When we adjusted the cytokine+ CD4+ T-cell frequencies for age-specific CD45RO+CD4+ memory cell frequencies 27, similar frequencies of total cytokine+, TNF-α-expressing, and polyfunctional CD4+ T cells were found between adolescents and children ( Table 2). The memory phenotype of the MVA85A-boosted CD4+ T-cell response was determined in adolescents by measuring expression of CD45RA and CCR7 on Ag85A or BCG-specific cytokine-expressing CD4+ T-cell subsets. CCR7 expression was detectable among total CD4+ T-cell populations, following incubation of whole blood (Fig. 4A). https://www.selleckchem.com/products/idasanutlin-rg-7388.html All Ag85A-specific cells exhibited a predominant effector memory phenotype (CD45RA−CCR7−). This was observed regardless of

time point or pattern of cytokine expression examined (Fig. 4). Ag85A-specific cells producing only IFN-γ showed a temporary increase in CD45RA expression at day 28 post-vaccination, when compared with day 7 and 56 post-vaccination (Fig. 4B). This was not seen for BCG-specific cells (Fig. 4C). In these two trials we showed that MVA85A is safe and immunogenic in adolescents and children from a TB-endemic buy Pifithrin-�� region in South Africa. Adverse events in these younger individuals were generally fewer, of shorter duration and were more likely to be localized to the vaccination site, compared with adverse effects previously shown in MVA85A-vaccinated adults from the same region 25. MVA85A

induced potent immunity that was dominated by polyfunctional CD4+ T-cell populations

co-expressing IFN-γ, IL-2 and TNF-α, or co-expressing these cytokines with IL-17 and/or GM-CSF. We did not expect to detect the Th1/Th17 population, as IL-17-expressing cells (Th17) are largely thought to be a subset separate to Th1 cells 20, 28, 29. Co-expression of IFN-γ and IL-17 has been reported, notably at autoimmune disease sites such as the gut in patients with Crohn’s Disease 19, 30. However, to our knowledge, this is the first description of a population co-expressing IFN-γ, IL-2, TNF-α and IL-17. At this stage, we do not know what role this population could play in protective immunity against TB or how these cells are induced. We also observed that most MVA85A-induced CD4+ T cells Clomifene co-expressing IFN-γ, IL-2 and TNF-α in children also expressed GM-CSF. These data are consistent with recent findings from a report showing GM-CSF co-expression with IFN-γ and TNF-α by M.tb-specific CD4+ T cells in children with TB or latent M.tb infection 16. The role of GM-CSF in anti-mycobacterial immunity is mostly unknown, but KO of this cytokine in the murine TB model results in impaired control of bacilli and increased mortality 15. Notably, M.tb-specific GM-CSF-expressing T cells have been identified in granulomatous tissue from individuals with latent M.tb infection 31, suggesting that this cytokine may contribute to anti-M.tb immunity.

This implies that thymically derived natural Treg cells may also play a role in controlling the overall size of the GC response, or upon systemic TGF-β neutralization, other factors or cytokines may partially compensate leading to nominal induction of iTreg cells. The potential role of IL-10 was also examined by repeated administration of a blocking anti-IL-10R mAb. Mice were injected i.p. on day 0 with 1 mg of anti-IL-10R (1B1.3a) mAb or control rat IgG. Starting AZD6738 in the second week, 500 μg of anti-IL-10R mAb

or rat IgG was injected twice weekly and continued until the mice were killed. The SRBC were given i.p. on day 0. Similar to anti-TGF-β-treated mice, blockade of the IL-10R resulted in an inability to control the balance of IgM+ to switched GC B cells in the spleen. Although not evident at days 8 and 12, this imbalance became marked at days 18 and 24 and reflected a significant increase in both the frequency and Palbociclib order total number of IgM− GC B cells (Fig. 9b). Examination of the frequency and number of total B220+ PNAhi B cells showed little difference between anti-IL-10R mAb and control-treated mice, except at day 24 (Fig. 9a). This is again similar to the result observed after TGF-β neutralization, and may likewise reflect the activity of natural Treg cells or the ability of other cytokines to partially compensate.

Finally, to ensure that anti-IL-10R mAb treatment did not directly modulate responding B cells, the GC population was tested for expression of IL-10R. As shown in the Supplementary material, Fig. S3, no expression above background was detected. A large number of studies have documented the role of Treg cells in controlling antibody responses.16–46 Using either in vivo disruption (anti-GITR mAb) or depletion (anti-CD25 mAb) protocols, investigators have shown that loss of Treg-cell activity results in enhanced humoral

responses to experimental antigens,16–22 pathogens23,24 and auto-antigens.17,25–29 In all of these reports, antibody levels directed against the specific 4-Aminobutyrate aminotransferase antigen or infectious agent were significantly elevated, including IgG,16–27,29 IgA18,25 and even IgE.19,26 Additional studies examined whether adoptive transfer of polyclonal21,30–32,35,37–40 or TCR transgenic33,34,36,41 Treg cells could dampen antibody responses to defined allo-antigens or auto-antigens. In all cases, the transfer of Treg cells significantly lowered or even eliminated serum antibodies directed against these antigens. As GCs serve as the basis for T-cell-driven humoral responses, the current study examined the behaviour of primary splenic GC reactions induced to a number of antigens in mice treated with an anti-GITR mAb (Figs 1–4). After disruption of Treg-cell activity, total SRBC-induced GC B-cell numbers were increased at all time-points examined (days 8–24). A higher proportion of IgM− switched B cells within the GC compartment largely accounted for this increase.

[106] Healthy first degree relatives of lupus patients have more pronounced serum IFN activity and the levels are more abundant in younger individuals.[107, 108] A combination of risk alleles in the type I signalling pathway (e.g. STAT4 and IRF5) may confer an additive predisposition of disease.[109] It can be inferred that the use of genetic mapping may help predicting the development and severity of disease in the future. Interferon-regulated

chemokines may be employed to monitor disease activity and organ damage.[110, 111] It has also been proposed that type I IFN-inducible mRNA can be used as pharmacodynamic markers to monitor treatment response of anti-IFN therapy in SLE.[112] The use of anti-IFN-α in the treatment of moderately active SLE was examined in a phase I multicentre double-blind randomized this website trial. In that study, the use of sifalimumab (an anti-IFN-α monoclonal antibody) led to a dose-dependent inhibition of type I IFN-induced

mRNA in whole blood and corresponding changes in related proteins in affected skin. Exploratory analyses showed consistent trends towards improvement in disease activity, less requirement of new or escalation of immunosuppressive treatments and fewer flares in sifalimumab-treated patients.[113] Tolerability profile was acceptable and comparable to patients receiving placebo. Tumour necrosis factor-α is expressed as a trimer on cell surface and in soluble form after the activation of macrophages and dendritic cells. Being described to have both protective and deleterious effects in SLE, Thiamine-diphosphate kinase its position in lupus pathogenesis remained controversial. In NZB/W mice, there was diminished www.selleckchem.com/products/Fulvestrant.html production of TNF-α.[114] In some mouse model, the deficiency of TNF-α appeared to provoke lupus-like autoimmunity. While TNF-α defective NZB/W mice develop severe disease manifestations, TNF-α intact NZB/W mice only show modest lupus activity.[115] Conversely, TNF-α concentration was elevated in both sera and renal tissue of MRL/lpr lupus mice and the levels of TNF-α correlated with the severity of kidney disease.[116] Moreover, even

in NZB/W mice, renal expression of TNF-α is escalated in conjunction with kidney inflammation.[117] In MRL/lpr mice, anti-TNF-α therapy led to improvement of joint and lung manifestations.[118, 119] Whether the controversial role of TNF-α in the pathogenesis of murine SLE could be related to the different animal models used remains unclear. The circulating TNF-α level in active SLE patients closely followed the disease activity and elaborated TNF-α expression was seen in the renal parenchymal tissue in patients with lupus nephritis.[29, 120] Nonetheless, conflicting evidence exists in subjects who had received anti-TNF-α therapy for other autoimmune disorders.[121, 122] These individuals developed lupus-like features coupled with elevated anti-nuclear factors, anti-dsDNA and anti-cardiolipin antibodies.

The baby received intensive phototherapy and was treated with intravenous piperacillin and tazobactam combination for suspected sepsis. The blood sample was collected aseptically on day 1 of admission and processed for bacterial and fungal pathogens. Also, double volume exchange transfusion and intravenous immunoglobulin were commenced. He developed thrombocytopenia and was infused platelet concentrates. Postexchange transfusion, total bilirubin level, dropped to 11.9 mg dl−1 on day 2 after which phototherapy was

stopped. On day 3 of admission, the blood cultures showed growth of yeast-like colonies, however, culture was negative for bacteria. Therefore, a presumptive diagnosis of fungaemia was considered and the baby find more was administered intravenous amphotericin B (0.6 mg kg−1 day−1) for 1 week. A repeat blood culture on day 6 of admission showed clearance of fungaemia. DAPT price The subsequent stay of the baby was uneventful and repeated blood cultures done twice were sterile. He was discharged on day 20 of admission with oral voriconazole (4 mg kg−1 per dose twice a day) as domiciliary treatment for 7 days. Currently, the baby continues to be healthy. The isolate was assigned an accession number VPCI 1049/P/12 and showed moist, yeast-like, tan-yellow and wrinkled colonies on Sabouraud’s glucose agar after 4 days of incubation at 37 °C (Fig. 1a). On

microscopic examination, lactophenol cotton blue mount showed fusiform spindle-shaped elongated blastoconidia and presence of hyphae (Fig. 1b). On CHROMagar Candida

medium (Difco, Becton Dickinson, Baltimore, MD, USA) the isolate formed rough green colonies after 48 h of incubation at 37 °C. However, germ tube test and chlamydospore formation were negative. The isolate showed a positive test for diazonium blue B (DBB), hydrolysed urea and was inhibited mafosfamide on 0.1% cycloheximide-containing medium. API ID 32C and VITEK2 compact (bioMérieux, Marcy I’Etoile, France) gave inconclusive profiles. The isolate assimilated sucrose, raffinose, soluble starch, trehalose, lactose, maltose and nitrate. Furthermore, molecular identification was done by the amplification and sequencing of the D1/D2 domain of the LSU region.[4] GenBank BLAST searches were performed for species identification. The sequence exhibited 99% identity with P. aphidis (GenBank accession no. HQ676615). The LSU sequence of the isolate was submitted to GenBank under the accession number KC812275. The isolate, VPCI 1049/P/12 has been deposited in the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands under the accession number CBS 12818. Antifungal susceptibility testing of the isolate was determined using the Clinical and Laboratory Standards Institute (CLSI) microbroth dilution method, following the M27-A3 guidelines.[5] The antifungals tested were amphotericin B (Sigma, St.

Recombinant TG2 protein (rTG2) was used as a reference in Western blot analysis. A fragment of 1·5 Kb long of the TG2 promoter region from Caco-2 cells was cloned in a firefly luciferase reporter vector pGL3 (Promega). Caco-2 cells

were plated in a 24-well plate and transfected transiently with TG2 promoter construction together with a Renilla luciferase vector. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 4·105 per well in a six-well plate. When cells reached 40–50% confluence they were washed with 2 ml Opti-MEM (Invitrogen) and incubated with a DNA-Lipofectamine Enzalutamide manufacturer (Invitrogen) mixture for 6 h in a humidified 5% CO2 environment. After 6 h of incubation the transfection medium was replaced

with fresh culture medium, and cells were incubated for 24 h. Subsequently, cells were incubated with cytokines alone (TNF-α 10 ng/ml, IFN-γ 200 UI/ml) or with the addition of inhibitors of signalling pathways (SP600125 20 µM, Ly294002 2 µM, sulphasalazine 10 µM) for another 24 h. Luciferase activity was measured in cellular lysates using the Dual-Luciferase Reporter Assay System Kit (Promega), according to the manufacturer’s protocol. For each sample firefly luciferase data were normalized to the Renilla luciferase internal control. Relative luciferase units (RLU) are referred to the non-stimulated control. To evaluate the expression of surface TG2, THP-1 cells were treated for 20 h with TNF-α 10 ng/ml and IFN-γ Epigenetic Reader Domain inhibitor 200 UI/ml, and inhibitors of signalling pathways. Cells were incubated with the anti-TG2 monoclonal antibodies (4E1G9, 2G3H8, 5G7G6 or 1H7H9) produced in our laboratory [16]. Then cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Ig)G (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Flow

cytometry analysis was performed in a fluorescence Methane monooxygenase activated cell sorter (FACS)Calibur flow cytometer (BD Biosciences), and data were analysed using FlowJo software (Tree Stars, Ashland, OR, USA). To investigate whether the proinflammatory cytokines TNF-α, IFN-γ, IL-15, IL-6 and IL-1 modulate TG2 expression, we measured TG2 mRNA levels by quantitative (q)RT–PCR in five human cell lines from different cell lineages (Caco-2 and HT29, intestinal epithelia; A549 and CALU-6, lung epithelia; THP-1, monocyte-like) stimulated for 24 h with the cytokines mentioned. In all cell lines tested, except for A549, IFN-γ was the most potent inducer of TG2 expression (Fig. 1). The highest induction of TG2 by IFN-γ was observed in THP-1 cells (fold increase = 20·2). In the two intestinal epithelial cell lines, Caco-2 and HT29, the up-regulation of TG2 transcript by IFN-γ was about 18-fold while TG2 levels were increased by 6·9- and 7·3-fold in A549 and CALU-6, respectively.

Although the α7 nAChR was expressed in human mast cells, this receptor is not likely to be functional in catestatin-induced mast cell activation. Although catestatin has been shown to stimulate rat mast cell release of histamine,23 to our knowledge, this is the first study demonstrating multiple functions of wild-type catestatin and its variants in human mast cells. Our findings suggest a new role for catestatin peptides in immunoregulation of the cutaneous immune system via mast cell activation. Eicosanoids and histamine are mainly secreted by activated mast cells, and are mediators of inflammatory

reactions.21 Both LTs and PGs are critically involved in inflammatory and allergic conditions, and PGD2 and PGE2 are abundant in allergic skin inflammation selleck compound such as contact hypersensitivity.24–26

Furthermore, intracellular Ca2+ is thought to play a key role in mast cell activation, including chemotaxis and release of histamine and eicosanoids.27,28 In this report, wild-type catestatin and its variants increased intracellular Ca2+ mobilization in mast cells and caused them to migrate, degranulate, and release inflammatory mediators. These observations suggest that catestatin peptides might participate in inflammatory reactions via mast cell activation. Overall, wild-type catestatin and its variants had almost equal potencies in activating human mast cells, except for the strongest PD-0332991 supplier activity of Pro370Leu in inducing LTC4 release, and the least stimulatory capacity of Arg374Gln in degranulating mast cells. This observation partially contradicts the literature relating to catestatin peptides, where wild-type catestatin and its variants display differential potencies Mirabegron in inhibiting catecholamine release and in inducing monocyte migration.9,11 This was not the result of artificial effects of catestatin peptides, because a control peptide had no effect on mast activation. Hence, the potencies of wild-type catestatin and its variants might vary following their specific activities,

and between cell types. Mast cells accumulate and become activated at sites of inflammation, and their numbers significantly increase during wounding,29 where the levels of catestatin have been found to be enhanced.4 Although the amount of catestatin has been estimated to 20 μm in normal murine skin,4 the precise concentration of an active catestatin in human skin is not yet known. However, because the levels of catestatin increase during skin injury or inflammatory conditions,4 one could expect that catestatin might reach its optimal levels at inflammatory sites or wound sites. In this study, the concentrations used for catestatin peptides ranged from 0·02 to 10 μm, doses that have been reported to display antimicrobial activities against skin pathogens4 and Plasmodium falciparum.

One candidate upstream component is the leucine-rich repeat (LRR)-containing G-protein-coupled receptor (GPCR) follicle-stimulating hormone receptor (FSHR-1), which was identified in a limited reverse see more genetic screen of 14 candidate transmembrane LRR receptors in C. elegans. RNAi directed against fshr-1 results in a high degree of susceptibility to killing by P. aeruginosa, Staphylococcus aureus and Enterococcus faecalis, but not in a reduced lifespan during infection by non-pathogenic E. coli[24]. Expression of FSHR-1 in intestinal cells is necessary and sufficient for its role in innate immunity. Genetic analysis

indicates that FSHR-1 functions in the intestine in a separate pathway from PMK-1 and DAF-2, the worm insulin receptor that is involved in stress responses (see below) [24]. Further, qRT–PCR analysis shows that FSHR-1 and the PMK-1/p38 MAPK cassette regulate the induction of overlapping, but non-identical, sets of P. aeruginosa-induced genes. Although transcriptional profiling data suggest that FSHR-1 regulates host response genes independently of PMK-1, it is unclear whether the PMK-1/p38 MAPK cassette may be involved partially in signal transduction downstream of FSHR-1 [24].

It is also possible that the FSHR-1 and PMK-1/p38 MAPK pathways function O-methylated flavonoid in parallel but converge on common sets of target genes in response to pathogen infection. How is FSHR-1 involved in mediating the C. elegans host response? Daporinad We currently lack evidence that FSHR-1 can sense infection directly, for instance by binding pathogen-associated molecular patterns (PAMPs). FSHR-1 is the sole C. elegans LGR-type

GPCR. In mammals the heterodimeric glycopeptide hormone FSHα/β is the canonical ligand for this class of GPCR. Worms do not have an identifiable FSHα subunit and the endogenous ligands, if any, have not been identified. As an LGR-type receptor, one might expect FSHR-1 to transduce signals through heterotrimeric G-proteins in the intestinal cell. Recent findings implicate at least one heterotrimeric G-protein in signal transduction events upstream of PMK-1 in a different tissue, the hypodermis (see below). Whether this or other G-proteins mediate FSHR-1 signal transduction in the intestine remains unknown. Recent findings show that the protein kinase Cδ (PKCδ) TPA-1 activates the protein kinase D DKF-2 upstream of PMK-1/p38 in the intestine [20]. The upstream signals that control TPA-1 activity remain unknown, although by analogy with other systems, a likely candidate is diacylglycerol (DAG, produced by phospholipase C).

In addition, grafted neuronal elements were closely

associated LDE225 supplier with microglial cells. However, microglia play a dual role and can exert both beneficial or detrimental effects on grafted neurones [78,82]. Resting microglia may have a beneficial role by providing neurotrophic support or sensing the environment to clear cell debris and misfolded proteins [78]. On the other hand, they can migrate to the site of injury and release various pro-inflammatory factors, which can become detrimental when delivered in a chronic and uncontrolled fashion [83–85]. It should be noted that similar observations were made in a transplanted PD patient where solid tissue grafts were also surrounded by an inflammatory response as early as 18 months after surgery [86]. Adequate trophic support is also necessary for graft survival [82,87]. Several studies in PD and HD animal models have repeatedly emphasized that only low number of cells survive transplantation [88–90]. The reason for this is not well understood but it has been hypothesized that deficient trophic support may be implicated. For example, both pretreatment

of cells derived from foetal ventral mesencephalon with basic fibroblast growth factor (bFGF) or glial cell line-derived neurotrophic factor (GDNF) and repeated parenchymal infusion of trophic factors in transplanted 6-hydroxydopamine (6-OHDA)-lesioned Bay 11-7085 rats significantly ameliorate dopaminergic cell survival and promote Silmitasertib mouse fibre outgrowth [91–93]. GDNF pretreatment of embryonic dopaminergic

cells implanted in PD patients showed good survival and led to beneficial effects clinically [94]. However, animal studies in 6-OHDA-lesioned mice have reported that implanted dopaminergic cells pretreated with brain-derived neurotrophic factor (BDNF) show a lower survival rate, albeit leading to improved behavioural recovery [95]. Importantly, treatment with trophic factors favour a TH phenotype in foetal cells in vitro [96,97]. BDNF has been shown to promote survival and to afford protection from excitotoxicity both in vitro [98,99] and in vivo [100]. In normal conditions, BDNF is highly expressed in the cortex, especially in layer V, and retrogradely transported to the striatum [101,102]. The expression of mHtt interferes both with normal BDNF transcription [103] and with the transport of vesicles along the microtubules [82,104]. As mHtt aggregates are found especially in layer V [43] of the cortex, which projects onto the graft p-zones [43,105], grafts may not receive adequate trophic support, making the grafted cells more susceptible to harmful factors derived from the diseased brain (Figure 1). In their report, Keene et al.

IEL, LPL or peripheral blood lymphocytes (1–5 × 106) were each diluted in 200 µl PBS containing 0·6 mM/ml Proteinase K (Sigma-Aldrich) and 200 µl lysis buffer AL (QIAamp DNA Blood Mini kit, Qiagen, Hilden, Germany), incubated for 10 min at 56°C and then stored at room temperature in lysis buffer AL until further use. IEL, LPL or intestinal mucosal biopsies (2 × 106) were also incubated in RNAlater

ACP-196 (Ambion, Austin, TX, USA) at 4°C overnight and then stored at −80°C. Peripheral blood and mucosal lymphocytes (1 × 106) in a volume of 30 µl were incubated at 4°C for 20 min with a cocktail of the following antibodies: anti-CD4-APC, anti-CD3-peridinin chlorophyll (PerCP), anti-CD62L-phycoerythrin (PE) and anti-CD45RA-fluorescein isothiocyanate (FITC) (BD multi-test for naive CD4+ T cells; BD Biosciences, San Jose, CA, USA). For analysis of extrathymic maturation of T lymphocytes in the intestinal mucosa, 1 × 106 LPL in a volume of 30 µl were stained with the following mouse anti-human antibodies CD2-PECy5, CD3-Pacific-blue (clone: UCHT1), CD5-APC (clone: L1712), CD7-FITC, CD16-PE and CD19-PE (all from BD Biosciences). Isotype controls were mouse immunoglobulin (Ig)G1-PE, mouse IgG2a-FITC, mouse IgG1-PECy5, mouse IgG2a-APC (clone: G155-178) and mouse

IgG1-Pacific blue (clone: MOPC-21) (all from BD Biosciences). For analysis of the check details frequency of proliferating T lymphocytes in peripheral blood the cells were prestained with anti-CD3 Pacific-blue, permeabilized and fixed with 100 µl fixation and permeabilization buffer (Nordic BioSite, Täby, Sweden), incubated at 4°C overnight and stained with Ki-67-PE or isotype control IgG1κ (Ki-67 PE-conjugated reagent set; BD Biosciences Pharmingen) in 50 µl permeabilization buffer (Nordic BioSite).

Flow cytometry was performed by acquisition of at least 20 000 lymphocytes, based on forward- and side light-scatter characteristics, on a BD LSR II (BD Biosciences) and subsequent analysis was performed using FlowJo software (Tree Star Inc., San Carlo, CA, USA). Genomic DNA from peripheral blood or mucosal lymphocytes was purified by the QIAamp DNA Blood Mini kit (Qiagen) according to the manufacturer’s instructions. Prior to the PCR, the DNA concentrations in all samples were determined by ultraviolet spectrophotometry diglyceride at 260 and 280 nm wavelengths and adjusted to a concentration of 30 ng/µl. The amount of TRECs relative to the amount of the reference DNA sequence, originating from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was determined by quantitative real-time PCR (LightCycler 1·2; Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany), using specific primers and the fluorescent dye SybrGreen I for detection of the specific products. The PCR primers were purchased from Scandinavian Gene Synthesis AB (Köping, Sweden).

To identify TBE virus-endemic areas, it is effective to conduct an epizootiological survey of wild rodents. The neutralizing test can be used for serological diagnosis of wild rodents, but it is time consuming and uses hazardous live viruses that require a high-level MAPK inhibitor biosafety facility. It is also known that non-infected wild rodents sometime indicated low neutralization antibody titers by the neutralization test. Therefore, a diagnosis which is more convenient for the epizootiological survey of wild rodents is required. In this study, we tried to develop ELISAs using two recombinant antigens

in the serological diagnosis of rodents for the first time. Domain III of the E protein was known to have the neutralizing epitopes (11) and was used for the serological diagnosis in several flaviviruses (13, 14). In this study, the recombinant domain III of the E protein was applied to the diagnosis ELISA for wild rodents. The EdIII-ELISA was shown to

have a relatively high sensitivity (27/35, 77.1%) and specificity (68/85, 80.0%) as compared with the neutralization test when the cut-off value for the ELISA was set at 0.64 (Fig. 2). Eight of 35 this website neutralization test-positive samples were negative in the EdIII-ELISA (Table 1). Several false-positive samples showed high reactivity to the negative control antigens, NusA (data not shown). In another study it was reported that a neutralizing response to West Nile virus in naturally infected horses was induced with epitopes within not only EdIII, but also other domains (25). It was suggested that these false-negatives were due to the lack of other domains and the Thiamine-diphosphate kinase conformational structure of the EdIII expressed in E. coli, and to the presence of antibodies that have high reactivity to NusA -Tag protein. In the flavivirus, co-expression of prM and E proteins in mammalian cells leads to the secretion of SPs to culture medium (19, 26, 27). The SPs have no viral

genome and do not produce progeny virus, and they have similar antigenicity and immunogenicity to the native virus. Therefore, SPs have been developed as a safe and useful alternative for live viruses as the antigen for serological diagnosis tests and vaccines (18, 20, 28, 29). In this study, the SPs were used as the antigens in ELISA to detect TBE virus-infected rodents. The SP-ELISA was shown to have a very high sensitivity (32/35, 91.4%) and specificity (85/85, 100%) as compared with the neutralization test when the cut-off value for the ELISA was set at a 0.089 (Fig. 4). In a recent study, it was reported that the antigenic structures of E proteins were disturbed when the ELISA plate was coated directly with the viral particles as solid-phase antigens (30). To avoid this, our SP-ELISA uses capture antibodies to coat the SP-antigen on the plate. And unlike infectious virions, the SPs do not require formalin inactivation, which affects the reactivity of several epitopes of the E proteins (31).