cells TG2 can activate NF-κB

by blocking the inhibitor function of IκBα via polymer formation [11]. Consequently, there is a complex cross-regulation between TG2 activity and the NF-κB pathway, a mechanistic link that can be observed in inflammation and cancer. TG2 expression in human liver cells [12], intestinal epithelial cells [13] and Cell press rat small intestine cells [14] can be induced by proinflammatory cytokines such as interleukin (IL)-1, tumour necrosis factor (TNF)-α and interferon (IFN)-γ, thus amplifying the inflammatory cascade. Therefore, the development of specific TG2 inhibitors with reduced in-vivo toxicity could represent a novel therapeutic approach with the aim of modulating TG2 activity and reduce, or even abolish, the disease processes where the enzyme activity is dysregulated [15]. To this end, more detailed information about the biology and molecular regulation of the TG2 gene in inflammatory settings is needed. In this study, we evaluated the regulation of the TG2 expression by proinflammatory cytokines in different cell lines and particularly in the intestinal mucosa. We found that IFN-γ is the most potent inducer of TG2 expression, and acts synergistically with TNF-α.

Endogenous peroxidase

activity was blocked by incubation for 5 min in peroxidase block, diluted in 0·03% hydrogen peroxide in 95% ethanol. Following three rinses with distilled water, 0·05% Tris-buffered saline (TBS) for 5 min and 1% bovine serum albumin (BSA) in TBS for 10 min, the sections were incubated for 60 min at room temperature with the primary antibodies (mouse anti-human) diluted in 1% BSA/TBS in the following dilutions: anti-CD4 (clone 4B12; 1:20) and anti-CD8 (clone 1A5; 1:20) obtained from Novocastra and anti-forkhead box P3 (FoxP3) antibody (clone 236 A/E7; 1:50), obtained from eBioscience (San Diego, CA, USA). After rinsing with TBS, a secondary antibody (EnVision+ click here kit K4004; Dako, Carpinteria, PF-2341066 CA, USA) labelled with horseradish peroxidase was applied for 30 min at room temperature. Enzymatic activity was revealed

by a 5–10-min incubation with 3, 3′-diaminobenzidine (DAB) + substrate-chromogen (EnVision+ kit K4007; Dako), which results in a brown-coloured precipitate at the antigen site. Counterstaining was performed with aqueous Mayer’s haematoxylin (Merck, Darmstadt, Germany). Negative controls were performed with omission of the primary antibody. The sections and antibodies were examined using an LSM 510 microscope (Carl Zeiss MicroImaging, Oberkochen, Germany). Biopsies taken from 17 individuals, seven patients with psoriasis, two of whom had a positive elicitation reaction and 10

healthy controls, five of whom had a positive elicitation reaction, were prepared for the microarray study. Before taking these skin biopsies the skin was frozen using a liquid nitrogen spray to inhibit RNA degradation. The skin biopsies were placed immediately in liquid nitrogen and transferred to a −80°C freezer. For RNA extraction, the frozen skin biopsies were ground in liquid nitrogen, transferred to lysis/binding buffer (Applied Biosystems, Rotterdam, the Netherlands) and homogenized with a rotor stator (Polytron PT3000; Kinematica AG, Buch Adenosine triphosphate & Holm A/S, Herlev, Denmark). Total RNA was then extracted using the mirVanaTM isolation kit (Applied Biosystems) following the manufacturer’s specifications. RNA concentration was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the RNA quality was assessed using an Agilent RNA 6000 nano kit on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA was stored at −80°C. The microarrays used for this study were Human Gene 1·0 ST arrays (Affymetrix Inc., Santa Clara, CA, USA) containing probe sets of approximately 26 000 genes. Generation of cDNA, biotin-labelled cRNA and GeneChip hybridization was performed by the RH Microarry Centre at Rigshospitalet (Copenhagen, Denmark).

Hong et al. assessed the risk factors of BPH in 641 South Korean men in a community-based cross-sectional study of male participants aged 50–79 years.21 Age was the only significant demographic risk factor of BPH. The presence of chronic bronchitis and a high prostate specific antigen (PSA) level increased the risk by threefold and twofold,

respectively. The risk decreased DNA Damage inhibitor as drinking frequency increased. Physical activity three to five times a week reduced the risk relative to being active less than twice a week; however, engaging in physical activity nearly every day increased the risk 1.7-fold relative to being active up to twice per week. Interestingly, the risk was decreased as drinking frequency was increased.

However, physical activity three to five times a week reduced the risk relative to less or too much activity. In other studies LUTS have also been associated with lifestyle factors. In the Massachusetts Male Aging Study, 1019 men without prostate cancer were followed up for a mean period of 9 years and it was revealed that high levels of physical activity (top vs bottom quartile kcals/day OR 0.5, CI 1.1–3.0), cigarette smoking (OR 0.5, CI 0.3–0.8) decreased the risk of BPH.22 Total or fat calorie intake, sexual activity STA-9090 cost level, alcohol intake, BMI, waist-hip ratio (WHR), diastolic blood pressure, history of diabetes, hypertension, vasectomy, or serum levels of androgens or estrogens did not individually predict clinical BPH. However, Rohrmann et al.23 reported that moderate alcohol consumption and physical activity had protective effects against LUTS in older men, but current cigarette smoking was not consistently associated in their studies from the Third National Health and Nutrition Examination Survey (NHANES III) on 2797 men aged ≥60 years. Data from NHANES III also showed a relationship Thalidomide between markers of MS and LUTS, defined as having three of four urinary symptoms (nocturia, incomplete bladder emptying, weak stream, hesitancy).9,23 There is much evidence that BMI or WHR (abdominal obesity) increase the risk of BPH.

The Boston Area Community Health (BACH) survey is a population-based epidemiological survey of a broad range of urological symptoms and risk factors in a randomly selected group of 1899 men.24 Using ATP III guidelines to characterize MS and American Urological Association (AUA) symptom index (AUASI) to assess LUTS, the authors found the interesting result that there is a significant association between MS and voiding symptoms rather than with storage symptoms of LUTS. In the present study, the prevalence of MS increased as AUASI score increased in the mild symptom range (2–7), but stabilized with higher scores (Fig. 1). According to the BACH survey, the overall prevalence of MS was 29% and demonstrated the association of each LUTS and individual components of MS.

MRFs simultaneously inhibit the expression of genes instructing alternative lineages such as other MRFs or cytokines instructing opposing lineages (Fig. 1). Furthermore, considering the heritable maintenance of Neratinib purchase most T-cell subsets, MRFs can potentially propagate chromatin and gene states, perhaps even in the absence of the original signals and ERF activation. In these ways, even with a seemingly small initial regulatory footprint, once induced, MRFs can dominantly influence cellular phenotype. Recent genomic studies provide important

examples of complex transcriptional control of cellular differentiation, and underscore the co-operative and networked action of several

transcription factors in immune cell differentiation.[12-14, 30, 31, 39, 40] Whereas we can appreciate the simple significance of cellular instruction through over-expression of factors like MYOD, FOXP3, and in iPS cells, OCT4, SOX2, KLF4 and c-MYC, studies such as those discussed here reveal that such activity occurs through extensive collaboration with supporting factors. Indeed, experiments establishing the sufficiency of many MRFs for lineage instruction relied on stimulation-dependent over-expression and the coincident activation of crucial Vemurafenib nmr ERF co-factors. The integration of information in the form of co-ordinated binding of environmental

response factors and nuclear master regulator transcription Gefitinib order factors to regulatory sites across the genome (Fig. 1) represents an elegant strategy for initial instruction and subsequent stabilization of immune cell phenotype in response to environmental cues. ERFs play a dominant and immediate role in altering chromatin state and initiating transcription, followed by induced MRF expression resulting in positive feedback transcription loops that stabilize the cellular phenotype. These consecutive steps during CD4 T-cell subset differentiation can be projected onto Waddington’s epigenetic landscape, to indicate the contribution of key transcription factors to the restriction and instruction of developmental potential (Fig. 2). Given the function of MRFs to stabilize cellular phenotype it will be interesting to assess the mechanisms conferring this activity. If not prominent in chromatin remodelling during initial differentiation, are MRFs involved in the maintenance of chromatin accessibility and gene state, in the absence of ERFs, either in quiescent or proliferating cells? Additionally, while early studies of MRFs and STATs focused on signature Th genes with established function, like ifng and the Th2 cytokines, we have little understanding of most of the hundreds to thousands of enhancers that are activated predominantly by ERFs.

These inflammatory processes take place in the synovial membrane [4], and are characterized by lymphocyte

and macrophage invasion [5, 6] and elevated proinflammatory cytokines [7]. Because there are currently no therapeutic approaches to halt OA progression, much hope has been expressed regarding the development of new therapeutic strategies, including cell-based approaches. In this context, mesenchymal stem or stromal cells (MSCs) have been investigated extensively throughout the past two decades mainly for their regenerative potential [8-10]. Their immunosuppressive competence has, however, become another important field of research (overview in [11] and [12]). Therefore, MSCs have been investigated in animal Depsipeptide ic50 models of multiple sclerosis [13], pulmonary fibrosis [14], renal failure [15] and myocardial infarction [16]. In a clinical setting, MSCs have been used successfully as an immunosuppressive treatment in patients with severe graft-versus-host disease [17]. MSCs were also identified to play a crucial role in modulating the inflammatory processes in rheumatoid arthritis [18]. In an Selleckchem LEE011 animal model of collagen-induced arthritis, MSCs reduced inflammation significantly in

the joints by reducing proliferation and modulating cytokine expression [19]. The mechanisms of MSC-mediated immunosuppression are unclear and still controversial [20, 21], while representing a promising target of cell-based therapies in diseases with important inflammatory processes. MSCs have been proved to suppress T cell proliferation successfully both in vitro and in vivo [22, 23]. Recent studies have also shown that MSCs regulate and recruit regulatory T cells (Tregs) in a co-culture approach [24-26]. Tregs themselves have been identified as key players in numerous diseases, among them rheumatoid arthritis [2, 27]; however, until recently they have not been associated with OA pathogenesis [28, 29]. this website Although an important number of Phase I/II

studies using MSCs in OA have been started (overview on [30]) and these cells have already been used in small patient series [31], the underlying processes of both the regenerative properties and, more importantly, the immunosuppressive capacities of MSCs in OA, are only poorly understood. The aim of this study, therefore, was to analyse the effect of human MSCs from OA patients on Tregs in an allogeneic lymphocyte co-culture model. We compared MSCs derived from the bone marrow of a joint-adjacent bone and from the synovium of the affected joint to investigate whether the synovial MSCs located within the tissue affected by inflammation exerted different immunomodulatory properties. MSCs were isolated from bone marrow and synovial membrane of 34 patients (age 68 ± 12 years, 19 female and 15 male) that had been collected during total hip arthroplasty for primary OA Kellgren grades III and IV.

We measured the expression level of Arg1, iNOS and Fizz1 in PBS-, chitin- or glass-exposed macrophages from WT, Stat6-deficient or MyD88/TRIF-deficient mice by quantitative RT-PCR. IL-4- or LPS-exposed macrophages from WT mice served as positive controls for AAM Gamma-secretase inhibitor or classically activated macrophages, respectively. As expected, Arg1 and Fizz1 were induced by IL-4, whereas Arg1 and iNOS were induced by LPS (Fig. 3C). Chitin exposure did not result in upregulation of Fizz1, whereas Arg1 and iNOS were both weakly induced by chitin in WT but not Stat6- or MyD88/TRIF-deficient mice (Fig. 3C).

We therefore conclude that chitin-exposed macrophages did not acquire an alternatively activated phenotype consistent with the finding that the inhibitory activity was also observed in cultures with splenocytes from Stat6-deficient mice (Fig. 3D). Addition of exogenous L-arginin to cocultures of T cells and chitin-exposed macrophages did not restore T-cell proliferation, leading us to conclude that the inhibitory activity

was not due to Arg1-mediated depletion of L-arginin from the culture medium (Fig. 3E). Nitric oxide concentrations in culture supernatants from chitin-exposed macrophages were not increased which demonstrates that the weak chitin-induced iNOS mRNA expression did not result in detectable iNOS enzymatic activity Erlotinib ic50 (Fig. 3F). To determine whether cell–cell contact was required for the inhibitory activity or whether inhibition was caused by factors in the culture supernatant, we stimulated splenocytes in the presence of chitin-exposed macrophages. T-cell proliferation was not inhibited by supernatants from chitin-exposed macrophages (Fig. 3G). Therefore, we conclude that the inhibitory activity requires cell–cell contact. The potent inhibitory receptor PD-1 is expressed on activated T cells and binds to the B7 family members B7-H1 (PD-L1) or enough B7-DC (PD-L2). To determine whether chitin-exposed

macrophages express either of these ligands, we stained chitin-exposed BM-derived macrophages (BMDM) with mAb against B7-H1 or B7-DC. B7-H1 was induced by chitin but not by glass beads, whereas no expression of B7-DC could be detected (Fig. 4A). The increased expression of B7-H1 correlated with the amount of chitin used to stimulate the macrophages (Fig. 4B). Since chitin has recently been shown to induce expression of IL-17A and IL-17 receptor in macrophages by a TLR2-dependent pathway 18, we determined the induction of B7-H1 expression in BMDM from TLR2-deficient mice and other knockout strains. Interestingly, B7-H1 expression was induced independently of TLR2, TLR3, TLR4, MyD88, TRIF and Stat6 which demonstrates that neither contamination with low amounts of LPS nor signaling via TLR or Stat6 was required for induction of B7-H1 (Fig. 4C).

Pregnant mothers admitted to the Labour and Delivery ward at McMaster University Medical Centre, Hamilton, ON, Canada provided informed consent before delivery Cilomilast concentration for CB donation. The CB samples were collected from otherwise healthy pregnant women as we were interested in investigating the mechanisms in CB CD34+ cells. Upon delivery, each CB sample was collected

in a 60-ml syringe containing 2 ml heparin (1000 units/ml; Sigma, Mississauga, ON) and stored at 4°C until processing. This study was approved by the Hamilton Health Sciences/McMaster Faculty of Health Sciences Research Ethics Board. Cord blood samples were depleted of erythrocytes using gravity sedimentation as previously described.[12] To enrich the sample for CD34+ cells, the pellet was resuspended at a concentration Pexidartinib of 5 × 107 cells/ml in RoboSep Buffer (PBS containing 2% fetal bovine serum and 1 mM EDTA; Stem Cell Technologies, Vancouver, BC). The cells were transferred to a 5-ml Falcon polystyrene round-bottom tube (Becton Dickenson 2058, Franklin Lakes, NJ) and EasySep Negative Selection Human Progenitor Cell Enrichment Cocktail with CD41 depletion (Stem Cell Technologies) at a concentration of 50 μl/ml cells was added. The solution was mixed

and incubated for 15 min at room temperature. The magnetic nanoparticles (Stem Cell Technologies) were added at a concentration of 50 μl/ml cells and incubated for 15 min at room temperature. The cell suspension was then brought to a total volume of 2·5 ml by adding RoboSep Buffer and the tube was placed inside the RoboSep Magnet (Stem Cell Technologies) for 10 min at room temperature. This sample was further enriched by placing the liquid portion in a new 5-ml tube and re-incubating the sample in the magnet for 10 min. The purity of CD34+ cells was between 85 and 90%. Lipopolysaccharide from

Escherichia Fludarabine supplier coli 0111:B4 was purchased from Sigma and used at the optimal concentration of 10 μg/ml as previously reported.[12] For stimulation studies, CD34+ enriched cells were stimulated with LPS overnight (37°C in 5% CO2) in tissue culture plates (Falcon Plastics, Oxnard, CA) supplemented with RPMI complete (RPMI-1640, HEPES, Penicillin/Streptomycin and fetal bovine serum). After overnight incubation, cells were centrifuged and resuspended in FACS buffer for flow cytometry staining. Immunofluorescent staining for GM-CSFRα and IL-5Rα expression were performed as previously described.[12] Analysis of intracellular proteins followed a protocol that was described previously.[16] Briefly, following incubation (37°C in 5% CO2) of enriched CB CD34+ cells with LPS for 5, 15, 30, 45 or 60 min, cells were fixed using PhosFlow CytoFix Buffer (BD Biosciences, Mississauga, ON, Canada), and then centrifuged for 10 min at 523.656 g.

5% bovine serum albumin; ELISA buffer) and incubated. Bound IgG antibodies were detected by adding 50 μL/well of peroxidase-conjugated anti-mouse IgG (1:2000 in ELISA buffer) and incubated at 37°C for 1 hr. The color reaction was developed by adding 100 μL/well of o-phenylenediamine dihydrochloride (Sigma, St Louis, MO, USA) in the presence of 0.07% H2O2 for 30 min at room

temperature, and the absorbance at 450–620 nm was measured. The mTOR inhibitor results for each serum sample were reported as the positive–negative difference (P–N), that is, the difference of the optical density (OD) with the positive antigen to the OD with the negative antigen; NusA -Tag protein was expressed from E. coli. Rabbit anti-TBE virus E protein IgG (23) was coated onto 96-well microplates (50 μL/well, 5 μg/mL in carbonate buffer). After overnight incubation at 4°C, the plates were washed five times with PBST. A blocking solution was applied (200 μL/well) and the plates were incubated at 37°C for 1 hr. The plates were washed before adding the SP antigen (50 μL/well, 1:150 dilution in ELISA buffer) and incubated at 37°C for 1 hr. After washing with PBST, the serum samples were added in duplicate (50 μL/well, 1:100 dilution in ELISA buffer)

and incubated at 37°C for 1 hr. Bound IgG antibodies were detected by adding 50 μL/well of ALP-conjugated anti-mouse IgG (1:5000 in ELISA buffer) and incubating at 37°C for 1 hr. The color reaction was developed by adding 100 μL/well of p-nitrophenyl phosphate and

incubating at 37°C for 60 min, and the absorbance http://www.selleck.co.jp/products/Adrucil(Fluorouracil).html at 405–620 nm was measured. The results for each serum sample were reported as the P–N, that Z VAD FMK is, the difference of the OD with the positive antigen to the OD with the negative antigen, which was prepared from the supernatant of non-transfected 293T cells. The OD values of each ELISA were compared with the results of the neutralization test. The sensitivity and the specificity of the ELISA were calculated corresponding to each cut-off value. The sensitivity was the ratio of the number of positive sera for ELISA and the neutralization test to the number of positive sera for the neutralization test. The specificity was the ratio of the number of negative sera for ELISA and the neutralization test to the number of negative sera for the neutralization test. The cut-off value that showed the minimum difference between the sensitivity and the specificity was used as the cut-off value of each ELISA. To prepare the recombinant antigen, we first attempted to express the whole E proteins of the TBE virus in E. coli, but the proteins were expressed as insoluble proteins and could not be applied to the ELISA (data not shown). Next, domain III of the E protein of the Oshima 5–10 strain was expressed as a fused protein with NusA -Tag protein (EdIII). To confirm and characterize the EdIII antigen, expressed proteins were analyzed by SDS-PAGE and Western blot (Fig. 1).