, 2006; Pamp & Tolker-Nielsen, 2007). Moreover, swarming motility has been shown to be part of a complex differentiation process, which

leads to increased production of virulence factors and antibiotic resistance (Overhage et al., 2008). Swarming is dependent on functional quorum sensing (which induces the production of rhamnolipid), type IV pili and flagella (Kohler et al., 2000; Deziel et al., 2003). We have demonstrated recently that ginseng extract https://www.selleckchem.com/products/LBH-589.html reduces the production of signal molecules of quorum sensing (BHL and OdDHL) in supernatants of P. aeruginosa PAO1 cultures (Song et al., 2010). This finding may partly explain our results from the swarming tests in this study. However, the molecular mechanism of inhibition of swarming motility and induction of swimming and twitching motility by ginseng extract is

still unknown and needs further studies. In our animal study, pretreatment buy Kinase Inhibitor Library with ginseng orally resulted in significantly higher phagocytosis rates and index in the BAL phagocytes from the wild-type P. aeruginosa PAO1-infected animals compared with saline-pretreated animals (Fig. 5a and b). In contrast, in the animals infected with flagella-deficient P. aeruginosa PAO1-filM, ginseng pretreatment did not improve the phagocytosis or the index. Clearly, the significantly increased phagocytosis rate and index in the PAO1-infected animals are due to the stimulation of P. aeruginosa PAO1 motility induced by ginseng in vivo. Previously, 3-oxoacyl-(acyl-carrier-protein) reductase we demonstrated in our animal models of chronic

P. aeruginosa lung infection that ginseng treatment results in faster bacterial clearance from the lungs and milder lung pathology when compared with the untreated animals (Song et al., 1997a, b, 1998). We also observed a significantly stronger neutrophil chemiluminescence in the blood, a shift of the immune response from a high anti-P. aeruginosa immunoglobulin G (IgG) response and local infiltration of mast cells in the lungs (T-helper type 2 response) to a TH1 immune response characterized by downregulation of IgG and upregulation of IgG2a levels, and improved functions of phagocytes by means of upregulated production of interferon-γ and downregulated interleukin-4 in the lung tissues and spleen (Song et al., 1997a, b, 1998, 2003, 2005). It has been well documented that a TH1 response favors host cleaning of infections by P. aeruginosa (Johansen et al., 1995, 1996., 1997; Moser et al., 1997, 2000, 2005). Our results from the present study suggest that ginseng induces increased bacterial motility in the biofilm-like alginate beads, resulting in the release of bacteria from the biofilm and loss of protective effects from the polymeric matrix, followed by an increased efficiency of the host immune system and antibiotics to clear the biofilm infection. The activation of the TH1 immune response induced by ginseng treatment and the increased motility of bacteria due to the effects of ginseng might exhibit a synergistic effect on the infection.

Titres Rapamycin purchase by human reference sera and monoclonal antibodies to OMV antigens with known bactericidal titres. Opsonophagocytic activity was measured as respiratory burst [36]. Live meningococci from strain 44/76 and B1723 were used as targets, and human polymorphonuclear leucocytes from

a normal human donor, probed with dihydrorhodamine 123 (Invitrogen, Oslo, Norway), as effector cells. A human serum, different from that used in SBA, served as complement source after passing through a Protein G-column. A positive human reference serum was included as control for complement activity and assay sensitivity. Twofold serial dilutions of the mouse sera were analysed, and respiratory burst

was analysed with a flow cytometer (Partec CyFlow® ML, Partec GmbH, Münster, Germany) gating on the polymorphonuclear population. Opsonic titres were recorded as log2 of the highest reciprocal serum dilution giving ≥50% respiratory burst of the polymorphonuclear leucocytes. Magnetic polystyrene beads (40 mg/ml) (Dynabeads® Talon®; Invitrogen Dynal, Oslo, Norway) were washed with 50 mm Na-phosphate, pH 8.0, 300 mm NaCl and 0.01% Tween-20 according to the manufacturer’s instructions and incubated for 10 min in the same buffer with his-tagged recombinant Omp85. Preliminary experiments showed that 4 mg of beads bound ≤40 μg Omp85, as no Omp85 protein was detected XAV-939 cost after SDS gel electrophoresis of the

supernatant. The recombinant Omp85 protein preparation showed one band in SDS gels of molecular mass about 90 kDa. Due to the small amounts of recombinant Omp85 available, serum pools were used for the adsorption experiments. Differences in antibody levels were analysed with Student’s t-test or Mann–Whitney rank sum test with a SigmaStat 3.1 program (Systat Software, Chicago, IL, USA). Correlations between Ixazomib mouse SBA and PorA antibody levels were assessed by the non-parametric Spearman’s rank-order correlation test. P-values < 0.05 were considered significant. After induction of transformed meningococci with IPTG, the genetically modified Omp85+ OMVs expressed fivefold higher levels of Omp85 (mean 5.2; range 4.4–7.2 of six determinations) relative to PorA in SDS gels and on blots compared with same levels in the wt 1 and wt 2 OMVs (Fig. 1A,B). The SDS gel also shows that the three OMV preparations contained different levels of the opacity proteins OpcA and OpaJ (Fig. 1A). Omp85+ OMVs expressed negligible levels of both proteins; OpaJ was modestly increased in the wt 2 OMV control, whereas a dominant OpcA band was observed in wt 1 OMVs, as previously reported [33]. These antigens might possibly affect the bactericidal activity of the OMV vaccines. However, OpaJ does not induce bactericidal antibodies in mice [37].

3). In addition, the detection limit is very low. With only two DNA copies, it has a higher sensitivity than the currently applied molecular methods, such as semi-nested PCR learn more (10 pg) (Prariyachatigul et al., 2003), PCR enzyme immunoassay (3.2 pg) (Lindsley et al., 2001), PCR hybridization (0.1 pg) (Vanittanakom et

al., 1998) and nested PCR (0.07 pg) (Zeng et al., 2009). The results of P. marneffei detection by LAMP in 23 paraffin wax-embedded clinical samples and 11 bamboo rat tissues were also highly specific. The etiologic agents of the 23 clinical samples were verified previously using culture and sequencing data. Twelve samples were histopathologically positive; all molecular identifications matched with the clinical diagnoses. Samples from penicilliosis and from the natural bamboo rat host were positive with LAMP, whereas all others, including healthy human skin, proved to be negative. Test results were not inhibited by nontarget

DNA. This makes the LAMP technique highly promising for evaluation and application in problematic clinical see more samples such as blood, urine and sputum. In this study, we have proved with the example of P. marneffei that LAMP is a very efficient method for the quick and sensitive identification of fungal pathogens and opportunists. The method can be applied not only to cultures but also to a variety of clinical samples. This can be of great significance to organisms that cause invasive or disseminated infections that are difficult to cultivate from such samples, such as the zygomycete species. A further application may be for detection without isolation of the fungi in the environment. In summary, in the current study, we proved that the LAMP technique enables specific detection of P. marneffei and excludes related biverticillate penicillia and Talaromyces teleomorphs. Similar results were obtained in Paracoccidioides (Endo et al., 2004), Candida (Inacio et al., 2008) and Ochroconis (Ohori et al., 2006). However, in Fonsecaea, identification was possible only at the generic level (Najafzadeh, 2009). An explanation for this phenomenon may be found in the fact that

Penicillium species are relatively distant from each other, with ITS barcoding gaps well over 1%, whereas in Fonsecaea ITS, interspecific Ureohydrolase differences are a few bases only, species delimitations being based on multilocus analyses. We thank Prof. Yokoyama (Center for Pathogenic Fungi and Microbial Toxicoses Chiba University, Chiba, Japan) for providing the reference strains taxonomically close to P. marneffei included in this paper. This study was supported partly by a grant (30770121/2007) from the National Natural Science Foundation of China. “
“Human parturition is associated with an intrauterine pro-inflammatory environment in the choriodecidua. Evidence that some mediators of this signaling cascade also elicit responses leading to labor prompted us to characterize the cellular sources of these mediators in the human choriodecidua.

The consequence of this altered accessory repertoire on R-DC is that cocultured T cells acquire a deep anergic state 12, 13. Here we demonstrate that R-DC induce suppressor function in cocultured T cells. The inhibitory effect was found to be caused by the culture supernatant (SN) of CD4+ and CD8+ peripheral blood T cells, activated with R-DC, but not with naïve T cells from cord blood (CB). We found that R-DC-induced Treg produced and released IL-35, which Selleckchem Palbociclib is responsible for the inhibitory effect of the Treg SN. Most importantly,

blocking of B7-H1 and sialoadhesin on R-DC with specific mAb against both receptors prevented the induction of IL-35. Thus, inhibitory signals delivered from R-DC

to T cells via B7-H1 and sialoadhesin were essential to the induction of human IL-35-producing Treg, defining a novel route of T-cell instruction. We have recently demonstrated that HRV is able to subvert the T-cell stimulatory function of human DC. Cocultured T cells acquire a deep anergic state 12. This study was aimed to investigate whether T cells stimulated with R-DC gain a regulatory function. The results presented in Fig. 1A demonstrate that addition of T cells prestimulated with R-DC strongly inhibited T-cell proliferation, induced by untreated DC. Such an effect AZD6244 did not occur when T cells were primed with untreated DC. Prior fixation of R-DC-induced Treg reverted the inhibitory function (Fig. 1B). Addition of the inhibitor WIN 52035-2 at the time of transfer, which specifically blocks HRV binding to its cellular receptor ICAM-1 14, did not remove the suppressive effect, which indicates that viral transfer is not involved in the inhibitory response (Fig. 1C). Depletion of R-DC from the coculture with T cells did not alter the results: the purified prestimulated T cells still Thiamet G showed

eminent inhibitory effects when added to an MLR (data not shown). We conclude that T cells prestimulated with R-DC are responsible for the inhibitory effect observed. FOXP3 is a forkhead family transcription factor important in the development of Treg; therefore we evaluated its levels in R-DC-induced Treg. Analysis of FOXP3 expression in CD25- T cells revealed that induction of FOXP3 does not differ between DC and R-DC stimulated T-cells, indicating that the suppressive capacity of R-DC stimulated T cells is not directly correlated with an increased induction of FOXP3. Also FOXP3 levels drop again after 48 and 96 h in T cells, stimulated with R-DC or DC (Fig. 1D). Activation-induced transient FOXP3 expression in human T cells has been frequently observed before 10, 15–17 and is not necessarily correlated with regulatory activity 7, 10, 18. In order to analyze whether the inhibitory effect was mediated through (a) soluble factor(s), we added the SN of the R-DC-induced Treg to T-cell/DC cocultures.

Mice were treated i.p. with anti-CCR3 in three different doses (30–300 μg/animal in 500 μl PBS) or isotype control (100 μg/animal in 500 μl PBS, rat IgG2b, clone R35-38; BD-Bioscience Europe, Erembodegem, Belgium) 1 hr before allergen exposure on the first day of exposure. Cytospin preparations from BM and BAL were stained for CD34 using a biotinylated rat anti-mouse CD34 mAb (clone

RAM34; BD Biosciences). Bound antibodies were visualized with a Vector Red Alkaline Phosphatase Substrate kit (Vector Laboratories Inc., Burlingame, CA). The slides Pembrolizumab cost were also stained with Luxol Fast Blue and counterstained with Mayer’s haematoxylin (DAKO) to identify these cells as eosinophil-lineage precursors. Five hundred cells were evaluated in random fields of view. Cytospins from BAL were stained with a rat anti-mouse CD34 mAb (clone RAM34; BD Biosciences). A rabbit anti-rat immunoglobulin

(DAKO) was used as a link antibody before incubation with alkaline phosphatase–anti-alkaline phosphatase (DAKO). Bound antibodies were visualized with the Vector Red Alkaline Phosphatase Substrate kit. Slides were then treated with a biotin blocking system (DAKO) and incubated overnight at 4° with a biotinylated rat anti-mouse Sca-1/Ly6 mAb (Clone 177228; R&D Systems). Next day, the slides were washed and incubated with streptavidin-β-galactosidase and X-Gal substrate (β-Gal Selleck Palbociclib staining set; Roche) and counterstained with Mayer’s haematoxylin. Four hundred cells were counted in random fields of view. All data are expressed as mean ± SEM. Statistical analysis was carried out using a non-parametric analysis of variance (Kruskal–Wallis test) to determine the

variance among more than two groups. If significant variance was found, an unpaired two-group test (Mann–Whitney U-test) was used to determine significant differences between individual groups. Wilcoxon signed rank test was used to analyse changes within the same group. P < 0·05 was considered statistically significant. Flow cytometric analysis for CD34+ CCR3+ cells in BM, blood, lung and BAL showed a significant increase of this PAK5 cell population in all three compartments of OVA-sensitized/exposed animals when compared with OVA-sensitized but saline-exposed control animals (Fig. 1a). Triple staining for CD34+ CCR3+ Sca-1+ on lung cells was performed to determine if a part of the CD34+ CCR3+ cells also expressed Sca-1. Allergen exposure induced a significant increase in the number of CD34+ CCR3+ Sca-1+ lung cells both in the SSChigh gated population (i.e. eosinophils) and in the SSClow gated cell population (i.e. eosinophil-lineage-committed progenitors) when compared with saline-exposed animals (Fig. 1b). CCR3+, Sca-1+ CCR3+ and CD34+ CCR3+ cells were also increased in the SSChigh and SSClow gated cell populations in allergen-exposed mice when compared with saline-exposed mice (Fig. 1c,d).

The stained cells were analyzed using a flow cytometer, Cytomics FC500 (Beckman Coulter Inc., Fullerton, CA). The concentrations of TNF-α in the BALF were measured by an enzyme-linked immunosorbent assay using capture and biotinylated developing antibodies (BD Biosciences). The detection limit was 5 pg mL−1. Anti-TNF-α or -Gr-1 (rat IgG) mAb was purified using the

protein G column kit (Kirkegaard & Perry Laboratories) from the culture supernatants of hybridomas [clone MP6-XT2.2-11 or RB6-8C5, respectively Ceritinib mouse (a kind gift from Dr Akio Nakane, Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan, or Dr Fujiro Sendo, Yamagata University, Yamagata, Japan, respectively)]. To neutralize the biological activity of TNF-α, mice were injected intraperitoneally with mAb against this cytokine at 150 μg on days −1, 0 and +2 after infection. To delete Gr-1+ cells, mice received intraperitoneal injections of mAb against this molecule at 100 μg on days −1 and 0 after infection. Rat IgG (ICN Pharmaceuticals Inc., Aurora, OH) were used as the control antibodies. After staining

Selleckchem Z VAD FMK with FITC-conjugated anti-Gr-1 mAb (clone RB6-8C5, BD Biosciences), Gr-1bright+ and Gr-1dull+ cells were purified from BALF cells using the FACSAria™ Cell Sorter System (Becton Dickinson, Mountain View, CF). The sorted cells were centrifuged onto a glass slide, stained by May–Giemsa and observed under a microscope. In some experiments, these cells were

stained with phycoerythrin-conjugated CD11b, CD11c or F4/80 mAb (clone M1/70, HL3 or BM8, respectively; BD Biosciences) or biotinylated anti-major histocompatibility complex (anti-MHC) class II (I-Ab) or CD80 mAb (clone AF6-120.1 or B7-1, respectively; BD Biosciences) and allophycocyanin-conjugated streptavidin, and the stained cells were analyzed using a flow cytometer (Cytomics FC500). The purified Gr-1+ cells were CYTH4 cultured at 1 × 106 mL−1 with or without S. pneumoniae in RPMI1640 medium (Nipro, Osaka, Japan) supplemented with 10% FCS, 50 μM 2-mercaptoethanol, 100 U mL−1 penicillin G and 100 μg mL−1 streptomycin (Sigma, St. Louis, MO) at 37 °C in a 5% CO2 incubator for 24 h. The culture supernatants were kept at −80 °C until measurement of TNF-α. Analysis of data was conducted using statview ii software (Abacus Concept Inc., Berkeley, CA) on a Macintosh computer. Data are expressed as mean±SD. Statistical analysis between groups was performed using the anova test with a post hoc analysis (Fisher’ PLSD test). Survival data were analyzed using the generalized Wilcoxon test. A P-value <0.05 was considered significant. Initially, to define the role of TNF-α in the host defense to pneumococcal infection, we examined the effect of neutralizing anti-TNF-α mAb on the clinical course of infection with this bacterium. As shown in Fig. 1a, none of the infected and PBS-treated mice died during the observation periods (14 days).

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 Alisertib research buy 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 MK-2206 in vivo 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 Methocarbamol 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 contrast, the finding that the Fc fragments of antibodies were sufficient to reproduce

the anti-inflammatory effects of IVIg suggested that this treatment operates primarily by inducing immune-modulating mechanisms, which is discussed below. A breakthrough in understanding how IVIg provides protection from autoimmune diseases was JNK inhibitor the discovery that the type of glycan attached to the Fc domain decisively determines its anti-inflammatory effect when used in a prophylactic setting in a model of antibody-induced arthritis [12]. All IgG molecules possess a conserved N-linked glycosylation site in their Fc domain that can accommodate one of 32 distinct glycans [13, 14]. These glycans engage in numerous noncovalent interactions with the IgG protein itself, which regulates the quaternary structure of the Fc domain and thereby shapes the interaction between IgG and Fc receptors [15, 16]. The glycosylation pattern of IgG antibodies is altered in some autoimmune diseases such as rheumatoid arthritis, with changes correlating with disease activity [17]. This suggests an association, and possibly a causative connection between antibody glycosylation and inflammation. It is now possible to modify the glycosylation of antibodies using various enzymatic reactions or enrichment methods in vitro. Noteworthy, upon complete

removal of its glycosylation, IVIg was shown to lose its ability to inhibit the inflammation caused in mice by the injection of arthritogenic antibodies [12]. In about check details 1–3% of the IgG in IVIg, the glycans attached to the Fc domain end in sialic acid moieties. The specific Lonafarnib removal of these terminal sialic acid residues by neuraminidase treatment suffices to abolish the protective effect of IVIg [12]. In contrast, enrichment of IVIg in sialic acid-containing IgG increases

their anti-inflammatory activities [12]. It is therefore believed that a prominent protective component in IVIg preparations consists of the Fc portions of IgG dressed with glycans terminating in sialic acid [12]. The fact that such sialylated IgG represent only a minor fraction of IgG in IVIg might explain the need to use such high doses of this preparation to achieve anti-inflammatory effects [18]. Indeed, IVIg is typically administered at around 2 g/kg of body weight for the treatment of autoimmune or inflammatory diseases, while patients with immunodeficiencies usually receive only 0.5 g/kg. The identification of the molecular patterns responsible for the anti-inflammatory effect of IVIg has permitted the production of a recombinant IgG1 Fc protein that is sialylated in vitro and recapitulates the anti-inflammatory activity of IVIg against antibody-mediated arthritis in vivo in mice [18]. Production of such an engineered protein could offer an attractive alternative to IVIg, whose use is constrained by cost and availability. The identification of the receptor for IVIg and the cell type(s) implicated in its anti-inflammatory effects are pressing issues to resolve.

Clearly, several approaches may be taken to enhance DC tolerogenecity. We previously check details demonstrated that genetic modification of immature DCs

with inhibitory cytokines such as IL-10, TGF-β or soluble TNF receptor could inhibit pathogenesis of autoimmune diseases or prolong allograft survival 14–16. In addition, exosomes derived from IL-10-treated or IL-4 gene-modified DCs could also suppress inflammation and attenuate progression of autoimmune diseases 17, 18. In spite of the existing findings, new approaches to enhance DC tolerogenecity or utilizing new subsets of tolerogenic/suppressive/regulatory DCs for the control of inflammation and autoimmune diseases with increased efficacy and identifying the underlying mechanisms remains a hot topic in immunology. Ligation of Fc receptors for IgG (FcγRs) by immune complexes selleck inhibitor (IC) may trigger two opposing signals, activating or inhibitory, depending on the FcγR subtypes 19. Three FcγR subtypes are currently known: FcγRI, FcγRIIa and FcγRIII that trigger cell activation by the presence

of an immunoreceptor tyrosine-based activation motif in their cytoplasmic fragments 19, and FcγRIIb that deliveries inhibitory signal through its intracellular domain containing an immunoreceptor tyrosine-based inhibition motif 20. Accumulating evidences have shown that inhibitory FcγRIIb is important for the maintenance of peripheral tolerance, PAK5 and FcγRIIb deficiency is associated with the pathogenesis of experimental autoimmune models and of systemic lupus erythematosus (SLE) 21, 22. SLE is characterized by high levels of autoantibodies in circulation. Tissue deposition of IC plays a major role in the pathogenesis of inflammatory injuries in SLE. Therefore, enhancement

of FcγRIIb expression and function may be useful to the prevention and treatment of inflammatory autoimmune diseases such as SLE. Disorders of DC subsets and functions are associated with the pathogenesis of SLE. High level of type I IFN from overactivated plasmocytoid DCs (pDCs) are also involved in the pathogenesis of SLE 23. SLE patients have significantly decreased expression of BDCA2, a negative regulator of TLR9-dependent activation and, accordingly, have markedly elevated IFN-α levels 24. Furthermore, the decrease in myeloid DCs with a tolerizing phenotype was reported in SLE patients 25. Given these lines of evidence, it is plausible that intervention of DC function may be another approach for the treatment of SLE.

The second encodes a factor with considerable homologies (50% identical, 66% similar residues) to the human ‘metastasis-associated-protein’ MTA3 which is a component of the nucleosome-remodelling and histone-deacetylase complex (105) and, like the human protein, contains one BAH (bromo-adjacent homology) domain, one GATA-type zinc finger domain and one classical

zinc finger domain (data not shown). As previously suggested (72), the antigen B cluster is formed of one copy each of AgB1, AgB2, AgB4 and AgB5, two identical genes encoding AgB3 and one slightly altered AgB3 gene (AgB3’). The only difference to the previously suggested cluster organization (72) is that in the newest assembly version the AgB5 locus and R788 solubility dmso one AgB3 locus have changed position (Figure 2). All genes of the cluster display the typical organization (103) of two exons, with a signal peptide encoded by exon 1, separated by a small intron. Transcriptome analyses on in vitro

cultivated metacestode vesicles further indicate that AgB1 is, by far, the most abundantly expressed isoform, followed Selleck Metformin by AgB3’ (20% of the expression level of AgB1) and AgB3 (10%). Only marginal expression could be detected for AgB2, AgB4 and AgB5 in the metacestode, and likewise, almost no expression was measured for any AgB isoform in the protoscolex (data not shown). In E. granulosus, the situation appears to be highly similar to E. multilocularis (Figure 2). Within a region of approximately the same size as in E. multilocularis, close orthologs of EmLDLR (EgLDLR) and EmMTA (EgMTA) are present and are flanking a cluster of seven loci with one copy each of AgB1, AgB2, AgB4 and AgB5, as well as three slightly differing copies of AgB3 (AgB3-1, AgB3-2, Florfenicol AgB3-3). Although care has to be taken in suggesting complete synteny between both species in this region, because the single E. granulosus contigs (flanked by ‘N’ in Figure 2) have been assembled into supercontigs using the E. multilocularis sequence as a reference, at least the E. granulosus

copies of AgB1, AgB4 and AgB3-2 are clearly assembled into one contig and display the same gene order and transcriptional orientation as in E. multilocularis (Figure 2). This makes it highly likely that the genome arrangement as suggested for E. granulosus in Figure 2 reflects the true situation. Apart from the AgB cluster, we could not detect any AgB-related sequences elsewhere in the genomes of E. multilocularis and E. granulosus, with one notable exception of an AgB-like gene on E. multilocularis scaffold_7, that is, however, not represented in EST databases, does not show a detectable transcription profile in RNA-seq data, contains inactivating mutations within the reading frame (data not shown), and thus most likely represents a pseudogene.