In summary, we have shown that Th17 cells can differentiate into IFN-γ-producing and FOXP3+ T cells after repetitive in vitro stimulation with OKT3 and PBMCs. We further demonstrated that this differentiation was due to TCR stimulation, resulting in epigenetic modification of FOXP3 and reprogramming of the gene expression signatures,

including lineage-specific transcriptional factors and cytokines. In addition to the expression of IFN-γ and FOXP3, we showed that these Th17 cells after differentiation into cells with a Treg phenotype mediated potent suppressive function. These results indicate that human Th17 cells exhibit substantial developmental plasticity and can differentiate into Tregs. In addition, our data provide novel information regarding T-cell-mediated immunity, which may have clinical implications for the development of target therapies. Tumor tissue samples of melanoma, LY294002 in vivo ovarian, breast and colon cancers and patient data were obtained from hospitalized

patients undergoing surgery at St. Louis University Hospital, as approved by the Institutional Review Board and ethics committee of the institution. R788 Buffy coats from healthy donors were obtained from the Saint Louis Red Cross. PBMCs were purified from buffy coats using Ficoll-Paque. Bulk and naïve CD4+ T cells were isolated by either positive or negative selection with microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD4+CD25+ Tregs

were further purified from CD4+ T cells by FACS sorting after staining with anti-CD25-PE antibody (BD Bioscience). Tumor-infiltrating lymphocytes (TILs) were generated from various tumor tissues, as previously described 28. Briefly, tissues were minced into small pieces followed by digestion with collagenase type IV, hyaluronidase and deoxyribonuclease. After digestion, the cells were washed in RPMI1640, and then cultured in RPMI1640 containing 10% human serum supplemented ifenprodil with L-glutamine, 2-mercaptethanol and 50 U/mL of IL-2 for the generation of T cells. The percentages of CD4+ Th17 cells were determined from bulk T cells by FACS analysis after intracellular staining for IL-17. Th17 cell clones were generated from TILs by a limiting dilution cloning method, as previously described 27, 28. Briefly, CD4+ TILs were diluted in U bottom 96-well plates at a 0.3-cell/well concentration and then co-cultured with irradiated allogeneic PBMCs in the presence of soluble anti-CD3 antibody (OKT3, 100 ng/mL) for 10–14 days. Th17 clones were screened by determining IL-17 secretion in cell supernatants by ELISA (eBioscience) after stimulation with plate-bound anti-CD3 antibody (2 μg/mL). The expression markers on T cells were determined by FACS analysis after surface staining or intracellular staining with specific anti-human antibodies conjugated with either PE or FITC.

Furthermore adult LTi-like cells, just like their counterparts from embryonic day 15 spleen, restore

a significant degree of B/T segregation in the spleen of LTα−/− mice, and up-regulate VCAM-1 and CCL21 protein expression on the stromal cells with which they are associated 6. Most recently, adult LTi-like cells were shown to induce lymphoid tissue formation in the intestine of CXCR5−/− mice 7. Although normal podoplanin (gp38) expression on T-zone stromal cells requires lymphocytes, 5-Fluoracil clinical trial LTi-like cells can provide lymphotoxin signals required for the expression of podoplanin and CCL21 on T-cell zone stroma, as injection of LTα−/− lymphocytes into RAG-deficient mice up-regulates podoplanin on T-zone stroma, and this is associated with B/T segregation and T-cell organization 8. Interactions between LTi-like cells and stromal cells continue into adulthood and are important for restoring SLO integrity and function after virus infection 9. The white pulp of spleen is compartmentalized into B and T zones where cellular and humoral immune responses Selleckchem Bortezomib are initiated. In B zones, B cells are intermingled with stromal cells, such as follicular DC 10. T zones contain T cells, DC and fibroblastic reticular cells (FRC) whose relationship to other stromal cells and effects on leukocytes are not fully elucidated 11. FRC ensheath a reticular

network serving as a conduit system for the transport of fluid and soluble substances of low-molecular weight from the blood to the white pulp 12. Soluble Ag and chemokines travel via this conduit system allowing Ag uptake by DC as well as lymphocyte migration within the spleen and other lymphoid tissues 13, 14. FRC express the glycoprotein marker podoplanin but appear to be a heterogeneous cell population, with the most prominent subset forming a dense network throughout the T zone where they produce the extracellular matrix scaffold of the LN 15, 16. Recent findings have

demonstrated that a stromal population of podoplanin+ T-zone reticular cells (TRC) regulates the homeostasis of naïve T cells but not B cells by providing survival factors including IL-7 and CCL19 in LN 17. Collectively, these data suggest that like T-lymphocytes, closely associating with heptaminol stroma, adult LTi-like cells interact with stromal cells to create distinct microenvironments in lymphoid tissues which facilitate effective immune responses. It is therefore important to identify the nature of the stromal cell subsets as well as the molecular pathways involved in LTi survival during the development of the immune system from embryo to adult. In this study, we investigated whether podoplanin+ stromal cells in the adult spleen provide survival signals for adult LTi-like cells. An obvious candidate for LTi survival is cytokine IL-7, whose receptor (IL-7Rα) is expressed on LTi.

003, Wilcoxon-test). Male-target cells pulsed with the control-peptide I540S did not influence T cell reactivity compared with naïve cells (I540S: 12–29/100,000; median: 23; P < 0.106 to P < 0.066). In vivo-primed female T cells recognized peptide-loaded T2-cells (W248: 85 ± 28/100,000 T cells; T368: 35 ± 12/100,000; K1234: 50 ± 17/100,000) being UTY-specific as indicated by Anti-MHC-I-antibody-blockage

(W248: 30 ± 10/100,000 T cells; T368: 26 ± 9/100,000; K1234: 10 ± 3/100,000; P < 0.026 to P < 0.018, Wilcoxon-test). In contrast, T2-cells alone or loaded with the I540S-control-peptide demonstrated only low unspecific-reactions (20–1/100,000 T cells, median: 9; P < 0.113 to P < 0.018, Wilcoxon-test). According to this website the in vitro experiments (Table 2, Fig. 3) in vivo primed female T cells mostly reacted with male-BM (<45 specific-spots/100,000 CD3+T cells)

followed by monocytes (<29 spots) and PBMCs (<15 spots) and in vivo immunogenicity of the hUTY-peptides was comparable with those in vitro: W248 exhibited the most immunogenic potential on T2-cells (85 spots/100,000 T cells > K1234 (50 spots) >T368 (35 spots)). We provide evidence that hUTY-derived male-peptides specifically expand T lymphocytes derived from female-DLA-identical-dogs either using autologous-peptide-pulsed-female DCs as APC in vitro or male-DLA-identical PBMCs in vivo. The expanded female T cells recognized HLA-A2-binding hUTY-derived endogenously presented peptides W248, K1234 and T368 only on buy BIBW2992 DLA-identical

male-cells (mostly BM) representing a male-specific restriction. Thereby, W248 appeared to be the most immunogenic-peptide. Importantly, no response against autologous- and female-DLA-identical cells, not expressing the male-specific-UTY antigen, was detected. Therefore, we conclude that the mHA UTY is very homologous Tenofovir in male-humans and dogs, and the canine-system could serve as a large-animal model to study T cell applications in terms of immunotherapeutic approaches after alloSCT in male patients with female donors. Consequently hUTY-(especially W248)-pulsed female DCs might be used in male hematopoietic-SCT recipients with female stem-cell donors [3, 6, 7]. CD8+T cell-proliferation was induced up to 3-fold within 3–4 weeks (Fig. 1). After in vitro stimulation expanded CD8+T cells specifically reacted against the hUTY-derived peptides presented on autologous-female DCs in up to 3.1% of all T cells (IFN-γ-ELISPOT assay, Fig. 2), but not against autologous-naïve DCs and monocytes. This proves that HLA-A2-restricted peptides selected from human-UTY protein bind to canine-DLA-identical molecule(s), and these peptides are immunogenic in dogs and can induce UTY-specific T cell reactivity. Detected amounts of reactive-UTY-specific CD8+T cells after in vitro culture with IFN-γ-ELISPOT and [51Cr]-release-assays were comparable. This is in accordance with findings by others, although both the assays address different CTL-mechanisms [41].

Fifty kDa γ-PGA was obtained from Bioleaders (Daejeon, Korea) and its purity was more than 97%. The levels of endotoxin and peptidoglycan Neratinib contained in a 40 µM γ-PGA solution were <0·01 EU/ml and <10 pg/ml when measured by the Limulus amebocyte lysate assay using E-toxate kits (Sigma-Aldrich, St Louis, MO, USA) and by the silkworm larvae plasma assay (Wako Pure Chemicals, Osaka, Japan), respectively. Lipopolysaccharide (LPS) derived from Escherichia coli 026:B6 was purchased from Sigma-Aldrich. IL-6, TGF-β, anti-TGF-β antibody and mouse IgG1 isotype control

antibody were from R&D Systems (Minneapolis, MN, USA) and IL-2 was from Peprotech (Rocky Hill, NJ, USA). Anti-interferon (IFN)-γ antibody (XMG1·2) and anti-IL-4 antibody (11B11) were obtained from BD Biosciences (San Jose, CA, USA). T cells were stained with an appropriate mixture of monoclonal antibodies (mAbs), as described [27]. Data were acquired on a BD FACSCantoII. The mAbs used were: anti-CD17A-phycoerythrin (PE) (BD Biosciences), and anti-CD4-allophycocyanin (APC), anti-CD25-PE, anti-FoxP3-fluorescein isothiocyanate (FITC), anti-IFN-γ-FITC, anti-RORγt-PE, anti-CTLA-4-PE, anti-CD11c-FITC, anti-CD44-PE and anti-glucocorticoid-induced tumour necrosis factor (GITR)-PE (all from eBioscience, San Diego, CA, USA). Single-cell suspensions were obtained from Selleck Wnt inhibitor the spleens and lymph nodes of mice, and erythrocytes were lysed in ammonium

chloride (ACK) solution [150 mM NH4Cl, 1 mM potassium

hydrogen carbonate (KHCO3), 0·1 mM ethylenediamine tetraacetic acid (EDTA)]. To sort naive non-Treg CD4+ T cells, cells were stained with a mixture of anti-CD4, anti-CD44 and anti-CD11c or a mixture of anti-CD4, anti-CD44, and anti-CD25, and sorted by FACSAriaIII (BD Biosciences). The purity of the CD4+CD44loCD11c– and CD4+CD25-CD44lo populations was >98%. CD4+ T cells were purified to >98% of the purity using anti-CD4 magnetic microbeads and columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% Dapagliflozin fetal bovine serum (FBS). For Th cell differentiation, purified whole CD4+ or naive CD4+ T cells (2 × 106 cells/ml) were stimulated with soluble 1 µg/ml anti-CD3 mAb (145-2C11; eBioscience) and soluble 1 µg/ml anti-CD28 mAb (37·51; BD Biosciences). Four ng/ml IL-2 was added for non-polarizing conditions (referred to as Th0) and 20 ng/ml IL-6, 5 ng/ml TGF-β, 5 µg/ml anti-IFN-γ mAb and 5 µg/ml anti-IL-4 mAb were added for Th17-polarizing conditions. After 4 days, IL-17 concentrations in culture supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems), and the cells were restimulated with 40 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in the presence of Golgi Stop (BD Biosciences) for 6 h and assayed by intracellular FACS methods.

The data also show that MPyV establishes a long-lasting infection in the brain and other organs of immunocompromised mice. Having shown that MPyV infected the brain of BALB/c and KSN mice, the next set of experiments was conducted to assess the spatial and temporal patterns of virus spread within the brain. After stereotaxic inoculation of the brain with

MPyV, the mice were perfused with chilled PBS as described above. The brain was removed and cut into 2-mm coronal slices using a precision brain slicer (Brain Matrix; Braintree Scientific, Braintree, MA, USA). Total DNA was extracted from each slice, and the amounts of viral DNA were determined by real-time PCR as described above. When BALB/c mice were inoculated CX-4945 with MPyV, Selleck Galunisertib the amounts of viral DNA increased predominantly at the local inoculation sites with a peak at 4 days p.i. and then declined after 6 days p.i. (Fig. 2a, A and B). At 30 days p.i., extremely low levels of viral DNA were detected in all regions of the brain (Fig. 2a, A to E). On the other hand, viral DNA was readily detected around the sites of inoculation from 2

to 30 days p.i. in the brains of KSN nude mice (Fig. 2b, A and B). In addition, viral DNA was persistently detected in some areas away from the inoculation site, even at 30 days p.i. (Fig. 2b, C and D). As the amounts of MPyV DNA in the brains of BALB/c mice rapidly decreased from around 4 to 6 days p.i. (Fig. 1a, Fig. 2a), the question arises as to whether innate immune responses in the brain are associated with these differences in the kinetics of MPyV infection between the two mouse strains. To answer this, the expression levels of cytokines and chemokines in the brains of MPyV-inoculated mice were determined using real-time PCR. To prepare standard cDNA, a cDNA pool was synthesized from RNA extracts of mouse brain as described previously (24, 25), and the standard cDNA for each target gene was generated by conventional PCR using specific primer sets (Table 1) and Ex Taq (Takara). IKBKE To examine gene expression patterns in the mouse brain, BALB/c and KSN mice were inoculated with MPyV and the brains were harvested at 5 days

p.i. as described above. Total RNA was extracted from coronal slices of the brain with a High Pure RNA tissue kit (Roche), and a cDNA pool was generated by using a PrimeScript 1st strand cDNA Synthesis Kit (Takara) following the manufacturer’s protocols. Real-time PCR was performed on each cDNA preparation using specific primers (Table 1), a Platinum SYBR Green qPCR SuperMix UDG Kit (Invitrogen) and a LightCycler (Roche) according to the manufacturers’ instructions. The relative amounts of each target cDNA were normalized with reference to those of GAPDH cDNA. In BALB/c mice, MPyV inoculation into the brain led to a statistically significant increase in the transcription of IFN-β and CCL5 genes, and the expression levels of IFN-α, IL-1β, IL-6, and CCL2 were similar to those seen in mock-inoculated mice (Fig. 3a).

33 Lassa fever, caused by infection with a arenavirus, showed a higher rate of case-fatality in pregnant women particularly in the third trimester.34 However, those are not the rule and may even be the exception; in general, pregnant women are resistant to viral infections including HIV. Thus, the obvious question is why pregnant

women are more susceptible to some viruses or to some specific microorganisms than non-pregnant women? Is the presence of the placenta affecting the sensitivity to specific infections? The trophoblast, the cellular unit of the placenta, not only MI-503 recognizes microorganisms and initiates an immune response as previously described, it may also produce anti-microbial

peptides and, therefore, actively protect itself see more against pathogens. Studies have demonstrated the expression of the anti-microbial human beta defensins 1 and 3 by trophoblast cells.35,36 Secretory leukocyte protease inhibitor (SLPI), which is a potent inhibitor of HIV infection37 and inducer of bacterial lysis,38 has also been found in trophoblast cells.35 The expression of TLR-3, TLR-7, TLR-8 and TLR-9 by trophoblast cells may explain how the placenta regulates the expression of these anti-microbial factors. Stimulation of first trimester trophoblast cells through TLR-3 with Poly (I:C) promotes the production and secretion of SLPI and IFN-β, two important anti-viral factors. These factors provide the first line of defense against viral infections and have the potential to activate multiple intracellular pathways.39 IFN-β and SLPI production by trophoblast cells, in response to a viral infection at the maternal-fetal interface, may represent a potential mechanism by which the placenta prevents transmission of viral

infection (e.g. HIV) to the fetus during pregnancy. These data suggest that the placenta represents an active immunological organ, (innate immune system), capable of recognizing and responding to pathogens. However, it also indicates that the placenta is prone to infections from microorganisms, which in its absence (non-pregnant) would never Phosphoglycerate kinase take place. Pregnant women are exposed to many infectious agents that are potentially harmful not only to the mother but also to the fetus. Risk evaluation has been focused on whether there is a maternal viremia or fetal transmission. Viral infections which are able to reach the fetus by crossing the placenta might have a detrimental effect on the pregnancy. It is well accepted that in those cases infection will lead to embryonic and fetal death, induce miscarriage or induce major congenital anomalies.40 However, even in the absence of placental transmission, the fetus could be adversely affected by the maternal response to the infection.

Soaking protocols were successfully applied in nematode parasites belonging to clade III [A. suum, O. volvulus, B. malayi and L. sigmodontis (111–116)] and improved for specificity and efficiency to reduce off-target effects, toxicity and costs. In contrast, successful RNAi in

worms of clade V has only been reported for a small percentage of genes that were investigated in this group of nematodes [for example (117)]. Silencing effects on different genes from T. colubriformis, H. contortus and O. ostertagi were often inefficient, difficult to reproduce and dependent Sirolimus mouse on delivery method used (118–121). In a more recent study, Lendner and colleagues failed to establish knock-down of tropomyosin in various life stages of H. polygyrus. High Content Screening In this study, dsRNA seemed not to be taken up efficiently by the parasite regardless of delivery by feeding, soaking or electroporation, with the latter even found to be lethal to L1 larvae (122). As most described techniques for dsRNA delivery involve the removal of the parasite from the host, one major obstacle for successful RNAi is the ability to maintain healthy, viable worms under in vitro culture conditions required for consistent silencing effects (123). Therefore, RNAi approaches are limited to certain life stages of the respective parasite. To circumvent difficulties associated with common RNA delivery techniques, Song et al. tested

a new approach to establish RNAi in B. malayi parasites targeting

genes in developing larvae within the intermediate host. Aedes aegypti mosquitoes were injected with dsRNA or siRNA targeting the B. malayi cathepsin L-like protease. Supplying the RNAi trigger in vivo to healthy worms in a host environment (‘in squito’) led to the highest reported specific reduction in target gene expression in B. malayi (83%) resulting in multiple phenotypes (124). These included reduced motility (69%) and growth retardation (48%) that lead to the prevention of larval development and reduced numbers of larvae migrating to the head of the mosquito, thereby abolishing parasite mafosfamide transmission, decreasing parasite burden and increasing host survival. The mechanism by which the siRNAs reach the parasite within the mosquito is unclear but rapid dissemination of Cy3-labelled siRNA after injection into the haemocoel indicated the creation of a scenario in vivo that is similar to the soaking technique in vitro (124). In addition, low efficacy in delivery of dsRNA or siRNA might also be attributed to the lack of molecules involved in RNA uptake and transport to allow for systemic spread of interfering RNAs. Recent EST database analyses revealed that H. contortus apparently lacks orthologs for rde-4, responsible for dsRNA recognition and binding, as well as sid-1, sid-2, rde-2 and rsd-2 orthologs, required for dsRNA uptake and systemic spread, whilst dicer and drh-1 involved in dsRNA processing are present (122).

DNA and RNA are then detected by, respectively, TLR7 and TLR9 and trigger MyD88- and IRF1-dependent responses. Interestingly, our data indicating that actin polymerization and phagocytosis are not required for dectin-1-dependent cytokine induction (e.g. for S. cerevisiae-induced TNF-α secretion) are in agreement with a recent report showing that dectin-1 can be activated by β-glucan immobilized on a nonphagocytosable surface (such as a culture plate), occurs prior to initiation of phagocytic cup formation and Cabozantinib supplier is not dependent on actin dynamics [55]. Altogether our data, pointing to the importance in anti-fungal defenses of the latter pathway, may be useful to better

understand the strategies used by C. albicans to evade the innate immune system and to devise alternative EGFR inhibitor therapeutic strategies. Knock-out mice were originally obtained from T. Taniguchi (IRF1−/−, IRF3−/−, and IRF7−/−), and S. Akira (TLR2−/−, TL3−/−, TLR4−/−, TLR7−/−, TLR9−/−, MyD88−/−, Mal−/−, TRAM−/−

and TRIF−/−) as previously described [29]. Dectin 1−/−, 3d and TLR7/9 double Ko (TLR7−/−/TLR9−/−) mice were provided by, respectively, G. Brown [11], B. Beutler [35] and S. Bauer. C57BL/6 WT mice, used as controls, were purchased from Charles River Laboratories (Calco, Italy). The mice were housed and bred under pathogen-free conditions in the animal facilities of the Elie Metchnikoff Department, University of Messina. All studies were performed in agreement with the European Union guidelines of animal care and were approved by the Ethics Committee of the Metchnikoff Department of the University of Messina (CESA) and by the relevant national authority (Istituto Superiore di Sanità). C. albicans (ATCC 90028) was purchased from the American Type Culture Collection. S. cerevisiae strain A11 was isolated in the clinical mycology

laboratory of the Elie Metchnikoff Department, University from of Messina [22]. For in vivo and in vitro experiments, these two strains were grown in a chemically defined medium as previously described [22]. CFU numbers used in each experiment were determined after plating on Sabouraud dextrose agar (Difco Laboratories). Heat killed C. albicans strains were prepared as previously described [22], followed by washing with PBS and resuspension to the original volume. Depleted zymosan (i.e. hot alkali-treated zymosan, which is devoid of TLR-dependent stimulating properties) and control stimuli (poly I:C, Escherichia coli ultrapure LPS, CpG B, CL264) were purchased from InvivoGen. Curdlan was purchased from Wako Pure Chemicals and detoxified using cold NaOH treatment [22]. Fungal cell extracts were obtained by vortexing of C. albicans (grown in the mid-log phase) in the presence of glass beads 425–600 μm in diameter (Sigma).

Forty animals were allocated into four groups according to the different times at 30 minutes (I), 24 hours (II), 72 hours (III), and 7 days (IV) after the operation. According to the different routes to give tracer, each group was further allocated into two subgroups of the artery injection and vein injection. For each animal, one hindlimb was assigned as Y-27632 mw the experimental

side, the contralateral side as control without giving tracer. The erythrocytes were separated, labeled with fluorescein isothiocyanate (FITC), detected, and injected into the artery or vein. Subsequently, the flaps were harvested 5 seconds after injection and immediately frozen, sectioned, and observed under microscope. In group I and II, the fluorescence was observed mainly around the vessel adventitia of the vein and artery and tunica intima of the artery. In group III, there was weak fluorescence observed in the lumen of vein. In group IV, fluorescence was distributed principally in the lumen of the vein. In addition, fluorescence

was not observed in the saphenous nerve in group I and there was mild fluorescence in the saphenous nerve in groups II, III, and IV. These findings suggest that the venous return is MI-503 order through “bypass route” in earlier period. In later period, the venous retrograde return is through “bypass route” and “incompetent valves route;” however, “incompetent valves route” becomes the main route. © 2009 Wiley-Liss, Inc. Microsurgery 2010. “
“Lymphatic fistula complicating lymphedema is thought to occur due to communication between lymph vessels and the skin, which has yet to be shown objectively. The objective of this case report is to show the pathology and treatment using simultaneous lymphatic fistula resection

and lymphatico-venous anastomosis (LVA). A 40-year-old woman underwent extended resection and total hip arthroplasty for primitive neuroectodermal tumor in the right proximal femur 23 years ago. Baricitinib Right lower limb lymphedema developed immediately after surgery and lymphatic fistula appeared in the posterior thigh. On ICG lymphography, lymph reflux toward the distal side dispersing in a fan-shape reticular pattern from the lymphatic fistula region was noted after intracutaneous injection of ICG into the foot. We performed simultaneous lymphatic fistula resection and of LVA. Pathological examination showed that the epidermis and stratum corneum of the healthy skin were lost in the lymphatic fistula region. Dilated lymph vessels were open in this region. The examinations provide the first objective evidence that the cause of lymphatic fistula may be lymph reflux from lymphatic stems to precollectors through lymphatic perforators. © 2013 Wiley Periodicals, Inc. Microsurgery 34:224–228, 2014.

flexneri and in a T3SS-dependent manner. Next, we evaluated whether ShET-2 is delivered into cells by intracellular Shigella. We used a reporter assay system based on translational fusion of the secreted proteins with mature TEM-1 β-lactamase (Charpentier & Oswald, 2004). Plasmids carrying translational fusions with sen gene (pTB-ShET-2–TEM-FLAG), ipaH9.8 gene (pTB-IpaH9.8–TEM-FLAG; positive control) or gst gene (pTB-GST–TEM-FLAG) were transferred into S. flexneri wild-type

strain 2457T or BS547 (T3SS-defective mutant). We confirmed the ability of ShET-2–TEM-FLAG to be secreted via I-BET-762 molecular weight the TTSS (data not shown). HEp-2 cells infected with S. flexneri wild-type strain 2457T expressing the translational fusions were loaded with CCF2-AM

and examined with a fluorescence microscope (Fig. 2). As we expected, uninfected cells and cells infected with 2457T/pTB-GST–TEM-FLAG (negative control) emitted green fluorescence as well as cells infected with BS547/pTB-IpaH9.8–TEM-FLAG or Pirfenidone concentration BS547/pTB-ShET-2–TEM-FLAG, indicating the absence of β-lactamase activity in these cells (Fig. 2). However, cells infected with 2457T/pTB-ShET-2–TEM-FLAG or 2457T/pTB-IpaH9.8–TEM-FLAG (positive control) emitted blue fluorescence. These data indicated that ShET-2–TEM-FLAG is delivered into the host cells by the intracellular Shigella. The ShET-2 coding gene sen is located downstream of the ospC1 gene (Fig. 3), which has been shown to be coexpressed with other genes related to T3SS function (Mavris et al., 2002).

The OspC1 protein has been implicated in Shigella-induced MEK/ERK pathway activation and PMN transepithelial migration (Zurawski et al., 2006). Expression of the ospC1 gene is controlled by the MxiE regulator via binding of the protein to a 17-bp MxiE-binding motif located in the promoter upstream region (Kane et al., 2002). Le Gall et al. (2005) suggested that both the ospC1 and sen genes might be part of the same operon based on macroarray analysis. We performed RT-PCR to determine whether sen was Nitroxoline cotranscribed with ospC1. Pairing primers downstream of ospC1 and upstream of sen, we found that the amplified products were consistent with the presence of a polycistronic ospC1-sen mRNA transcript (Fig. 3). The role of putative promoter sequences in the region between ospC1 and sen that might drive the expression of ShET-2 cannot be ruled out. Considering that ospC1 is regulated by MxiE, a regulator proposed to control the expression of virulence factors after internalization of the bacterium in the eukaryotic cell (Kane et al., 2002; Mavris et al., 2002), the data presented here suggest that ShET-2 might be regulated by MxiE and could also play a role in the intracellular stage of Shigella infection. Vaccine trials in humans using attenuated Shigella strains with mutations in the ShET showed a diminution of reactogenicity, defined as less diarrhea and fever (Kotloff et al., 2004, 2007).