2). Both techniques are compatible with CLSM (Haagensen et al., 2011; Weiss Nielsen et al., 2011). Static growth conditions Everolimus manufacturer are obtained by culturing cells in a growth chamber that is attached to a microscope slide (Fig. 2a). The static growth system has the advantage that it is easy to set up and the disadvantage that growth conditions

are not easily controlled. Flow cells are composed of a chamber through which medium flows and a cover slip on which biofilm forms (Fig. 2b). The flow-cell system has a continuous supply of nutrients that is easily changed, for example, for administration of antifungals with minimal biofilm disturbance (Weiss Nielsen et al., 2011). CLSM of biofilm formed in flow cells is a powerful tool to study gene regulation upon changing environmental conditions and can be used to study regulation of, for example, FLO genes by the use of FLO promoter-GFP fusions in the biofilm-forming cells. The CLSM flow-cell method can also be used to visualize phenotypic variabilities and bistabilities in the biofilm such as variation in repression of FLO5, 9, 10 and bistabilities in FLO11 expression generated by Hda1. While many bacterial biofilms are formed on glass surfaces, S. cerevisiae biofilms are observed on polystyrene surfaces (Reynolds & Fink, 2001). However, some polystyrenes are autofluorescent and interfere with CLSM recording. Polyvinyl coverslips are an optimal choice as a surface

for yeast biofilm development and CLSM imaging, as this plastic supports biofilms and is not autofluorescent in the range of the common fluorophores (430–610 nm) (Haagensen et al., 2011; Weiss Nielsen et al., 2011). Three-dimensional biofilm structures selleck compound can be quantified using comstat software, based on the stack of images acquired by CLSM (http://www.comstat.dk). Features calculated by COMSTAT include biovolume, learn more area occupied by cells in each layer, thickness, substratum coverage, fractal dimension, roughness, surface-to-volume ratio, number of microcolonies and microcolony size (Heydorn et al., 2000a, b). Although this software is mainly used for quantification of bacterial biofilms, it will be a valuable tool for objective quantitative

analysis of yeast biofilms (Seneviratne et al., 2009). Fluorescent markers for CLSM are relatively easily integrated in the S. cerevisiae genome. The high frequency of homologous recombination allows for one-step gene replacement between a DNA cassette and a corresponding genomic sequence with as little as 35 bp of genomic homology (Rothstein, 1983; Wach et al., 1994). This unique feature and others have led to the synthesis of two complete deletion strain collections of S. cerevisiae (Giaever et al., 2002; Dowell et al., 2010), and GFP fusions to most S. cerevisiae gene products (Huh et al., 2003). A powerful resource for identification of genes involved in biofilm development is an almost complete collection of deletion mutants in the biofilm-forming S.

We further demonstrate that CD4+CD25+Foxp3+ TREG cells readily inhibit these responses and mediate disease protection, which correlates with their accumulation in the draining LN and lamina propria. Moreover, TREG cells can directly suppress γδ T-cell expansion and cytokine production in vitro and in vivo, suggesting a pathogenic role of γδ T cells in intestinal inflammation. Thus, functional alterations in TREG cells provoke dysregulated CD4+ and γδ T-cell responses to commensal

antigens in the intestine. The gastrointestinal tract represents a major site where immune tolerance mechanisms assure a homeostatic Selleck PLX4032 equilibrium between the mucosal immune system and commensal microorganisms 1, 2. Given the permanent co-existence of harmless and pathogenic bacteria that constantly trigger local immune responses, the intestinal mucosa must maintain tolerance in these sites. A disturbance in immune homeostasis of the human gut may provoke inflammatory bowel diseases (IBDs) like Crohn’s

disease (CD) and ulcerative colitis, both characterized by Torin 1 mw an abnormal accumulation of activated lymphocytes in the gut resulting in chronic intestinal inflammation 1–5. CD4+Foxp3+ TREG cells are widely recognized as dominant mediators responsible for the control of peripheral tolerance 6–10. Functional abrogation of these cells results in over-activation and uncontrolled inflammatory responses towards tissue-derived antigens and commensal bacteria, leading to the development of various chronic inflammatory disorders 10–13. Our current understanding of the role of Foxp3+

TREG cells in the prevention of IBD development is largely derived from mouse models where intestinal inflammation is induced by adoptive transfer of CD4+ T effector (TEFF) cells into lymphocyte-deficient nude, Reverse transcriptase SCID or RAG−/− hosts 14. Collectively, these studies show that CD4+Foxp3+ TREG cells prevent colitis development or even cure established disease by restraining pathogenic CD4+ T-cell and DC immune responses 15–18. However, other cellular targets of suppression in vivo remain ill-defined. Recently, increasing evidence points to a significant multi-faceted role for non-CD4+ lymphocytes, including γδ T cells, in the maintenance of intestinal homeostasis 19–21. More specifically, it has been shown that γδ T cells readily accumulate in inflamed tissues of IBD patients 22–25, although, in murine studies, γδ T cells have been shown to either potently reduce 26–28 or exacerbate inflammation 29–33. Some studies also identify γδ T cells as a source of rapidly activated T cells with Th17-like effector properties providing the first line of defense against pathogens 34–36.

Bacterial isolates were stored at −80 °C in brain heart infusion broth +20% glycerol. Isolates were cultured on chocolate agar plates with incubation at 35 °C +5% CO2 for 18–24 h and all tests were performed on a subculture of a single isolated colony. Identities of all isolates were confirmed by standard biochemical tests (Kilian, 2003) and 16S rRNA gene sequencing (Lau et al., 2004). www.selleckchem.com/products/PD-0332991.html Biotypes were assigned according to Kilian’s biotyping scheme based on three biochemical reactions: urease, indole and ornithine decarboxylase (Kilian, 1976). The nontypeable nature

of all 125 isolates was confirmed by slide agglutination test using antisera against all six serotypes purchased from commercial sources (Difco, Oakville, ON, Canada; Denka Seiken, Tokyo, Japan). The absence of both the serotype-specific and the capsule transport, CB-839 bexA, genes was confirmed by PCR using primers described by Falla et al. (1994). β-Lactamase production was detected using Dryslide Nitrocefin (BBL, Becton Dickinson, Oakville, ON, Canada). Disc diffusion test was carried out as described by the Clinical Laboratory Standards Institute (CLSI, 2008). The following antibiotics (Oxoid, Nepean,

ON, Canada) were tested: ampicillin (2 and 10 μg), amoxicillin–clavulanic acid (30 μg), cefaclor (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin oxyclozanide (5 μg), clarithromycin (15 μg), moxifloxacin (5 μg), sulfamethoxazole–trimethoprim (25 μg), azithromycin (15 μg), imipenem (10 μg), levofloxacin (5 μg) and tetracycline (30 μg). Detection of β-lactamase-negative ampicillin-resistant (BLNAR) strains was accomplished using two concentrations of ampicillin (Karpanoja et al., 2004). Hi BLNAR strain ATCC 49247 was used as a control in

each experiment. MLST was carried out by PCR amplification of seven housekeeping genes according to the previously described method (Meats et al., 2003), and the assignment of STs was conducted using the Hi MLST website (http://haemophilus.mlst.net/). Genetic relationships between isolates based on MLST data were also analysed by eburst (Feil et al., 2004) and concatenated sequences of the seven housekeeping gene loci using software available from the Hi MLST website cited above. Seventy isolates (56%) were from invasive disease cases and were recovered from normally sterile body sites (blood, 61 isolates; CSF, eight isolates; liver abscess, one isolate). The other 55 isolates (44%) were from the respiratory tract. The breakdown of the invasive isolates by year is as follows: eight isolates from 2000, eight from 2001, four from 2002, three from 2003, 13 from 2004, 20 from 2005 and 14 from 2006. Invasive isolates were from patients whose ages ranged from 1 day to 94 years.

Informed consent was obtained from all participants. Promastigotes of L. braziliensis (MCAN/BR/98/R69) and L. amazonensis (IFLA/BR/67/PH8) were cultured in Schneider’s medium supplemented with antibiotics (200 IU penicillin and 200 µg streptomycin/ml) and 10% inactivated fetal calf serum (all from Sigma-Aldrich, St Louis, MO, USA). Stationary phase promastigotes were washed three times in phosphate-buffered saline (PBS), selleck kinase inhibitor and disrupted by 10 freeze and thaw cycles, followed by ultrasonication (Ultra-tip Labsonic

System; Laboratory-Line, Melrose Park, IL, USA), at 40 watts for 15 min in an ice bath, to generate the crude extracts of L. braziliensis (LbAg) and L. amazonensis (LaAg). All antigenic preparations were adjusted to 1 mg/ml protein nitrogen in PBS and stored Everolimus at −70°C until use. PBMCs were isolated from heparinized venous blood by Ficoll–Hypaque gradient centrifugation (Sigma). After being washed three times in PBS, the PBMC were resuspended in RPMI-1640 medium (Sigma) supplemented with 10% human AB serum, 10 mM HEPES, 1·5 mM l-glutamine, 0·04 mM 2-mercaptoethanol and antibiotics (200 IU/ml penicillin and 200 mg/ml streptomycin) (all from Sigma). Cells were adjusted to

3 × 106 cells/ml, added to 24-well plates and kept unstimulated or were stimulated with 50 µg/ml of each Leishmania crude antigen or 20 µg/ml of concanavalin A (ConA; Sigma) for 5 days at 37°C, in a 5% CO2 incubator. After this time, the supernatants were collected

and stored frozen at −70°C until analysed for IFN-γ production by a commercial ELISA kit (BD Pharmingen, San Diego, CA, USA). The procedures were performed according to the manufacturer’s instructions. Samples were tested in duplicate and concentration was analysed using the SOFTmax®PRO version 4·0 program (Life Sciences Edition; Megestrol Acetate Molecular Devices Corporation, Sunnyvale, CA, USA). Results were expressed as picograms per millilitre. The minimum IFN-γ level detected was 7·8 pg/ml. A total of 3 × 106 PBMCs of each individual were kept at rest, unstimulated, or were stimulated with 50 µg/ml of either Leishmania crude antigens in the presence of 2 µg/ml antibody to CD28 (e-Bioscience, San Diego, CA, USA) for 2 h at 37°C, in a 5% CO2 incubator. ConA was also used as a positive control (20 µg/ml; Sigma). Brefeldin A (BFA; Sigma) was added to all cultures at a final concentration of 10 µg/ml and cells were incubated for an additional 12 h before staining.

Candida species are the most common fungi isolated from catheter, denture and voice prosthesis-associated

infections, and also are commonly isolated from contact lens-related infections (e.g. fungal keratitis). These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Drug resistance in fungal biofilms is multifactorial and phase-dependent, e.g. efflux pumps mediate resistance in biofilms during early phase whereas altered membrane sterol composition contributes to resistance in mature phase. Both substrate type and surface coatings play an important role in the pathogenesis selleck of device-related fungal biofilms. Microarray and proteomic analyses have identified the differentially expressed genes and proteins in Candida biofilms, and recent studies demonstrate that microbial biofilms interact with host immune cells. In this review, we will summarise recent advances in research on fungal biofilms and their relevance to device-associated infections. “
“The current study was conducted to know the incidence, predisposing factors, spectrum, clinical profile and antifungal susceptibility (AFS) of fungal wound infection (FWI) in burn patients. Of a total of 71 patients, 20 (28.2%) emerged with the diagnosis of FWI. Fungal pathogens

in this study were Candida tropicalis (14%), Candida parapsilosis (5.6%), Aspergillus niger (2.8%) and one each of Candida albicans (1.4%), Candida glabrata (1.4%), Syncephalestrum (1.4%) and Fusarium solani (1.4%). All patients with mould infections expired before the mycological culture results could be Selleckchem BGB324 conveyed to clinicians. Of the yeasts isolated in the study, one each of C. tropicalis and C. albicans showed cross-resistance to azoles. All the moulds were susceptible to amphotericin B. This study depicted

that fungal invasion is associated with a high mortality, burn size 30–60% and high incidence of inhalational injury. Fungal invasion was detected on an average of 14 days after injury. Association of use of four classes of drugs – aminoglycosides, imipenem, vancomycin and third generation cephalosporins and use of total parenteral nutrition was observed. Expedient laboratory diagnosis Acyl CoA dehydrogenase of FWI and appropriate systemic antifungal therapy guided by AFS may improve outcome for severely injured burn victims. “
“Onychomycosis is a common fungal infection most often affecting the toenails. If untreated, it can cause discomfort sufficient to reduce quality of life. To evaluate efficacy and safety of bifonazole cream vs. placebo in onychomycosis treatment after non-surgical nail ablation with urea paste. Fifty-one study centres randomized 692 subjects with mild-to-moderate onychomycosis to receive bifonazole 1% cream or placebo for 4 weeks following non-surgical nail ablation with urea 40% paste over 2–4 weeks.

Other investigations have reported several Gr-1+ mononuclear cells affecting immune responses (Bronte et al., 2000; Nakano et al., 2001; Delano et al., 2007). Bronte et al. (2000) found Gr-1+CD11b+CD31+ macrophages in the secondary lymphoid organs of immunocompromised mice that suppress the function of CD8+ T cells (designated as inhibitory macrophages). Nakano et al. (2001) reported Gr-1+CD11b−CD11c+ cells found in mouse lymph node and spleen that display characteristics of plasmacytoid

dendritic cells and produce interferon-α (IFN-α) Proteasome inhibitor after stimulation with the influenza virus. Delano et al. (2007) have recently demonstrated the dramatic increase of Gr-1+CD11b+ cells with heterogenous morphologies in the spleen, lymph nodes and bone marrow during polymicrobial sepsis, which produce inflammatory cytokines and chemokines including TNF-α, and contribute to the suppression of antigen-specific CD8+ T cell IFN-γ production and a shift from Th1- to Th2-type antibody responses. At present, the relationship between these reported cells and Gr-1dull+ cells described in the current study remains unclear. Although both Gr-1dull+ cells and neutrophils showed intracellular expression of TNF-α in a flow cytometric analysis, it is not clear as to which cell populations contributed more to the production of this cytokine in the lungs during infection with S. pneumoniae.

In this respect, the sorted Gr-1bright+ cells (neutrophils) BMN 673 in vitro did not or marginally secreted TNF-α Tobramycin in an in vitro culture. However, these findings may not necessarily exclude their possible contribution to the in vivo synthesis of this cytokine. In our hands, in vivo depletion of Gr-1+ cells by the specific mAb did not lead to the complete inhibition of TNF-α synthesis detected in BALF, which suggested that TNF-α production was not completely ascribed to neutrophils and Gr-1dull+ cells. We also observed the expression of this cytokine in F4/80+ cells at an earlier stage of pneumococcal infection before Gr-1dull+ cells appeared. Considered collectively, these results suggested that alveolar

macrophages may contribute in part to the synthesis of TNF-α in lungs. In conclusion, we revealed the possible involvement of neutrophils and Gr-1dull+ CD11c+ macrophage-like cells in the production of TNF-α in lungs at an early stage of infection with S. pneumoniae. TNF-α was shown to play pivotal roles in recruiting neutrophils and protecting mice from this bacterial pathogen, suggesting that this unusual subset may contribute to the host defense by inducing this cytokine. Thus, the present study provides important implications for our understanding of the pathogenic mechanism of pneumococcal infection and development of more effective vaccine strategies. Further investigations will be necessary to define the more detailed characteristics of this population and its precise role in the host-protective responses.

5A and data not shown). OVA-specific Th2-cell dependent IgG1 and IgE were detected in the serum of mice upon alum/OVA sensitization and antigen challenges. Surprisingly, no change was detectable Pritelivir chemical structure at the OVA-specific Ig levels when mice were pretreated with the differentially matured and OVA-loaded DCs (Fig. 5B). Together, MyD88-dependent T. brucei-derived VSG antigens or nonTLR-dependent TNF conditioning of DCs did not alter subsequent Th2-cell driven allergic asthma. EAE serves as a common murine model for the early phases of multiple sclerosis, which can be achieved by immunizing

mice with the auto-antigen MOG in CFA. Mice develop MOG-reactive pathogenic Th1 and Th17 cells, which then infiltrate into the PI3K inhibitor CNS and cause inflammatory edema leading to the reversible paralysis symptoms 43. Previously, we have shown that repeated injections of DCs stimulated with TNF and loaded with MOG-peptide suppressed EAE, partially by creating a Th2/Tr1 cytokine environment including immune deviation and IL-10-mediated suppression 23, 33. We therefore wanted to analyze how the partial DC maturation stages induced by TLR-dependent or independent

stimuli would modulate the autoimmune disease EAE. To detect whether the DC injections ameliorate or worsen the disease, we switched the amounts per DC injection from 3 to 3.5×106 cells, which is the fully protective protocol 23, 33, 44 to 2–2.5×106 cells, which leads to about 50% reduced clinical score 44. Three i.v. injections of suboptimal amounts of MOG-loaded TNF-matured DCs protected mice partially from EAE as 10 out of 15 mice cAMP developed clinical symptoms and mice only reached a mean maximum score of 1.850±0.944 (Fig. 6A and B). Surprisingly, mice which received three injections of DCs matured with the T. brucei antigens mfVSG or MiTat1.5 sVSG were also partially protected from EAE as 8 out of 12 and 13 out of 19 mice developed signs of EAE, respectively (Fig. 6A and B). Together, our data indicate that all partially mature DCs protected mice

to a similar extent from EAE. As published previously 33, protection from EAE by TNF-matured DCs required activation of IL-10+ IL-13+ cytokine-producing CD4+ Th2/Tr1 cells. IL-4 is also produced but immediately consumed in normal mice and only detectable in IL-4R-deficient mice 33. We therefore assessed how the differentially matured DCs influenced the T-cell cytokine profile of the spleens as detected after MOG peptide restimulation and cytokine analysis. The cytokine profile of T cells from untreated mice typically consists of high amounts of proinflammatory IFN-γ and IL-17 but low amounts of IL-10 and IL-13. In contrast, this pattern becomes inverted in mice, which received repetitive injections of TNF-matured DCs 23, 33.

In fact, there has never been a more opportune time for research aimed at uncovering biomarkers check details in T1D: an ever-growing number of clinical studies of new-onset type 1 diabetes should provide unprecedented access to potentially large numbers of clinical specimens. Relevant clinical laboratory assay developments, along with recent developments in high-throughput technologies, now provide the means to assay large numbers of specimens rapidly and affordably. One challenge facing biomarker studies, however, is the lack of defined standards, not only among laboratory protocols for the various assays but also in handling and

preparation ABT-888 mw of clinical specimens, which can have considerable influence on assay results [23]. Another challenge is our lack of knowledge as to how much

individual T cell responses fluctuate over time in a given individual – subjects are usually tested only a few times per year, but effector T cell and regulatory T cell (Treg) activities might change multiple times during this period. Indeed, a recent study published by Diabetes TrialNet’s Mechanistic Outcomes Committee showed that, while assays measuring overall T cell reactivity against islet autoantigens could distinguish between patients with T1D and healthy controls relatively reliably, those assays that measured individual epitope-specific responses detected variable responses over time [24]. The last challenge is that, as yet, we have no solid data that indicate how T cell responses would be expected to change in a beneficial way in one individual following re-establishment of tolerance to β cells. Animal models tell us what to expect, but do not always correspond to the human case [25]. Thus, precise tracking during clinical interventions is required to develop reliable correlations between T cell responses and clinical outcomes. The potential benefits of biomarkers of tolerance in T1D are many [26]. They could speed

clinical assessments by providing surrogate end-points, permit more robust analysis of trial data through PFKL stratification of patients and facilitate personalized medicine by informing treatment decisions. Such benefits argue strongly for the creation of a coordinated biomarker discovery effort that, by establishing common procedures across all new-onset trials, permits comparison of data obtained in trials of varying agents and ultimately the identification of robust immunological markers of disease state and immune tolerance. The ITN has been working actively to advance such a goal for the past decade by integrating a biomarker discovery programme into each of its clinical trials.

p.m. versus 3000 c.p.m.; P < 0·03). From these data, along with

those shown in Figs 2 and 3, we speculate that eosinophils not only present antigens to CD4+ T cells in an MHC class II pathway, but also present antigens to CD8+ T cells by using their MHC class I molecules. To test this hypothesis, experiments were performed to determine whether the induction of C. neoformans-primed T-cell proliferation was caused by the presentation of ALK inhibitor antigens by eosinophils in conjunction with MHC class I and MHC class II molecules. C. neoformans-pulsed eosinophils were treated with anti-MHC class I or anti-MHC class II mAbs before incubation with C. neoformans-primed CD4+ and CD8+ T cells. The blocking of MHC molecules on the eosinophil surface was found to suppress the ability of C. neoformans-pulsed eosinophils to stimulate C. neoformans-primed T-cell proliferation (Fig. 6d). Moreover, the suppression seen Birinapant cost in the lymphocyte proliferation was more pronounced with anti-MHC class II, which coincided

with the higher proliferation of CD4+ T cells shown in Fig. 6c. In conclusion, C. neoformans-pulsed eosinophils stimulated C. neoformans-primed MSCs and T cells (CD4+ as well as CD8+) in an MHC class II- or class I-dependent manner. This stimulation of proliferation, however, was not observed for naive T cells or when C. neoformans-pulsed Mφ were used as APCs. To characterize and differentiate the T-cell profile seen after co-culture with C. neoformans-pulsed eosinophils, C. neoformans-primed purified T cells (CD4+ and CD8+) were analyzed Bay 11-7085 by flow cytometry to determine the intracellular expression levels of IFN-γ and IL-4 after 4 days of culture with C. neoformans-pulsed eosinophils or medium alone. Figure 7 shows a significant increase in the percentage of IFN-γ-producing cells when T cells were incubated with C. neoformans-pulsed eosinophils compared with T cells cultured in medium alone (6·56% versus 1·61%; P < 0·02). With regard to the IL-4-producing T-cell population, the percentage

with C. neoformans-pulsed eosinophils (2·42%) was similar to that for medium alone (2·35%). These results allowed us to conclude that C. neoformans-pulsed eosinophils were able to induce the expansion of IFN-γ-producing Th1 cells, but not of IL-4-producing Th2 cells. To analyze the production of cytokines by CD4+ and CD8+ T cells in supernatants, the concentrations of IFN-γ, TNF-α, IL-4, IL-10 and IL-13 were measured after 4 days of culture. The results presented in Fig. 8(a,b) show that there was a significant increase in the production of IFN-γ and TNF-α generated by C. neoformans-primed T cells cultured with C. neoformans-pulsed eosinophils compared to the cytokine production by T cells cultured in medium alone, with fixed yeasts of C. neoformans or with unpulsed eosinophils. In contrast, no differences in the levels of IL-4, IL-13 or IL-10 were detected in supernatants of C. neoformans-primed T cells cultured with C.

MALDI-TOF mass spectra were acquired using a Bruker Reflex INCB024360 mw mass spectrometer (Bruker-Daltonik, Bremen, Germany) in the positive ion reflector mode with an accelerating voltage of 20 000 V, grid voltage of 75%, guide wire voltage of 0·002% and a 400-ns delay time. Monoisotopic masses were calculated after internal calibration with autolytic tryptic peaks. Peptide mass fingerprints were searched on 23 October 2008 using Mascot search engine (http://www.matrixscience.com). Algorithms were used for protonated monoisotopic masses, with one missed trypsin cleavage and a tolerance in the mass measurement of 100 ppm, complete modification of cysteine by carbamidomethylation,

and partial modification of methionine by oxidation in the search settings to search all the entries of NCBI database as described previously (17). The criteria for matched

proteins included the number of match score and the sequence coverage. Statistical analysis was carried out using the Student’s t-test with all replicate gel and BYL719 mouse animals in each group. To confirm overexpression of known proteins, liver protein preparation and separation were performed as described above. Proteins were then transferred onto Hybond P polyvinylidene difluoride (PVDF) membranes (GE Healthcare) using a Mini Trans-Blot system (Bio-Rad) for 3 h at a constant current of 190 mA. Membranes were incubated with blocking buffer [Tris-buffered saline (TBS) containing 5% skim milk] at 37°C for 1 h or at 4°C overnight.

Then membranes were incubated with rabbit polyclonal anti-peroxiredoxin 6 (Prdx6) antibodies (1 : 1000; Abcam, Cambridge Science Park, Cambridge, UK) for 1 h at room temperature. After washing four times in TBS containing 0·1% polyoxyethylene sorbitan monolaurate (Tween-20; TBS-T), membranes were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG antibody (1 : 1000; Zymed Laboratory, San Francisco, CA, USA) for 1 h at room temperature. Immunodetection was accomplished using an ECL Western Blot Detection System (Amersham Biosciences). Chemiluminescence signals and band volumes were measured using an ImageQuant400 system (GE Healthcare). To examine the increase in Branched chain aminotransferase Prdx6 expression in response to O. viverrini infection, 20 μg of liver protein was separated by 1D 12% SDS-PAGE under sulphydryl reducing condition and transferred onto PVDF membrane. Immunoblot was conducted as described above, but including the detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using mouse monoclonal anti-GAPDH (1 : 2000; Millipore, Billerica, MA, USA). Bands were scanned using a gel document system (Amersham Biosciences) and band intensities analysed using a computer-assisted imaging densitometer system (Scion image; Scion Corporation, Maryland, USA). To determine the expression of Prdx6 mRNA, total RNA was isolated from approximately 150 mg of the hamster liver using TRIZOL™ (Invitrogen) according to the manufacturer’s instructions.