Monocyte-derived DCs were generated from PBMCs as previously desc

Monocyte-derived DCs were generated from PBMCs as previously described with some modifications [51]. Briefly, CD14+ monocytes were enriched by positive selection using CD14 Microbeads (Miltenyi Biotec). Monocytes were cultured in the presence of 20 ng/mL GM-CSF (Immunex, Seattle, WA, USA) and 20 ng/mL IL-4 (R&D systems) in RPMI1640 supplemented with 2.5% fetal calf serum. Medium was replaced by fresh medium containing cytokines 3 days later. On day 6, cells were harvested and used for subsequent experiments. The concentration of IL-12p70 and IL-10 was measured by ELISA Kit (eBioscicence) according to the instruction provided by the manufacturer. Statistical significance was evaluated

by Student’s t-test; p values less than 0.05 are considered significant. This article is dedicated to Sorafenib chemical structure the memory of Lloyd J. Old, M.D. We thank Drs. T. Takahashi and J. B. Wing for critical reading of the manuscript, and L. Wang, C. Brooks, E. Krapavinsky, E. Ritter, and D. Santiago for technical support. This study was supported by Grant-in-Aid for Scientific Research on Priority Areas (No. 17016031, H. Shiku, and No. 20015019, H. Nishikawa) and Grants-in-Aid for Scientific Research (B) (No. 23300354, H. Nishikawa), the Cancer Research Institute Investigator

Award (H. Nishikawa) and Cancer Vaccine Collaborative Grant for PLX-4720 Immunological Monitoring (S. Gnjatic, G. Ritter and L.J. Old), Cancer Research Grant from Foundation of Cancer Research Promotion (H. Nishikawa), Takeda Science Foundation (H. Nishikawa), Kato Memorial Bioscience Foundation (H. Nishikawa), the Sagawa Foundation for RVX-208 Promotion

of Cancer Research (H. Nishikawa), and Senri Life Science Foundation (H. Nishikawa). MH is a research fellow of the Japan Society for the Promotion of Science. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Figure S1. (A) Preparation of NY-ESO-1 and 146HER2 proteins complexed with cholesteryl pullulan (CHP): Recombinant NY-ESO-1 and 146HER2 proteins for clinical use were prepared, and the nano-particles consisting of CHP and the NYESO-1 protein, and CHP and the HER2 complex were formulated. (B) Study design of the clinical trial. (C) Patient characteristics in this study. Figure S2. (A) DCs were prepared from four healthy individuals as described in Materials and Methods. TNF-⟨ (100 ng/ml), LPS (1 mg/ml), or OK-432 (1 ìg/ml) was added in the culture of 1 × 105 immature DCs on day 6. After 48 h, supernatant was collected and cytokine production was analyzed with ELISA. (B) Summary of cytokine secretion in from four healthy individuals.

The ratio of the frequencies of IFN-γ+ CD8+ T cells to IFN-γ+TNF-

The ratio of the frequencies of IFN-γ+ CD8+ T cells to IFN-γ+TNF-α+ CD8+ T cells was significantly higher after JEV SA14-14-2 immunization compared with WNV infection for JEV S9 and WNV S9 (p<0.05, Mann–Whitney U test) (Fig. 2D). No significant difference in this ratio was detected between the JEV S9 and WNV S9 variants in either JEV SA14-14-2 immunized or WNV-infected mice. Of note, IFN-γ+TNF-α+ CD8+ T cells from WNV-infected mice produced more TNF-α on a per cell basis than those from JEV SA14-14-2 immunized mice, while levels of IFN-γ from this population were similar for JEV

and WNV (Supporting Information Fig. 2). Since JEV SA14-14-2 is an attenuated virus, we used a pathogenic JEV (Beijing strain) to determine if DAPT manufacturer differences in cytokine profiles between JEV and WNV Erastin nmr could be explained on the basis of the pathogenicity of the infecting virus. We infected mice with a low dose (103 pfu – comparable dose to WNV) or high dose (106 pfu – comparable dose to JEV SA14-14-2) of JEV Beijing. Similar to JEV SA14-14-2, infection with either low- or high-dose JEV Beijing induced a significantly higher frequency of IFN-γ+ CD8+ T cells than IFN-γ+TNF-α+ CD8+ T cells compared to WNV infection (p<0.05, Mann–Whitney U test) (Fig. 2B and C). These findings

indicate that the infecting virus (JEV versus WNV) determined the altered cytokine profile. To ascertain whether the differences in the cytokine profiles are related to different

CD8+ T-cell kinetics, we measured epitope-specific dimer+ CD8+ T cells 5, 7 and 10 days post-infection. Rapid expansion of CD44hidimer+ CD8+ T cells occurred between days 5 and 7 with peak levels occurring at day 7 for all infections with the exception of high-dose JEV Beijing, which peaked at or before day 5 post-infection (Fig. 3 and Supporting Information Fig. 3A). For JEV SA14-14-2 and low-dose JEV Beijing, an approximately four- to eight-fold contraction in frequency and absolute cell number (data not shown) of JEV S9 dimer+ CD8+ T cells occurred between days 7 and 10 while only a one- to two-fold contraction in frequency and absolute cell number (data not shown) of WNV www.selleck.co.jp/products/BAY-73-4506.html S9 dimer+ CD8+ T cells occurred in WNV-infected mice. Similar to the pattern seen for cytokine production, infection with JEV induced a higher proportion of cross-reactive WNV S9 CD8+ T cells than cross-reactive JEV S9 CD8+ T cells seen in WNV infection. Although the peak CD8+ T-cell response for high-dose JEV Beijing occurred earlier, there was no difference in the frequency of IFN-γ+ and IFN-γ+TNF-α+ CD8+ T cells at day 7 for all JEV infections. These results suggest that the kinetics of epitope-specific cells are not related to the altered cytokine profiles seen. Effector CD8+ T-cell activation depends on many factors, including antigen stimulation and inflammatory conditions 20.

This, however, is in contrast with previous studies, which report

This, however, is in contrast with previous studies, which reported that eosinophils mainly secrete Th2-type cytokines in response to parasite antigens and allergens.33,34 The GM-CSF is a cytokine expressed by a variety of cells, including activated T cells, Mφ, fibroblasts and epithelial cells. GM-CSF

is required for the recognition of pathogens, the timely development and proper compartmentalization of the immune response and the control of pulmonary growth of C. neoformans.35 Furthermore, GM-CSF stimulates the functional activity of eosinophils and maintains the maximum viability of cells,13 and GM-CSF-activated selleck chemical eosinophils have been reported to be capable of acting as specific APCs to a T-cell BAY 73-4506 mw clone derived from mice infected with Mesocestoides corti.27 The results of the present study showed that

GM-CSF only modified the MHC class II expression levels on eosinophil surfaces cultured with C. neoformans. Moreover, C. neoformans-pulsed eosinophils in the presence of GM-CSF expressed threefold more MHC class II than C. neoformans-pulsed eosinophils in the absence of this stimulating factor (Fig. 2b). In contrast, GM-CSF did not modify phagocytosis of the fungus, the expression of MHC class I, CD80 or CD86, cytokine production or the fungicidal molecules released by eosinophils incubated with the fungus. Related to this, Feldmesser et al.19 have demonstrated that short-term incubation with IL-5, GM-CSF and lipopolysaccharide (LPS) did not appear to enhance eosinophil phagocytosis. Phagocyte–microbe contact is accompanied by intracellular signals that trigger cellular processes as diverse as cytoskeletal rearrangement, alterations in membrane trafficking, activation of microbial killing mechanisms, production of pro- and anti-inflammatory cytokines and chemokines, activation of apoptosis and the production of molecules required for efficient antigen presentation to the adaptive immune system.36,37 In this regard, it has been shown that eosinophils are able to produce H2O2 in response to phagocytosis

of heat-killed Staphylococcus aureus38 and excretory–secretory products (ESP) from interacting with Fasciola hepatica.8 In addition, Phipps et al.39 suggests that eosinophil-derived NO contributes to innate protection against the respiratory syncytial virus. In fact, in cryptococosis, the generation of NO is required 4��8C for resistance to primary fungal infections. Moreover, mice deficient in inducible nitric oxide synthase (iNOS) did not survive a primary infection.40 Snelgrove et al.41 have shown that NADPH oxidase-deficient mice elicited a heightened Mφ-driven Th1 response with the containment of cryptococci within pulmonary granulomatous lesions. They also observed improved clearance of pathogen in lung and airways, with reduced dissemination to the brain. In the present study, opsonized C. neoformans down-regulated NO and H2O2 synthesis by eosinophils in an FcγRII-dependent manner.

Histamine-mediated signals affect the ability of DC to induce the

Histamine-mediated signals affect the ability of DC to induce the maturation of T cells along Th1 or Th2 pathways. Histamine appears to be involved in the Th-switch: Th1 cells express H1R, while H2R is found on both Th1 and Th2 cells, as well as DC. H2R also appears to play a critical role in the induction of immune tolerance. Histamine has many important, but still poorly understood immune-related functions, highlighting the need for additional animal models, including histamine receptor gene knockout

mice. Mast cells play critical, but undefined, immunoprotective roles in bacterial and helminth BMS-354825 cell line infections. Studies from the laboratory of Richard Stevens (Boston, MA) led to the identification of two major serine mast cell tryptases, mouse mast cell protease (mMCP)-6 and mMCP-7, that are critical factors in protection from bacterial and helminth infection. Dr. Stevens and colleagues demonstrated that mast cell-deficient

W/Wv mice can successfully combat a Klebsiella pneumoniae pulmonary infection when pre-treated with physiologic amounts of recombinant mMCP-6 or its human ortholog hTryptase-β 17. Dr. Stevens and Dr. Adachi created transgenic mice that lack both mMCP-6 and mMCP-7 18. They then showed that these tryptase-deficient animals have a markedly reduced ability to combat K. pneumoniae infection of the peritoneal cavity and an impaired ability to combat Trichinella spiralis infections. The mechanisms by which mast cell-restricted tryptases INK 128 cost are beneficial in varied infections remain to be determined at the molecular level, but it appears that they play important roles in orchestrating the accumulation of granulocytes in tissues. K. Frank Austen (Boston, MA) addressed an unexpected

role of mast cell proteases in the response to ischemia reperfusion injury. In mouse models of ischemia reperfusion injury, the heightened exposure of self-Ag leads to Ag recognition by natural IgM and subsequent complement activation. This results in immune mediated injury that depends on specific mast cell-derived proteases, as evidenced by the fact that mast cell-deficient mice are protected from injury. In hind limb ischemia reperfusion injury, mice Rebamipide lacking the elastase mMCP-5 are significantly protected. The same mechanistic principles apply to a second-degree burn model in which mice deficient in mast cell chymase/elastase (mMCP-4/5), but not tryptase (mMCP-6/7), are protected from ulceration and scarring. Dr. Austen proposes that mast cell-derived proteases such as mMCP-4/5 play a critical role in the tissue damage following injury. Stephan C. Bischoff (Stuttgart, Germany) observed that much of the mast cell literature is based on data obtained in animal species that, in nature, do not suffer from mast cell-mediated allergic diseases.

An ANOVA, Sex of Participant (female versus male) × Age of Partic

An ANOVA, Sex of Participant (female versus male) × Age of Participant (6–7 months versus 9–10 months), revealed only a significant effect of sex, C646 mouse F(1, 44) = 18.25, p < .001, indicating that the mean novelty preference for males was reliably higher than

that for females. In addition, as shown in Table 2, t-tests comparing preference scores to 50% (chance responding) revealed that in both age groups, males preferred the mirror image significantly above chance, whereas as a group, females showed no preference. Examined from the perspective of individual infants, at 6–7 months of age, 10 of 12 males displayed novelty preference scores above 50%, p < .04, whereas only 5 of 12 females did so, p = .77. Similarly, at 9–10 months of age, 11 of 12 males displayed novelty preference scores above 50%, p < .01, whereas only 6 of 12 females did, p = 1.0. For the high throughput screening compounds two age groups combined, the proportion of infants preferring the mirror image was greater for males than females, Fisher’s exact test, p < .005. Both the group and individual data show that males, more strongly than females, generalized familiarization to the novel rotation of the familiar stimulus and preferred the novel mirror

image stimulus. Quinn and Liben (2008) familiarized 3- to 4-month-olds with varying rotations of the number one (or its mirror image) and then tested with a novel rotation of the familiar stimulus paired with its mirror image. Males were more likely to prefer selleck chemical the mirror image, whereas females were more likely to divide attention between the test stimuli. This performance difference suggested that a sex difference in mental rotation ability is present as early as 3 months of age (see also Moore & Johnson, 2008, 2011, for additional evidence that the difference is manifested in the initial months of life). In Experiment 1, we investigated an alternative explanation for the Quinn and Liben (2008) result, one in which the performance difference between females and males can

be attributed to females being more sensitive than males to the various rotations of the familiarized stimulus. The 3- to 4-month-olds in the current study were presented with a discrimination task in which each female and a corresponding male were tested with randomly selected familiarization and novel test rotations of the number one (or its mirror image) from the Quinn and Liben study. Both females and males discriminated between the different rotations at equivalent levels of above-chance performance. This finding suggests that the performance difference in the Quinn and Liben task is unlikely to be attributable to females being more sensitive to the angular rotations than males. In Experiment 2, we used the Quinn and Liben (2008) procedure to determine whether a sex difference in mental rotation is also present in 6- to 7-month-olds and 9- to 10-month-olds.

Supernatants of NK cells (1×106/mL) incubated in the presence of

Supernatants of NK cells (1×106/mL) incubated in the presence of 1 μg of HPV16-VLPs or with positive and negative controls were stored at −80°C and were analyzed for TNF-α and IFN-γ production in a specific capture ELISA according to the manufacturer’s instructions (BioSource, Merelbeke, Belgium). NK cells (2×105/200 μL) were incubated with 10 μg of CFSE-VLPs or LYNX-VLPs in complete RPMI for 1 h at 4°C (binding step). After washing, cells were placed at 37°C for different incubation times. For the experiments investigating the entry pathway,

different reagents (Sigma) were added 1 h before the see more incubation with labeled VLPs: 2 μM of cytochalasin D, 25 μg/mL of chlorpromazine for the clathrin-dependent pathway, 25 μg/mL of nystatin for the caveolin-dependent pathway, or 1 U/mL heparinase Kinase Inhibitor Library cell assay II. For blocking experiments with anti-CD16 mAb (BD Biosciences, 1 μg/mL), cells were incubated with this antibody for 40 min before the addition of labeled VLPs. After incubation with fluorescent VLPs, 0.5×106 cells were incubated for 1 h with Hoechst 33342 DNA stain (10 μM, Acros Organics, Geel, Belgium). After washing, cells were deposited on a polylysine-coated coverslip, fixed with PAF (4%) and cell membranes were stained with phalloidin 633 (Invitrogen) for 45 min at room temperature in the dark. Then, coverslips were fixed with 20 μL of Mowiol (Hoechst GmbH, Frankfurt, Germany). Images were acquired using an

Olympus Fluoview FV1000 confocal system (Olympus, Aartselaar, Belgium) equipped with an Olympus IX81 inverted microscope (objective UPLSAPO 60X/NA 1.35). To check VLP conformation, 10 μL of each VLP pool were deposited on copper grids coated with a carbon film (EMS, 3-oxoacyl-(acyl-carrier-protein) reductase Brussels, Belgium). Grids were stained twice with 2% uranyl acetate (Fluka, Bornem, Belgium) for 1 min and washed with filtered demineralized water. To analyze VLP entry, NK cells were incubated with VLPs as described above for 10 min to 18 h. Cells were then centrifuged, fixed at room temperature in 4% glutaraldehyde (Laborimpex, Brussels,

Belgium) and post-fixed in 1% osmium tetroxide (Laborimpex) for 1 h at 4°C. Pellets were dehydrated with ethanol solutions (VWR International, Leuven, Belgium) and embedded in Epon (Serva, Breda, The Netherlands) and propylene oxide (Laborimpex) at 60°C. Ultrathin sections were stained with uranyl acetate (Fluka) and lead citrate (Leica, Groot Bijgaarden, Belgium). Grids were examined using a transmission electron microscope EM Jeol 100 CX II (Jeol, Zaventem, Belgium). A fluid-uptake assay with FITC-dextran was performed on cells (2×105/200 μL) incubated in the presence of HPV16–VLPs (10μg/mL) or positive and negative controls in complete RPMI containing FITC-dextran (1 mg/mL, average mol wt. 20 000, Sigma) at 37°C. After incubation, cells were washed three times and fixed (PAF 1%). Cells treated with 2 μM of cytochalasin D (Sigma) for 30 min before the incubation of VLPs were also used as controls.

We note, however, that the proportion of inter-population variati

We note, however, that the proportion of inter-population variation differs depending on the genetic system: it is around 15% for allozymes,24 most DNA markers,22,23 and HLA-DPB1,25,49 and is slightly lower for the other HLA loci (∼ 10% on average), but is notably higher for GM (∼ 46%, including ∼ 39% among geographic groups and ∼ 7% among populations within geographic groups).12 This may be the result, in the

case of GM, of a bias in frequency estimation because of serological typing (as discussed above), although the effect of positive selection cannot be totally ruled out. In the case of HLA, we can conclude that balancing selection lowers inter-population variation although this effect is not selleck very pronounced. Immunogenetics is therefore an informative tool in anthropology, despite the effect of natural selection, which is clearly demonstrated for HLA but appears to be weak. Moreover, the study of immunogenetic markers may provide important novel information for anthropological studies. Indeed, what is often considered to be a disadvantage in anthropological studies – a non-neutral mode of evolution of the studied polymorphisms – may

be highly relevant to understanding Selleck Lumacaftor complementary aspects of human evolution, like environmental changes. Relevant results obtained through computer simulation have recently been obtained by Currat et al.,91 who estimated an unequal coefficient of selection for HLA-DRB1 in Southwest European (0·7%) and Northwest African (1·9%) populations separated by the Strait of Gibraltar. This difference can be seen as a genetic signature of heterogeneous environments in the past, i.e. different pathogen richness or prevalence of specific infectious diseases in the two regions. Also, the case of Amerindians would deserve deeper investigation to understand Vitamin B12 the evolution of their peculiar HLA genetic profiles. This could also be carried out by simulating different

scenarios taking into account both the initial settlement of America and its recent history marked by European colonization, which brought many new pathogens to this continent. The study of polymorphisms of important molecules for immune responses opens crucial areas of research in the field of human evolution, such as gene–pathogen co-evolution. This work received financial support from the Swiss National Science Foundation (SNF, Switzerland) grants no. 3100A0—112651 and 31003A—127465 (A.S.M.), the ESF (Europe) COST grant of Action BM0803 ‘HLA-NET’ (A.S.M.), the Oslo University Hospital Rikshospitalet, and Medinova (E.T.), and the US National Institute of Health Grant no. AI067068 (J.A.H. and S.J.M.). The authors declare no conflicts of interests.

2b) Immunohistochemistry also shows that the sham-injured urethr

2b). Immunohistochemistry also shows that the sham-injured urethral sphincters are composed of distinct muscle tissues containing numerous myoglobin- (Fig. 2c) and SMA-positive cells (Fig. 2d). In contrast, the

7-day-old freeze-injured internal urethral orifices appear to be relaxed, creating a larger orifice (Fig. 2e). The injured urethral sphincters show reactive changes, including loss of muscle mass and relative disorganization of the remaining muscle tissues (Fig. 2e). Accompanying these changes is the loss of the majority of the striated and smooth muscle cells (Fig. 2f) and the absence of most myoglobin- (Fig. 2g) and SMA- positive cells (Fig. 2h). These findings of induced ISD-related urinary incontinence are similar to other models of urinary incontinence4,48–50 with respect to loss of striated and smooth muscle and reduced leak point

pressures. The selleck compound urinary sphincters of patients with post-surgical urinary incontinence are irreversibly damaged. However, this appears not to be the case in our model system. The cell-free injected control rabbits show a weak but natural selleck products recovery of striated and smooth muscle cells that is accompanied by a slight increase in leak point pressure. These results are not entirely surprising. Rabbits may have inherently different regenerative powers than humans. Additionally, and of possibly greater importance, the rabbits are young and in good health, in contrast to patients with ISD-related urinary incontinence, who are typically elderly and not in

good general health. In our rabbit model, we intentionally avoided more severe and serious sphincter damage that would have produced irreversible incontinence because of the potential for urethral stricture or perforation, followed by death. Thus, our model is considered to be an Clomifene acute incontinence of relatively short duration. Ten days after harvesting the bone marrow cells and placing them in culture, and 7 days after freeze-injury operation, we divide the rabbits into cell implantation and cell-free injection control groups.3 For the cell implantation group, we implant the 0.5 × 106 autologous bone marrow-derived cells suspended in 100 µL culture medium. A total of 2.0 × 106 cells are injected through a 29-gauge syringe needle into the injured regions at the 3-, 6-, 9-, and 12-o’clock positions. For the cell-free injection control group, we similarly inject 100 µL of cell-free culture medium. The number and volume of the implantation cells are chosen to avoid further damaging the host tissues or the implanted cells due to shear stress. At each operation, the retention of small swellings containing the implanted cells or control media is visually confirmed. At 7 days after cell implantation, the leak point pressure of the cell-implantation group, 13.15 ± 2.

The apoptotic cells are rapidly engulfed and digested by phagocyt

The apoptotic cells are rapidly engulfed and digested by phagocytes such as macrophages and immature dendritic cells. The swift engulfment of cell corpses by phagocytes prevents the release of noxious or immunogenic debris from dying cells into the circulation. In the process of apoptosis, the dying cells expose phosphatidylserine on their external membrane in a caspase-dependent manner. This externalization of phosphatidylserine is one of the hallmarks of apoptosis and acts as an “eat me” signal for phagocytes selleck 3. Recently, several molecules

that recognize phosphatidylserine have been identified 4–7. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by multiple genetic and environmental factors 8. Patients with SLE develop a broad spectrum of clinical manifestations affecting the skin, kidney, lungs, blood vessels, and/or nervous system. SLE is also characterized by the presence in sera of autoantibodies against nuclear components (anti-RNP

and anti-DNA antibodies). Unengulfed apoptotic cells can be found in the germinal centers of the lymph nodes of some SLE patients, and macrophages from these patients show a reduced ability to engulf apoptotic cells 9. Furthermore, circulating DNA or nucleosomes can also be found in the sera of SLE patients 10, 11. These results suggest that a deficiency in the clearance of apoptotic cells is one of the causes of SLE. Milk fat globule-EGF factor 8 (MFG-E8) is a glycoprotein. At the N-terminus, it has a EGF-like Casein Kinase inhibitor repeat(s), and at the C-terminus, there are two discoidin domains that bind phosphatidylserine. It was originally identified as a component of milk fat globules that bud from the mammary epithelia during lactation. But it is now known to play

important roles in various systems such as involution of mammary glands, adhesion between sperm and egg, repair of intestinal mucosa, and angiogenesis 12. MFG-E8 is secreted by activated macrophages and immature dendritic cells 13, and it promotes the engulfment of apoptotic cells by working as a bridging molecule between apoptotic cells and phagocytes 7. In MFG-E8-knockout mice, many apoptotic Selleck Rucaparib cells are left unengulfed in the germinal centers of the spleen 14. The MFG-E8−/− mice produce autoantibodies including anti-cardiolipin and anti-dsDNA antibodies and suffer from an SLE-type autoimmune disease. Human MFG-E8 is maintained at the optimal concentration to support the engulfment of apoptotic cells; in excess, MFG-E8 inhibits phagocytosis and causes autoimmune diseases 15, 16. In this report, we analyzed the human MFG-E8 gene of SLE patients, and found in two female patients an intronic mutation that caused aberrant splicing of intron 6, resulting in the inclusion of a cryptic exon in the transcript.

The eight strains isolated from Jiangsu Province in 1998 were als

The eight strains isolated from Jiangsu Province in 1998 were also included (six from patients and two from diseased pigs) (Table 1) (9, 15). All of the strains were screened using PCR targeting virulence-associated

genes including MRP (mrp), suilysin (sly) and EF (epf) (16). All of the isolates were also characterized using single enzyme PFGE with SmaI (9) and a MLST scheme (11). The complete genome sequence of five S. suis serotype 2 strains including GZ1 (ST1) (8), SC84 (ST7, NC_012924), 05ZYH33 (ST7) (17), 98HAH12 (ST7) (17), and P1/7 (ST1) (http://www.sanger.ac.uk) were analyzed for potential VNTR loci using TRF, (version 2.02) (18) and the Ceritinib clinical trial Tandem Repeat Database (http://minisatellites.u-psud.fr/) using alignment parameters as follows: two matches, three mismatches, and five indels

where 50 was the minimum alignment score; 500 bp was the maximum array size of the repeat unit. Where multiple repeat patterns existed in a given locus, the repetitive unit pattern with the highest match rate was selected. To avoid missing a locus where only one copy was in a given sequenced strain whereas multiple repeat copies occur in other strains, Protein Tyrosine Kinase inhibitor the TRF output generated from the P1/7, GZ1, SC84, 05ZYH33 and 98HAH12 genomes was compared. Primer Premier 5.0 was used to design the PCR primers targeting the VNTR loci within the flanking regions. Overlapping or adjoining tandem repeats were co-amplified with a single set of primers (19). Strains were cultured on sheep Columbia blood agar plates at 37°C in 5% CO2

for 24 hr. A single isolated colony was inoculated into 5 ml Todd-Hewitt broth and incubated overnight. Total genomic DNA was isolated using QiaAmp DNA isolation columns (Qiagen Gene, Beijing, China) and following the manufacturer’s instructions for Gram-positive bacteria. Standard PCR was performed per the manufacturer’s directions using Taq DNA polymerase (TaKaRa, Beijing, China) in 25 μl reaction mixtures: 2.5 μl buffer (10×), 5 U Taq DNA not polymerase, 200 μM each deoxynucleoside triphosphate, 1 μl bacteria genome DNA (10 ng/μl), 0.5 μM each oligonucleotide primer, and RNase-free water. The PCR reaction was performed using a thermal cycle PTC-200 DNA Engine (MJ Search, Beijing, China). The PCR regimen consisted of an initial denaturing at 95°C for 10 min followed by 30 cycles of amplification: 95°C for 1 min, annealing temperature for 1 min, and extension at 72°C for 1 min; with a final extension at 72°C for 10 min. The amplified products (1.5–2.5 μl) were resolved using electrophoresis on a horizontal 1.5% agarose gel (Amplisize, Bio-Rad, Hercules, CA, USA) at a voltage of 6 V/cm for approximately 4 hr using 0.5×TBE buffer (10×TBE is 890 mM Tris base, 890 mM Boric acid, and 20 mM EDTA; pH 8.0). The gels were stained with ethidium bromide (0.