[16] CD4+ T cells labelled with CFSE were cultured with anti-CD3 antibody (0·5 μg/ml) Fostamatinib chemical structure for 48 or 72 hr (Fig. 2f). At each time-point examined, SD-4+/+ and SD-4−/− T cells showed almost identical patterns of cell division (as reflected from diffusion of CFSE fluorescent intensity).

Similar results were also noted with lower concentrations (0·1 and 0·3 μg/ml) of anti-CD3 antibody (see Supplementary material, Fig. S2). We then examined the effect of SD-4 deletion on the intrinsic response triggered by concanavalin A, wihch activates T cells in a non-specific manner (Fig. 2g). Again, there was no significant change in T-cell proliferation. Hence, lack of SD-4 expression does not alter the intrinsic responsiveness of T cells to TCR-dependent or non-specific Buparlisib stimulation. These features distinguish SD-4 from PD-1 and BTLA, whose respective deletions augment T-cell responses to anti-CD3 stimulation.[20, 21] Using the mixed lymphocyte reaction, we examined the impact of SD-4 deletion on T-cell reactivity in response to allogeneic DC-HIL+ APC (Fig. 3a,b). CD4+ T cells

(varying numbers) isolated from WT or KO C57BL/6 mice were co-cultured with DC (constant number) prepared from BM cells of BALB/c mice. T-cell activation was measured by secreted IL-2 (Fig. 3a) or by proliferation (Fig. 3b). SD-4−/− T cells produced IL-2 at a four-fold greater level and proliferated at a two-fold higher level, respectively, than SD-4+/+ T cells. We next used a defined antigen model of gp100 (melanoma-associated antigen).[22] SD-4 gene deficiency was introduced into the pmel-1 TCR transgenic mice (in which all CD8+ T cells express the same TCR specific to a particular gp100 antigen peptide).[23] With respect to relative proportions of leucocyte sub-populations in lymphoid organs, there was no significant difference between SD-4+/+ and SD-4−/− pmel-1 mice (data not shown). We then assayed the reactivity of T cells to gp100 peptide-loaded APC. Spleen cells isolated from SD-4+/+ or SD-4−/− pmel-1 mice were

stimulated by increasing doses of antigen and measured for proliferation (Fig. 3c). SD-4+/+ pmel-1 spleen cells proliferated and produced IL-2 in response to gp100 antigen in a dose-dependent manner. Similarly, SD-4−/− pmel-1 spleen cells Baricitinib responded to antigen, but with significantly elevated levels (more than twofold greater responses by SD-4−/− pmel-1 T cells) at almost every single dose of antigen. To more rigorously examine the impact of SD-4 deletion, BMDC were prepared from WT mice and allowed to stimulate SD-4+/+ or SD-4−/− CD8+ T cells (Fig. 3d). SD-4−/− CD8+ T cells produced greater levels of IL-2 than SD-4+/+ CD8+ T cells (up to twofold), consistent with the previous data (Fig. 3c). As SD-4 is also expressed by DC (unpublished data), we examined the possibility that contaminant APC in the T-cell preparation from KO mice contributed to hyperactivation (Fig. 3a).

Our understanding of the basic immunobiological properties of DC has been significantly advanced over the years. This has not only provided good explanations for the problems encountered, but also stimulated many new

ideas regarding the potential ways forward aimed to improve DC therapy in a more fundamental way. The important issues lie within DC heterogeneity and functional plasticity, and hence their immunogenic versus tolerogenic properties or potentials. see more It has gradually become clear that DC are not a homogeneous population, and questions have also been raised about the origin and nature of the monocyte-derived, DC-like cells generated in vitro 27. The ability of these cells to provide activation signals, of both antigen-specific and non-specific triggers, can vary vastly among DC subsets or lineages, and depends on their functional status 28–31. Among them, a unique human DC subset (CD11c+CD141+), with superior antigen cross-presentation capacity and expressing the XC chemokine

receptor 1 (XCR1+), has recently been identified by several groups as the homologue of mouse CD8α+ DC 32–35. As with their murine counterparts, this type of DC was found to be effective activators of CD8+ cytotoxic T cells, which selleck screening library may have important implications in the design of new human DC vaccines. Moreover, in addition to subset-dependence, the functional properties of DC are also associated with the maturation status of the cell. Immature DC are in a so-called “antigen-uptake mode”, with low cell surface expression of MHC class I and class II molecules, which

can be rapidly enhanced upon exposure to maturation or activation signals, acquiring subsequently the “antigen-presenting mode”. The low MHC expression may therefore affect the ability of immature DC to present antigen to T cells. Under certain conditions, DC can even exert tolerogenic effects by producing immunosuppressive molecules, Cobimetinib molecular weight or by inducing regulatory T cells, to inhibit the immune system 1, 8, 24, 36. The concept of tolerogenic DC has become far more appreciated. It is now recognised that while immunogenic DC play an important role in host defence, their tolerogenic counterparts are crucial for the maintenance of self-tolerance, being part of a built-in mechanism to avoid autoimmunity 37. It has been demonstrated that, under the tumourigenic microenvironment, the host DC possessed a typical tolerogenic, or regulatory, phenotype 38. DC, as a double-edged sword, can therefore induce either active immunity or tolerance depending on their functional conditions. The types and functional status of DC, hence the immunogenic “quality” or nature of the cell vectors employed for tumour vaccine delivery, are therefore of critical importance. Various attempts have subsequently been made in order to generate DC with a highly immunogenic phenotype.

These findings suggest that VSL may have both domain-general and domain-specific associations with language learning. “
“Recent work has shown that young children can use fine phonetic detail during the recognition of isolated and sentence-final words from early in lexical development. The present study investigates 24-month-olds’ word recognition in sentence-medial position in two experiments using an Intermodal Preferential Looking paradigm. In Experiment 1, French toddlers detect word-final voicing mispronunciations (e.g., buz [byz] for bus [bys] “bus”), and they compensate for native voicing assimilations (e.g., buz devant toi [buzdəvɑ̃twa] “bus in front of you”) in the

middle of sentences. Similarly, English toddlers detect word-final voicing mispronunciations (e.g., sheeb for sheep) in High Content Screening Experiment 2, but they do not compensate for illicit voicing assimilations (e.g., sheeb there). Thus, French and English 24-month-olds can take into account fine phonetic detail even if words are presented

in the middle of sentences, and French toddlers show language-specific compensation abilities for pronunciation variation caused by native voicing assimilation. “
“Infants start pointing systematically to objects or events around their first birthday. It has been proposed that infants point to an event to share their click here appreciation of it with others. In this study, we tested another hypothesis, according to which infants’ pointing could also serve as an epistemic request directed to the adult. Thus, infants’ next motivation for pointing could include the expectation that adults would provide new information about the referent. In two experiments, an adult reacted to 12-month-olds’ pointing gestures by exhibiting “Informing” or “Sharing” behavior. In response, infants

pointed more frequently across trials in the Informing than in the Sharing condition. This suggests that the feedback that contained new information matched infants’ expectations more than mere attention sharing. Such a result is consistent with the idea that not just the comprehension but also the production of early communicative signals is tuned to assist infants’ learning from others. “
“Non-verbal referential communication is impaired in children with autism spectrum disorders (ASD). However, the development of difficulties with referential communication in the younger siblings of children with ASD (High-Risk Siblings)—and the degree to which early referential communication predicts later autism symptomatology—is not clear. We modeled the early developmental trajectories of three types of referential communication: responding to joint attention (RJA), initiating joint attention (IJA), and initiating behavioral requests (IBR) across 8, 10, 12, 15, and 18 months of age in High-Risk Siblings (n = 40) and the infant siblings of children without ASD (Low-Risk Siblings; n = 21).

Following incubation with the respective antibodies (20 min, room temperature),

cells were analyzed by FlowJo® (Tree Star, Ashland, OR, USA) software. Results are expressed as mean fluorescence intensity (mean of all) in the appropriate gate. Ten thousand cells were counted. T3M4 (5 × 105) cells in 2 mL medium were seeded into six-well culture plates and transfected with two different E-cadherin-specific siRNA (siRNA: Hs_CDH1_12 and Hs_CDG1_13; Dabrafenib molecular weight Qiagen, Hilden, Germany). Nontargeting scrambled siRNA (Ambion Applied Biosystems, Darmstadt, Germany) served for mock-transfection of the cells. Cells were transfected according to the manufacturer’s recommendations, using 450 ng of specific siRNA or scrambled siRNA and 12 μL Hiperfect transfection reagent (Qiagen) per subset. The siRNA and the scrambled siRNA were preincubated with serum-free medium and the respective transfection reagent for 15 min, and then added into the experimental subsets. After 24 h, medium was replaced, and the cells were incubated for another 24 h. The outcome of the transfection procedure was tested by cytofluorometry. Proteins from 3 × 106 T3M4 cells with or without treatment of neutrophil elastase (3 μg/mL for 2 h), respectively, after siRNA transfection were isolated using the ProteoExtract™-kit

(Calbiochem/Merck, Darmstadt, PD-0332991 cost Germany) for the isolation of subcellular compartments (membrane, cytoplasm, nucleus, cytoskeleton), according to the manufacturer’s recommendation. Selleck Pembrolizumab Protein samples were heated for 10 min at 95°C and separated by SDS-PAGE (7%). After blotting to a nitrocellulose transfer membrane (Whatman, Dassel, Germany), a rabbit polyclonal Ab to E-cadherin (Santa Cruz; 1:2000), or mouse mAb to β-catenin (BD Pharmingen, Heidelberg, Germany; 1:2000) diluted in 5% BSA, 1× TBS, and 0.1% sodium azide (Calbiochem/Merck) was added (at 4°C over night). After

washing, membranes were incubated using a goat antirabbit IgG POX, respectively, goat antimouse IgG POX (BD Biosciences, Heidelberg, Germany) as the secondary Ab (room temperature for 30 min). To control for equal loading, β-actin or in case of nuclear extracts p84 was determined using antiactin or anti-p84, respectively (both obtained from Abcam, Cambridge, UK). For detection, Amersham ECL plus Western Blotting Detection System (GE Healthcare, Munich, Germany) was used. Soluble E-cadherin in cell culture supernatants was determined using a commercially available ELISA kit (Quantikine ELISA Kit, R&D Systems, Darmstadt, Germany) according to the manufacturer’s instructions. All samples were at least measured in duplicate. Invasion assays were performed using a standardized Matrigel invasion chamber (Biocoat Matrigel™ Invasion chamber, 8 μm pore size; BD Biosciences) according to the manufacturer’s instruction.

IFN-γ has been shown to control C. abortus growth in ovine cells in a dose-dependent manner.26 The amount of IFN-γ that infected cells are exposed to is critical, because concentrations of around 50 U/mL or lower can induce persistent infection whereas concentrations of 200 U/mL or greater can eradicate the infection.26 Immune-mediated persistence of C. abortus has Pritelivir mouse important implications for OEA pathogenesis and epidemiology, because persistence in

non-pregnant sheep permits pathogen survival within the host outwith periods of reproduction.18 The mechanism by which IFN-γ controls the growth of Chlamydia is not uniform across species, and furthermore there is evidence for evolution of host–pathogen interactions and evasion of the IFN-γ response by Chlamydiae

as is the case for Chlamydia muridarum in mice.27 In sheep, we know that IFN-γ-mediated control of C. abortus occurs through the activation of indoleamine 2,3-dioxygenase (IDO), an enzyme that degrades intracellular pools of tryptophan.28C. abortus lacks all of the five genes (trpA–trpE) that comprise a functional operon required for synthesis of tryptophan from chorismate and is therefore highly susceptible to IFN-γ-induced IDO expression.29 Hence, this fits the prediction of the paradigm that host control of C. abortus is mediated through an IFN-γ TH1-type response, although to reiterate the point aforementioned, it is not yet clear whether CD4+ve T cells are the principal producers of this cytokine in immune

sheep. Placental IDO expression was first conclusively demonstrated as Rapamycin concentration a mechanism for tolerizing maternal T cells to the foetus in a series of experiments involving administration of an IDO inhibitor to mice carrying syngeneic or allogeneic concepti and adoptive transfer of allospecific CD8+ve T cells.30 In contrast cAMP to the intracellular IDO host defence pathway described earlier, placental IDO expression was not induced by IFN-γ but instead was found to be constitutively expressed by foetal trophoblast cells. This is not unique to mice. Constitutive IDO expression has also been described in syncytiotrophoblast of human, rhesus monkey and marmoset placenta at term.31,32 However, to date, there are no definitive reports in the literature of placental IDO expression in sheep (or other ruminants). Therein is a key question: is foetal trophoblast IDO expression associated with placental structure, particularly to the degree of foetal trophoblast invasiveness into maternal uterine tissue? The evolutionary processes that have driven the different shapes and structure of mammalian placentas remain controversial, so the answer to the IDO question may help identify factors that have influenced mammalian placental development and immunological materno–foetal interactions.

We previously reported that a single nucleotide polymorphism (SNP), rs2268338, within the gene encoding ACCβ was associated with susceptibility to diabetic nephropathy in Japanese patients with type 2 diabetes. Although subsequent functional analyses suggested that increased expression of ACCβ in the kidney contributed to susceptibility to the disease, its pathological significance has not been fully elucidated yet. Methods: To know the role of ACCβ in the pathogenesis of diabetic

nephropathy, we examined the effect of ACCβ overexpression on podocyte injury using podocyte-specific ACCβ transgenic (TG) mice and ACCβ-overexpressing cultured murine podocytes. Results: TG mice showed normal renal manifestation under non-diabetic condition. However, 12 weeks after induction of diabetes C646 by streptozotocin injection, the increase of urinary albumin excretion was exacerbated in TG mice, high throughput screening compounds accompanied by a decrease in the expression of synaptopodin in podocytes,

compared to wild-type mice. In cultured murine podocytes infected with adenovirus vectors encoding ACCβ, the expression of synaptopodin and podocin decreased under high glucose condition, but not under normal glucose condition. Furthermore, overexpression of ACCβ under high glucose condition resulted in reorganization of stress fibers, increased production of cytokines such as MCP-1, IL-6, TNF-α and VEGF, and induction of apoptosis in the murine podocytes. AMP-activated protein kinase (AMPK) is the main kinase regulator of ACCβ, which inactivates ACCβ through the phosphorylation

of serine residues on ACCβ. The AMPK activation by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) ameliorated ACCβ-induced decrease in the expression of synaptopodin and podocin, reorganization of stress fibers, increased production of cytokines, and induction of apoptosis under high glucose condition in the murine podocytes. Conclusion: From these observations, it is suggested that excess of ACCβ contributes to exacerbation of podocyte injury in diabetic nephropathy, and the regulation of AMPK/ACCβ pathway may be a new therapeutic strategy to prevent podocyte injury in patients with diabetic nephropathy. JHA JAY C1,2, GRAY STEPHEN P1, WINGLER KIRSTIN3, SZYNDRALEWIEZ Idelalisib manufacturer CEDRIC4, HEITZ FREDDY4, COOPER MARK E1,2, SCHMIDT HARALD HHW3, JANDELEIT-DAHM KARIN A1,2 1Diabetic complications division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia; 2Department of medicine, Monash university, Melbourne, Australia; 3Department of Pharmacology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Netherlands; 4Genkyotex SA, Geneva, Switzerland Introduction: Chronic kidney disease is a major complication of diabetes. However, the underlying causes remain unclear.

4B) of Hax1−/− mice were decreased for the CD4+ the CD8+ T-cell population (Hax1−/−: 6.55±1.86×106 and WT: 17.20±2.44×106 for CD4+ cells; p<0.001; Hax1−/−: 2.72±0.69×106 and WT: 7.76±1.79×106 for CD8+ cells; p<0.001). To evaluate the response of Hax1−/− B cells to key B-cell mitogens and

growth factors, splenic resting B cells of Hax1−/− and WT mice were isolated, labelled with CFSE and stimulated with anti-IgM F(ab’)2 plus anti-CD40, IL-4 plus anti-CD40 or LPS alone (Fig. 5B). In parallel, splenic CD4+ T cells were stimulated with anti-CD3/anti-CD28 (Fig. 5C). LPS-induced proliferation was slightly increased in Hax1−/− mice, while all other stimuli, for both B and T cells, showed no difference between Hax1−/− and WT mice. Next, we asked selleck compound whether Hax1−/− B cells were able to produce serum immunoglobulins at normal levels. We determined the

levels of IgM, IgG1, IgG2a and IgE in the serum of 7- to 8-wk-old naïve mice and found that the selleck inhibitor levels in Hax1−/− mice resembled those from WT littermates (Fig. 5A) except for the IgG2a levels, which were slightly but significantly lower in Hax1−/− mice. We next asked Whether the observed defects in B lymphocyte development were of B-cell-intrinsic or -extrinsic origin. Therefore, we performed adoptive transfer experiments using the congenic CD45.1/CD45.2 system. Lin– bone marrow cells from Hax1−/− and WT mice were transferred i.v. to reconstitute lethally irradiated CD45.1+/+ BALB/c mice. Analysis of the peripheral blood by flow cytometry 6 wk after transfer showed a weak increase in the percentage of circulating B220+ cells

and a parallel reduction in TCR+ cells in recipients of Hax1−/− cells compared to controls. Twelve weeks post transfer, this difference in the composition of the peripheral blood became negligible (Fig. 6A). Fourteen to sixteen weeks after transfer, the cell numbers of spleen, thymus and bone marrow from recipients of Hax1−/− and WT bone marrow cells, others respectively, were basically indistinguishable (Fig. 6B). Flow cytometric analysis of the bone marrow from recipients (Fig. 6C; primary gating history is shown in Supporting Information Fig. 2) demonstrated that the transfer of Hax1−/− bone marrow cells into a HAX1+ environment gave rise to normal levels of B220+ cells and functional B-cell subsets (Hax1−/−: 7.88±1.61×106 and WT: 7.26±3.16×106 for B220+; Hax1−/−: 2.11±0.45×106 and WT: 1.80±0.61×106 for B220+CD43+; Hax1−/−: 5.73±1.15×106 and WT: 5.41±2.53×106 for B220+CD43−; Hax1−/−: 0.46±0.08×106 and WT: 0.46±0.18×106 for Fr. A; Hax1−/−: 1.02±0.28×106 and WT: 0.69±0.22×106 for Fr. B; Hax1−/−: 0.47±0.10×106 and WT: 0.49±0.19×106 for Fr. C; Hax1−/−: 3.02±0.42×106 and WT: 2.85±1.22×106 for Fr. D; Hax1−/−: 1.35±0.37×106 and WT: 1.09±0.53×106 for Fr. E; Hax1−/−: 0.45±0.17×106 and WT: 0.47±0.26×106 for Fr. F). Accordingly, no differences were observed in splenic B-cell subsets (Fig. 6D; primary gating history is shown in Supporting Information Fig.

This work was supported by the Royal Netherlands Academy of Arts and Sciences SPIN projects, (KNAW grant 05-PP-35), European Commission contracts INCO-CT-2006-031714, INCO-CT-2006-032436 and Food-CT-2005-517812 and a VENI-grant from the Dutch Foundation of Science (NWO 016.066.093 to H. S.). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“In the MOG35–55 induced

EAE model, autoreactive Th17 cells that accumulate in the central nervous system acquire Th1 characteristics via a T-bet dependent mechanism. It remains to be determined whether Th17 plasticity and encephalitogenicity are causally related to each other. Here, we show that IL-23 polarized T-bet−/− check details Th17 cells are unimpaired in either activation or proliferation, and induce higher quantities of the chemokines RANTES and CXCL2 than WT Th17 cells. Unlike their WT counterparts, T-bet−/− Th17 cells retain an IL-17hiIFN-γneg-lo cytokine profile following adoptive transfer into syngeneic hosts. This population of highly polarized Th17 effectors is capable of mediating EAE, albeit with a milder clinical course. It has previously been reported that the signature Th1 and Th17 effector cytokines, IFN-γ and IL-17, are dispensable AZD0530 research buy for the development of autoimmune demyelinating disease. The current study demonstrates that the “master regulator” transcription factor, T-bet, is also not universally

required for encephalitogenicity. Our results contribute to a growing body of data showing heterogeneity of myelin-reactive T cells and the independent mechanisms they employ to inflict damage to central nervous system tissues,

complicating the search for therapeutic targets relevant across the spectrum of individuals with multiple sclerosis. EAE is a CD4+ T-cell-mediated autoimmune disease of the central nervous system (CNS), widely used as an animal model of multiple sclerosis (MS). Despite substantial progress in elucidating pathogenic pathways that drive EAE, the mechanisms employed by autoreactive T cells to initiate inflammatory demyelination and, hence, the effector functions that are critical for their encephalitogenicity, are largely unknown. We and others have previously shown that IL-12-polarized second Th1 and IL-23-polarized Th17 cells specific for the same myelin antigen are independently capable of inducing EAE following adoptive transfer into naïve syngeneic hosts [1, 2]. Surprisingly, full blown disease occurs in the absence of the signature Th1 and Th17 cytokines, IFN-γ, and IL-17A/F, either alone or in combination [3-5]. More recently, the master regulatory transcription factor, T-bet, was identified as a critical molecule in the programming of encephalitogenic Th17 as well as Th1 cells [6]. T-bet was originally described as a driver of Th1 differentiation via direct activation of the IFN-γ gene and upregulation of the IL-12 receptor β2 chain [7, 8].

In a previous study, C. jejuni 11168-GS, whose genome has been completed [17], was shown to have the form of a straight rod with polar flagella and significantly impaired motility [18], whereas its original clinical isolate (11168-O) had a spiral body with polar flagella with high motility [18]. However, in this study, C. jejuni KB3439, which is a straight rod with polar flagella, was highly motile, similarly to spiral C. jejuni with polar flagella, strongly suggesting that the spiral shape

is not essential for high-speed motility in C. jejuni in vitro. Cup-like structures were present in C. 5-Fluoracil manufacturer jejuni non-motile strain KB3449, indicating other impaired steps related to flagella formation. In this

study, it was found that C. fetus, which grows at low temperatures (25°C) but not at higher temperatures (42°C), has a flagellum at only one pole (except for dividing [long] cells, which have flagella at each pole), unlike C. jejuni, C. coli, or C. lari. Nevertheless, C. fetus has high-speed motility that is strictly temperature dependent (similar to C. jejuni). However, the polar cup-like structures of C. fetus seem to be composed of two parallel Venetoclax in vivo membranes (an inner membrane and an inside [third] membrane, located immediately inside and parallel to the inner membrane). For three other Campylobacter (C. jejuni, C. coli, and C. lari), the inside structure (of their

cup-like structures) remain uncertain. During this study, Chen et al. described the flagellar motor architecture of C. jejuni [19]. Their analysis by an electron cryotomographical survey focused on a small inner-outer membrane region, associated with the flagellar motor, and demonstrated two unique disk-like densities in the periplasm: the first disk (outer radius, 48 ± 9 nm) below the outer membrane (and connecting to the P-ring) and the second (radius, 32 ± 7 nm) Leukotriene-A4 hydrolase beneath the first (probably connecting to the M/S-ring). These two disks may correspond to the funnel shape we identified in this study. The cup-like structures, located immediately beneath the inner membrane at the pole-side (over 200 nm in length), have not been analyzed by Chen et al. [19]. The molecular structure in the flagellate polar region, factors (other than temperatures) which affect motility speed (such as serum concentrations or origin of serum) and inhibitors of motility are under continuing investigation in our laboratory. We thank Akemi Kai (Tokyo Metropolitan Institute of Public Health, Tokyo, Japan) for C. fetus and C. lari strains and Akihito Nishiyama (Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan) for discussion.

After 3 days, HSCs were isolated from the bone marrow. After 10 days in culture, 1×105 cells of two different HSC populations were injected into Rag-2/γC−/− mice expressing either H-2Kd or H-2Kb. Mice were analyzed 4–5 wk after HSC transfer. Animal experiments were done in compliance with the guidelines of German law and the Max-Planck-Institute of Palbociclib mouse Immunobiology and Epigenetics. HSCs were grown in Iscove’s medium (Biochrom) supplemented

with 2% of heat inactivated FCS (PAN Biotech), 10 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin (GIBCO), 50 mM 2-mercaptoethanol, 0.03% primatone (Sigma-Aldrich), 4.2 mg/mL insulin (Sigma-Aldrich), IL-6, IL-3 and c-kit-ligand. The expression of H-2d and H-2b was determined by flow cytometry using the specific monoclonal antibodies H-2Dd-PE and H-2Kb-FITC

(BD). Cells were stained with anti-B220/CD45R-PerCP (RA3-6B2, BD), anti-CD43-PE (S7, BD), anti-CD19-PE/-PerCP (1D3, BD), anti-CD21-APC (7G6, BD), anti-CD23-PE/biotin (B3B4, BD/PharMingen), anti-IgM-Cy5 (Jackson Immunoresearch) and anti-idiotype 54.1 (kindly provided MAPK Inhibitor Library in vivo by D. Nemazee). Flow cytometric analysis was performed with FACS-Calibur (BD). Statistical analysis was performed with the GraphPad Prism 4 software using Student’s t-test as the statistical hypothesis test. The authors thank U. Stauffer, N. Joswig and C. Johner for mouse work and further assistance. They thank E. Hobeika for the mb1-lox-GFP mice, P. Nielsen, D. Nemazee and M. Reth for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB620 and SFB746). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”.

Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Two outbreaks of Streptococcus suis ST7 occurred in humans in 1998 and 2005 in China. PFGE of GPX6 chromosome restriction fragments found all ST7 isolates to be indistinguishable. Due to the genetic homogeneity of ST7 isolates, development of a rapid sub-typing method with high discriminatory power for ST7 isolates is required. In this study, a novel method, MLVA, was developed to type S. suis serotype 2 strains. Further, this method was used to analyze outbreak-associated ST7 strains in China. A total of 144 ST7 S. suis isolates were sub-typed into 34 MLVA types. Among these, eight isolates from the 1998 outbreak were sub-typed into five MLVA types, of which four MLVA types were also detected in Sichuan in 2005. These data indicate that the pathogens responsible for the two outbreaks had the same origin. In addition, some observations also provided molecular evidence for the transmission route, possibly indicating that the MLVA method has usefulness in epidemiology. The developed MLVA scheme for S.