Scores above 50 in either category indicate the patient has no disability. Scores under 50 indicate increasing levels of disability selleck chemical compared to the general population (40–50 = mild disability, 30–40 = moderate disability, <30 = severe disability).[8] FFR is a valuable reconstructive option in high-risk patients with success rates as high as 80%.[9] Beyond successful limb salvage, we showed that the ability to ambulate significantly increased one's physical HRQoL and that ambulatory patients could achieve a HRQoL comparable to that of the general population. Factors such as the development of either immediate

or late complications did not influence HRQoL. The physical HRQoL scores as measured by the SF-12 in our patient cohort showed only mild disability compared with the general population when ambulation was achieved (82% of patients). This was in contrast to decreased physical HRQoL for nonambulatory patients post-operatively. Mental HRQoL was comparable with the general population for both ambulatory and nonambulatory patients. Another important factor influencing

HRQoL was amputation. We showed that patients had a higher Selleckchem PLX3397 physical HRQoL (comparable with that of the general population) when they did not undergo an amputation. However, this value continued to be influenced by the ambulatory status of the patient. Ambulatory patients showed only mild disability regardless of amputation status, and there was no difference between the physical HRQoL of ambulatory amputees and nonamputees. However, the HRQoL decreased dramatically for both amputees and nonamputees when these patients were not ambulatory. Interestingly, although both groups showed severe CHIR-99021 research buy disability, the HRQoL was significantly higher for ambulatory amputees than nonambulatory nonamputees, further suggesting that the ability to ambulate was the main factor influencing HRQoL. This cohort of patients required a high rate of revisional surgeries (61% of patients) to achieve a successful outcome. Although the great majority of these additional surgical procedures were minor, subjecting patients to multiple surgeries could conceivably reduce their satisfaction with

the initial procedure. Despite this concern, we found that 95% of patients would choose to undergo FFR again if given the choice, with average patient satisfaction of 4.89 on a 5-point scale. The high level of HRQoL in ambulatory patients is a desirable result after FFR of the lower extremity. Although various other studies have previously reported evidence of patient satisfaction or HRQoL outcomes following FFR, none has so far employed the use of a validated questionnaire in this patient cohort.[10, 11] The evidence has thus far been sporadic and largely anecdotal. Of course, there are limitations to this study as well, such as the potential for self-selection bias. However, the near-equal response rate between ambulatory and non-ambulatory populations is reassuring.

Therefore, SIGNR1 is widely involved in immune responses to pathogens in cooperation with other PRRs. In this study, we investigated LY294002 ic50 the roles of SIGNR1

in recognizing and inducing cellular responses to zymosan, HK- and live C. albicans. We found that SIGNR1 enhanced Syk-dependent oxidative burst response possibly in cooperation with Dectin-1. We first examined the binding to microbe particles using soluble forms of SIGNR1 and Dectin-1 tagged with an N-terminal Strep-tag II sequence. When tetramers were formed by preincubating with PE-Strep-Tactin at 37°C, soluble SIGNR1 (sSIGNR1) tetramer bound more to the microbes than that at 4°C, although soluble Dectin-1 (sDectin-1) bound equally to HK-C. albicans (Fig. 1A). Based on these observations, tetramers formed at 37°C were used in the subsequent experiments. Although both SIGNR1 and Dectin-1 recognized zymosan, as reported 23, 27, the amount of sSIGNR1 binding was much higher than that of sDectin-1 (Fig. 1B, left panels). Moreover, sDectin-1 bound comparably to zymosan and HK-microbes, but much less to live C. albicans, as reported 27. In contrast, sSIGNR1 equally bound not only to zymosan and HK-C. albicans but also live microbes (Fig. 1B, left panels). Furthermore, the binding of sSIGNR1, but not sDectin-1, was EDTA- and mannan-sensitive (Fig. 1B, right panels and data not shown). Less binding of sDectin-1 to live microbes NVP-AUY922 ic50 was also confirmed by immunofluorescence

microscopy, in which sDectin-1 bound to the surface of killed microbes, but stained mainly budding scars and occasionally showed a spotty staining pattern on live microbes (Fig. 1C). Since oxidative burst is crucial for Mϕ functions in response to microbes, we measured the oxidative burst response using RAW264.7 cells transfected with SIGNR1 cDNA Edoxaban (RAW-SIGNR1) or control plasmid (RAW-control). Parental RAW264.7 cells lack SIGNR1 expression. First, RAW-SIGNR1 and RAW-control cells were confirmed to express comparable levels of Dectin-1 (Fig. 2A). RAW-SIGNR1 cells showed a markedly higher response than the RAW-control cells (Fig. 2B). Although

this elevated response in the RAW-SIGNR1 cells was partially reduced by depletion of zymosan, and TLR2 ligand, PAM3CSK4 was ineffective in either inducing the response by itself (Fig. 2B) or elevating the response by depleted zymosan (Fig. 2C). Antagonistic anti-TLR2 mAb (T2.5) showed no effect on the oxidative burst of RAW-SIGNR1 to zymosan or depleted zymosan (Fig. 2D). These results implied that SIGNR1 plays a role in the induction of the oxidative burst independently of TLR2, this being consistent with previous reports 13, 14. Considering the role of Dectin-1 in oxidative burst 13, 14, it is possible that SIGNR1 utilizes the Dectin-1-dependent pathway, although both of these lectins can independently recognize zymosan/HK-C. albicans. To confirm this possibility, the effects of various inhibitors were examined in response to HK-C. albicans, since HK-C.

Mature PDC can activate as well as inhibit T cell responses. On one hand, mature PDC can prime productive CD4+ and CD8+ T cell responses [1], and on the other hand they possess a capacity to induce generation of CD4+ and CD8+ regulatory T cells (Treg) from naive CD4+ or CD8+ T cells, respectively [2-7]. Recently, we showed that human PDC preferentially induce generation of a unique type of CD8+ Treg, but not CD4+forkhead box protein 3 (FoxP3)+ Treg, when both CD4+ and CD8+ T cells are present [8]. Importantly, these CD8+CD38+lymphocyte activation gene (LAG)-3+ CTLA-4+ Treg were not only able to inhibit naive T

cells, but also memory T cell responses. JQ1 supplier Indeed, in vivo, depending on the experimental animal model, PDC either induce effective T cell immunity [9-11] or inhibit T cell responses by driving differentiation of Treg in vivo [12-14]. A recent study in which PDC were eliminated selectively from mice showed that PDC can simultaneously suppress and stimulate T cell responses in vivo [15]. Recently, it has been shown that the selective mammalian target of rapamycin (mTOR)-inhibitor rapamycin inhibits production of interferon (IFN)-α and proinflammatory cytokines

by TLR-activated mouse PDC, and reduces MK-8669 supplier their capacity to stimulate CD4+ T cells. Rapamycin was found to block the interaction of TLR with myeloid differentiation primary response gene 88 (MyD88), resulting in reduced interferon regulatory factor-7 (IRF-7) phosphorylation [16]. However, important questions regarding the effects of

rapamycin on PDC functions have still be to be resolved. First, the effect of rapamycin on the ability of PDC to generate Treg has not been studied. Secondly, Cao et al. studied mouse PDC and, whereas they recapitulated the inhibitory effect of rapamycin on IFN-α secretion on human PDC, it remains to be established whether Janus kinase (JAK) and how rapamycin affects the T cell stimulatory capacity of human PDC. These questions are clinically highly relevant, because the indications for rapamycin treatment are expanding. Used originally as an immunosuppressive drug in transplant recipients, rapamycin and rapamycin analogues are now increasingly being evaluated as an anti-proliferative drug in cancer treatment [17]. Moreover, studies have been initiated to determine its efficacy in autoimmune diseases such as systemic lupus erythematosus (SLE) [18], which are caused mainly by overproduction of IFN-α by PDC [19, 20]. Therefore, the aims of the present study were to determine systematically the effects of a clinically relevant concentration of rapamycin on cytokine production, T cell stimulatory capacity and CD8+ Treg-generating capacity of human PDC.

A possible strategy to overcome Treg-cell suppression focuses on OX40, a costimulatory

molecule expressed constitutively by Treg cells while being induced in activated effector T cells. OX40 stimulation, by the agonist mAb OX86, inhibits Treg-cell suppression and boosts effector T-cell activation. Here we uncover the mechanisms underlying the therapeutic activity of OX86 treatment dissecting its distinct effects on Treg and on effector memory T (Tem) cells, the most abundant CD4+ populations strongly expressing OX40 at the tumor site. In response to OX86, tumor-infiltrating Treg cells produced significantly less interleukin 10 (IL-10), possibly in relation to a decrease in the transcription factor interferon regulatory factor 1 (IRF1). Tem cells responded to OX86 by selleck chemicals llc upregulating surface CD40L expression, providing buy 5-Fluoracil a licensing signal to DCs. The CD40L/CD40 axis was required for Tem-cell-mediated in vitro DC maturation and in vivo DC migration. Accordingly, OX86 treatment was no longer therapeutic in CD40 KO mice. In conclusion, following OX40 stimulation, blockade of Treg-cell suppression and enhancement of the Tem-cell adjuvant effect both concurred to free DCs from immunosuppression and activate the immune response against the tumor. The

accumulation of Treg cells at the tumor site is one of the mechanisms developed by tumor cells to elude the immune system 1, through suppression of both innate and adaptive immune responses 2. Their inhibition is thought necessary for the establishment of a successful cancer immunotherapy. Several pieces of evidence indicate OX40 as a potential mediator of Treg-cell inactivation. Tangeritin OX40 is a costimulatory molecule constitutively expressed by Treg cells and expressed upon activation by T effector (Teff) cells. Triggering of OX40 has opposite

effects on these two T-cell populations: Treg cells are inhibited in their suppressive functions 3–6, while Teff cells are stimulated to proliferate, survive and gain memory phenotype 7–11. Treatment of different types of mouse transplantable tumors with the mAb OX86, the agonist of OX40, favors tumor rejection thanks to its double effect on Treg and Teff cells 3, 12. The tumor microenvironment is characterized by an immunosuppressive cytokine milieu, which promotes immune tolerance and tumor growth. Treg cells secrete interleukin 10 (IL-10), which plays a critical role in suppressing immune responses and in particular the maturation of fully competent DCs 13–15. Among tumor-infiltrating Teff cells, the subpopulation of effector memory T (Tem) cells is the most abundant.

Our data indicate that adoptive transfer of donor-derived T-cell receptor Bortezomib concentration (TCR) αβ+CD3+CD4–CD8–NK1.1– (double negative, DN) Treg

cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4+ and CD8+ T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4+ and CD8+ T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen. Injection of donor bone marrow (BM) was first reported to induce skin allograft tolerance by establishing chimerism in neo-natal hosts [[1]]. GPCR & G Protein inhibitor Thereafter, induction of mixed chimerism by BM transplantation has been considered

promising among the numerous methods developed for tolerance induction in transplantation. Mixed chimerism refers to a state in which allogeneic hemato-poietic cells coexist with recipient cells, resulting in a state of tolerance toward both the donor and the host, thus avoiding chronic rejection and side effects of any drug treatments in transplantation [[2]]. Although mixed chimerism has produced clinical benefits in transplantation [[3, 4]], sustained chimerism in patients and large animal models has not

yet been achieved. In addition, GVHD is still a major obstacle in BM transplantation. Obviously, this approach needs further improvement to be practical in the clinic. Regulatory T (Treg) cells, being able to suppress CD4+ and CD8+ T cells, as well as NK cells and dendritic cells (DCs), play an important role in regulating immune responses in models of autoimmunity, Dichloromethane dehalogenase infection, inflammatory disease, and transplantation [[5-7]]. Aside from the extensively studied FoxP3+ Treg cells, we have identified a novel immune Treg cell with phenotype TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) that plays an important role in the development of transplant tolerance by specifically eliminating antidonor CD4+ T cells, CD8+ T cells and B cells and prolonging graft survival [[8-13]]. Coherently, others have reported that DN Treg cells can downregulate CD8+ T cell-mediated immune responses in autoimmune or infectious disease models [[14, 15]]. The CD4+ T cell-converted DN T cell is highly potent in suppressing alloimmunity both in vitro and in vivo and adoptive transfer of this cell could prolong islet graft survival [[16]].

CD4+CD25hi T cells were isolated by MACS using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec GmBH, Bergisch Gladbach, Germany). According to the protocol recommended by the manufacturer, a two-step isolation was performed, firstly isolating CD4+ cells and secondly enriching for CD25hi T cells using a (suboptimal) concentration of CD25 MicroBeads. CD4+CD25−/low T cells and CD4− cells together were considered as Treg-depleted PBMC. For

the total PBMC populations, the obtained cells were added back (mock depletion). For three donors the depletion was not successful Selumetinib price (no decrease in Treg frequency after depletion) and these donors were excluded for analysis of depletion effects. Mean depletion was 62.9%

(range 20.9–100%). To analyze Treg phenotype, PBMC were fixed and permeabilized with a FOXP3 Staining set (eBioscience, San Diego, CA, USA) and stained with fluorochrome labeled KPT330 anti-CD3, anti-CD4, anti-CD25, anti-CTLA-4 (BD Biosciences, Franklin Lakes, NJ, USA), anti-FOXP3 (Miltenyi) and anti-GITR (R&D Systems, Minneapolis, MN, USA) Ab. To monitor proliferation BrdU incorporation was assessed using the BrdU Flow Kit (BD). Total and CD4+CD25hi depleted PBMC were cultured in RPMI 1640 (Gibco, Invitrogen, Carlsbad, CA, USA) supplied with 10% FBS (Greiner Bio-One GmbH, Frickenhausen, Germany) and 10 μM BrdU. BCG (Bio Farma, Bandung, Indonesia, 0.5 μg/mL), 1×106P. falciparum pRBC or 1×106 uninfected DNA ligase RBC (uRBC) were used for stimulation. After 96 h cells were fixed in 2% formaldehyde (Sigma-Aldrich, CA, USA) and preserved

at −80°C. After thawing, cells were permeabilized and incubated with DNase (Sigma-Aldrich), labeled with anti-BrdU, anti-CD4 and anti-CD25 Ab (BD), acquired and analyzed. Proliferation of effector T cells was defined as the percentage of BrdU-positive cells within the CD4+CD25+ T-cell population. Cytokine production was assessed using the Multiplex Bead Immunoassay for IFN-γ, IL-5, and IL-13 according to the supplied protocol (Biosource, Invitrogen, Carlsbad, CA, USA). Samples were acquired with Luminex 100™ xMAP technology (Luminex, Austin, TX, USA). Half the detection limit supplied by the manufacturer was used, relevant background values (control medium for BCG, uRBC for pRBC) were subtracted and zero or negative values were set at 1 pg/mL. Statistical analysis was performed in SPSS 14.0. Comparisons of basic phenotypes and responses were tested with Mann–Whitney test for data not normally distributed. For total versus depleted samples paired analysis was done using Wilcoxon Signed Ranks Test. In the multiplex cytokine analysis Bonferroni correction was applied by multiplying the p-values by the number of non-correlated measurements. We acknowledge the technical expertise of Marga van de Vegte-Bolmer in production of P. falciparum culture material.

Previous results with N-acetylcysteine indicate a positive https://www.selleckchem.com/products/abt-199.html impact on microcirculatory flow during smoking, particularly in habitual smokers [37]. Capillary blood flow velocity increased after oral treatment with both antioxidants.

There was a prompt reduction in CBV in response to smoke inhalation. After two weeks of treatment with ascorbate or vitamin E, there was still a significant reduction in CBV (p < 0.0004 and p < 0.000008 respectively) in response to smoking, indicating the absence of a modifying effect of either antioxidant on this variable. It is plausible that naturally compensatory mechanisms might operate to maintain blood flow velocity within certain limits. The contribution of additional antioxidants through formation and preservation of vascular antioxidative defense may be sufficient to increase CBV in the resting state, but the acute high oxidative stress by free radical generation induced by smoking — such

as superoxide anions or hydroxyl radicals — not sufficiently counteracted by the immediately available increased antioxidative capacity of the endothelial interface. Free radicals are thought to inactivate eNOS and one possible mechanism by which antioxidants may serve to preserve endothelial function is to increase the bioavailability of nitric oxide [32,66]. NO may not necessarily directly mediate reactive hyperemia in the skin, but might possibly act in conjunction with other agents such as blood cells, hormones, Rucaparib endothelial adhesion molecules, prostaglandins, neural control, signal transduction pathways, and endothelium-dependent hyperpolarizing factors buy Olaparib to mediate reactive hyperemia. Furthermore, skin microcirculation is a main tool for thermoregulation with dual sympathetic neural control mechanisms and with a high vasodilatory capacity in response to various stimuli such as thermal, metabolic, and pharmacological

stimuli, also affected by reactive oxygen species [27]. Cigarette smoke contains free radicals and other oxidants in abundance, both in the gas phase and in the tar phase [47]. As vitamin C, but not vitamin E, affects TtP strongly, it suggests an important contribution by aqueous-phase reactive oxygen species in the immediate changes induced by cigarette smoke, whereas there is no prediction of effects on later stages of the sequence of mechanisms induced by the smoke inhalation. Although vitamin E has been shown to protect endothelial cells from reactive oxygen species, oxygenated lipids, lipoxygenase products, adhesion of leukocytes, and upregulation of adhesion molecules [35], there are several reports with the same finding as in our study, i.e., a positive effect of vitamin C, but not that of vitamin E [32]. Smoking results in an intense oxidative stress on the circulation and its effect on the microcirculation is of particular interest as it is one of the strongest risk factors in the development of cardiovascular disease.

It is recommended that a panel of investigators with a proven track record of using well-characterized animal models of T1D for disease reversal should be assembled with a mandate to develop a consensus on which animal models should be used and how precisely experiments should be carried out to meet FDA requirements for study approvals. Preclinical studies are carried out ideally at more than one site to circumvent local animal colony-related artefacts. In order

to assure uniformity when making comparisons between studies, standard operating procedures should be defined and standardized positive controls (e.g. anti-CD3) should buy Everolimus be instituted so that data from multiple laboratories could be obtained and be directly comparable. Such a consortium could consist of geographically diverse laboratories employing the same preclinical models in a standardized manner to examine combination therapies that are recommended by the ITN–JDRF Type 1 Diabetes Combination Therapies Assessment GPCR Compound Library Group. This would allow at least three laboratories to test the same combination therapy independently and simultaneously. In general, all tests should be conducted in models of recent-onset diabetes, wherein the blood glucose values and age of each mouse at inception of the intervention have to be tracked as independent variables

that are likely to affect the outcome of the treatment. To this end the ITN, in co-operation with JDRF, has begun developing a consortium of laboratories to carry out preclinical evaluations of combination therapies in Cetuximab type 1 diabetes. The consortium will consist of ∼6 geographically diverse, independent laboratories that will, in parallel, assess toxicology, pharmacodynamics and efficacy of potential combinations. All laboratory protocols will be standardized and all therapeutics would come from a standardized central source, preferably ‘good manufacturing practice’ (GMP)-grade material. The goal of this initiative is to provide an infrastructure that generates high-quality preclinical data rapidly to stimulate clinical assessments of novel combination therapies in T1D.

It is recommended that the above-mentioned preclinical studies also attempt to identify suitable biomarkers. One major gap is that animal studies notoriously track cells in tissues such as the pancreatic draining lymph node, whereas human studies will naturally have to use peripheral blood. As it is known that there can be substantial homing differences between different lymphoid compartments, it would be optimal to generate peripheral blood data during the preclinical animal studies so that precursor numbers and changes in lymphocyte subsets over time can be estimated more accurately. These efforts should then be aligned with current attempts to identify biomarkers within clinical trials in new-onset T1D, for example, at an annual biomarker meeting of participating entities.

Interestingly, MFIs for IFN-γ and IL-2 from multifunctional T cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation (Fig. 2c). Although this last result corroborates the ELISA data for IFN-γ protein detection in Leishmania antigen-stimulated PBMCs, we could not find any correlation between protein levels in the culture supernatants and the IFN-γ MFIs of multifunctional

triple-positive CD4+T cells after LaAg or LbAg stimulation. The same lack of correlation was observed when we compared the ELISA data with the total percentages of multifunctional T cells or the iMFIs of total IFN-γ-producing CD4+T cells. Because in the ELISA technique supernatants are analysed after 5 days of antigen stimulation, with the participation of other Selleckchem FDA approved Drug Library cell types besides CD4+ or CD8+T lymphocytes that can also produce IFN-γ, this lack of correlation could be expected.

In experimental leishmaniasis it has been shown that subcutaneous injections with LaAg alone can significantly increase the susceptibility of Rhesus monkeys to experimental infection Proteases inhibitor with L. amazonensis, despite the enhanced IFN-γ production and increased delayed-type cutaneous hypersensitivity [56]. Similarly, intramuscular LaAg was found to increase the susceptibility of BALB/c mice to cutaneous leishmaniasis, in a manner associated with up-regulated Interleukin-2 receptor transforming growth factor

(TGF)-β overcoming the increased IFN-γ[57]. In humans, L. amazonensis whole-cell extract has also been tested for both immunoprophylaxis and immunotherapy. As observed in experimental models, although capable of eliciting T cell-mediated responses in immunized volunteers and the production of expressive amounts of IFN-γ[58–60], a vaccine candidate composed of killed L. amazonensis promastigotes from the same strain utilized in the present work failed to induce protection in a Phase III clinical trial [61]. Conversely, the same preparation was shown to be extremely suitable for immunotherapeutic practice, especially in individuals who are resistant to the usual antimonial therapy and those with counterindications such as cardiopathy and nephropathy [62,63]. As IFN-γ single-positive CD4+T cells are short-lived [22,23], our results can offer a possible explanation for the diverse results observed in the prophylaxis and immunotherapy studies with L. amazonensis whole-cell extracts. LaAg stimulation induces a substantial amount of IFN-γ single-positive CD4+T cells, which may not be sufficient to induce long-term and good-quality protection against reinfection, but could be effective when a rapid and transient Th1 response is needed, as in the case of immunotherapeutic interventions.

, 1993; Eslava et al., 1998; Schubert et al., 1998; Czeczulin et al., 1999; Henderson et al., 1999; Tarr et al., 2000; Doughty et al., 2002; Scaletsky et al., 2005; Dudley et al., 2006). However, little has been reported concerning the presence of these virulence genes in EAST1EC. In the current study, we investigated the presence of a panel of non-typical virulence genes in EAST1EC strains isolated in Akita prefecture, Japan, from 2007 to 2009, selleck products to detect putative pathogenic determinants other than EAST1 in a collection of EAST1EC strains derived from diarrheal patients. A total of 2168 E. coli strains derived from diarrheal patients, defined as putative DEC, were collected from medical institutions in Akita prefecture,

Japan, from 2007 to 2009. These isolates were serotyped using a commercially available kit (Denka-Seiken, Tokyo, Japan). Differentiation of DEC was done using PCR-based identification of astA with stx, eaeA, est, elt, invE,

and aggR as described previously (Ito et al., 1992; Itoh et al., 1992; Yatsuyanagi et al., 2002), and the strains which detected no virulence genes except astA were defined as EAST1EC. Template DNA was isolated from EAST1EC strains by alkali treatment and subjected to PCR analysis. Twelve virulence genes were probed: eight genes associated mTOR inhibitor with adhesin (iha, lpfA, ldaG, pilS, pic, daa, aah, and aid), three genes encoding different toxins from EAST1 (pet, cdtB, and hlyA), and one gene encoding a bacterial siderophore called yersiniabactin (irp2). Primer sequences and PCR conditions for the amplification iha (Szalo et al., 2002), lpfA (Doughty et al., 2002), ldaG (Scaletsky et al., 2005), pilS (Dudley et al., 2006), pic (Czeczulin et al., 1999), pet (Gioppo et al., 2000), irp2 (Czeczulin et al., 1999), daa (Vidal et al., 2005), aah (Niewerth et al., 2001), aid (Niewerth et al., 2001), cdtB (Tiba et al., 2008), and hlyA (Yamamoto et al., 1995) have been described previously. PCR products were separated on 2% (w/v) agarose gels. Amplified DNA fragments 3-mercaptopyruvate sulfurtransferase of specific sizes were purified with a QIAquick Gel Extraction kit (Qiagen, Tokyo, Japan) according to the manufacturer’s

instructions, after staining with ethidium bromide, and visualized on a UV transilluminator. PCR amplicons were confirmed by DNA sequencing analysis with the primers used for PCR and the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan) on an ABI-3130 apparatus (Applied Biosystems). Between 2007 and 2009, a total of 2168 putative DEC strains were isolated in Akita prefecture, Japan, 35 (1.6%) of which were EAST1EC strains (Table 1). There was a variety of DEC serogroups among the EAST1EC strains, including O166, which was the cause of a previous outbreak (Zhou et al., 2002). During the 3-year period, 141 (6.5%) EHEC (or STEC), 35 (1.6%) EPEC, 18 (0.8%) ETEC, and 29 (1.3%) EAggEC strains were also detected in the 2168 putative DEC strains; no EIEC strains were detected.