Bioluminescence images were acquired with a 7-cm FOV, medium binn

Bioluminescence images were acquired with a 7-cm FOV, medium binning factor and exposure time of 10–30 s. Quantitative analysis was performed by measuring the luminescence signal intensity per well using the ROI settings of the living image 3.0 software. ROI measurements are expressed Acalabrutinib cost in total flux of photons. Per cent inhibition was calculated by the following formula; 1 – (average bioluminescence in immune plasma sample/average bioluminescence in naive plasma sample)* 100%. In all experiments and assays, comparisons between two groups were performed by a Mann–Whitney U-test

using prism software version 5.0 (Graphpad, San Diego, CA, USA). P < 0.05 is considered statistically significant. Overall comparisons over three groups or more was performed by Kruskal–Wallis test. Calculations of sample sizes were performed (power 0.85; α = 0.05) by estimation of differences between IV and ID groups. To compare protective efficacy conferred by ID or IV immunization, mice immunized by either RAS or CPS protocols were challenged by infectious mosquito bites. Irrespective of the immunization protocol, ID immunization induced lower protection in BALB/cByJ (50%) and C57BL/6J (7–13%) mice as compared to 90–100% protection after IV immunization

(Table 1). Development of blood-stage parasites in unprotected ID immunized mice showed no significant delay compared to control mice. To evaluate whether infection by IV or ID routes resulted in different magnitude of Exoribonuclease liver infection, we measured in vivo parasite liver Fostamatinib concentration loads in C57BL/6 mice by real-time imaging after IV or ID injection of identical doses of fresh PbGFP-Luccon sporozoites. Mice that received IV injection showed a clear bioluminescent signal originating from the site of the liver as from 30 h post-infection

onwards. This signal subsequently further increased covering the whole liver area at 44 h post-infection (Figure 1a). In contrast, ID injection did not result in a bioluminescent signal distinct from background at 30 and 35 h post–infection, while a weak signal was visible at 44 h. After ID injection, mice showed approximately a 30-fold lower parasite liver load (P < 0.0001) compared to IV injected mice (Figure 1b). These data show a strong association [P < 0.001 (χ2 = 49.08, (d.f. = 1)] between the number of parasites reaching the liver in this experiment and the level of protection conferred by different routes of sporozoite administration as shown in preceding immunization experiments. We next assessed cellular immune responses after IV or ID immunization of C57BL/6j mice. Following RAS or CPS IV immunization, proportions of CD8+ T cells with effector memory phenotype (Tem) were significantly increased in both liver (P = 0.008) and spleen (P = 0.008). With the exception of one CPS mouse, this expansion of CD8+ Tem cells was not observed in any of the ID immunized mice, remaining at baseline levels similar to naïve mice (Figure 2a).

However, it seems most likely that a difference in the immunising

However, it seems most likely that a difference in the immunising regime offers

the most plausible explanation. In the 1980s, 2000 T. circumcincta L3 were given to the previously infected sheep 5 days a week whereas in the recent series of trials this dose was administered only three times per week, i.e. the recent sheep received only 60% of the dose given in the 1980s. Exposure to the heavier immunising infection appeared to confer a more solid immunity to subsequent challenge in yearlings and yet make the lambs more susceptible (Table 2). There was no evidence from the recent SAHA HDAC purchase trials with the lighter trickle infection to support the idea that one or more components of the immune response

were defective in lambs. This includes examination of the abomasal histology where for example mast cell numbers were in the normal range (data being prepared for publication). We therefore hypothesise that only older, more resilient sheep were able to respond adequately following the heavier trickle, whereas the growing lambs, being less able to cope with the pathological effect of the BTK inhibitors library greater parasite load, were only able to mount a weak, relatively ineffective response post-challenge. In conclusion, we suspect that age and acquired immunity in ovine gastrointestinal nematodiasis is more likely to be due to the lack of resilience to infection on the part of lambs than to a specific immunological deficiency. The authors would like to thank Frank Jackson’s laboratory at Moredun for supplying parasites, Stephen Smith and Andy Greer for technical assistance, Mara Rocchi for assistance with the FACS analysis and Jill Sales of BIOSS for statistical Branched chain aminotransferase analysis. We would also like to thank Roy Davie, David Kennedy and Manus Graham for help with surgery. This work was funded by a Veterinary Training Research Initiative from the Department of Environment, Food and Rural Affairs and by the Scottish Government Rural and Environment Research and Analysis Directorate. “
“Mycobacterium

tuberculosis (TB) often causes persistent infection and many immune cell subsets and regulatory mechanisms may operate throughout the various stages of infection. We have studied dendritic cell (DC) subsets, regulatory T cells (Treg) and the expression of activation and apoptosis markers on CD4+ and CD8+ T cells in blood from patients with active TB (n = 20), subjects with positive QuantiFERON-TB GOLD (QFT) test (LTBI, latent TB infection) (n = 20) before and after 3 months of preventive anti-tuberculous therapy and from QFT-negative controls (n = 28). The frequency of CD4+CD25+CD127− Treg was highest in the group with active TB (P = 0.001), but also increased in the LTBI group (P = 0.006) compared to controls.

Successful fluorochrome incorporation was confirmed by native pol

Successful fluorochrome incorporation was confirmed by native polyacrylamide gel electrophoresis generating a single band at about 600 kDa corresponding to toxin A protein dimer [29, 30] under non-denaturing

conditions, which exhibited fluorescence during illumination with UV light. Toxin A488 was also shown to induce morphological changes in Vero cells and Caco-2 cells identical to that seen for unlabelled toxin A (treated as per labelled toxin A without the addition of click here the label), confirming that labelling had not compromised receptor-binding ability. To confirm that fluorescence in flow cytometry was because of toxin A488 only, without any contribution from free label that may have either not been removed following the labelling procedure or become detached from the toxin during storage or binding studies, toxin A488 was preincubated with PCG-4-conjugated beads prior to incubation with Caco-2 cells. A complete loss of Caco-2 cell-associated fluorescence was seen after incubation with the toxin A-depleted preparation (Fig. 1), confirming that all fluorescence was toxin A specific. In initial studies, following incubation of PBMNCs with toxin A488 at 37 °C for up to 24 h, monocytes were distinguished from lymphocytes by their forward- and side-scatter characteristics. In contrast to toxin A488-exposed lymphocytes, toxin

A488-exposed monocytes showed significant fluorescence at all time points up to 5 h, with selleck chemicals llc a peak at 1 h (Fig. 2A). Drop

in monocyte-associated fluorescence from 1 h onwards after exposure to Torin 1 manufacturer toxin A488 was associated with loss of events in the monocyte gate (Fig. 2B). The fluorescence level of toxin A488-exposed lymphocytes remained low, with no significant change (compared with control lymphocytes) over the 24 h period of study (Fig. 2A). Thus, at 24 h, there was no significant difference in fluorescence between lymphocytes incubated (at 37 °C) in control medium, compared with those cultured with toxin A488. In contrast to monocytes, the number of events in the lymphocyte gate (in toxin A488-exposed PBMNCs) did not change significantly from cells exposed to control medium over the 24 h period of study (Fig. 2B). When studied after 48-h incubation at 37 °C, fluorescence of toxin A488-exposed lymphocytes was marginally, but significantly greater than lymphocytes cultured with control medium (5.35 versus 4.97; P < 0.01). By contrast, following incubation at 4 °C, the difference in fluorescence between toxin A488-exposed and control lymphocytes fell short of statistical significance (5.0 versus 4.85; P = 0.07). The ability of trypan blue to quench fluorescence of monocytes exposed to toxin A488 at 37 and 4 °C was subsequently investigated. Initial studies, using PBMNCs labelled with anti-CD45 antibody, followed by labelling with Alexa Fluor 488-conjugated anti-mouse antibody, showed that trypan blue quenched 87.27 (±4.7)% of cell surface–associated fluorescence.

6B) The serum concentration of self-DNA

in patients with

6B). The serum concentration of self-DNA

in patients with DNA-related autoimmune diseases is higher than that in healthy subjects 7–9. In addition, circulating CpG DNA has been reported to be a pathogenic factor of SLE 23, 24. However, it is unclear whether the augmented self-DNA in serum affects the immune response to CpG DNA in autoimmune diseases. In the present study, we clearly demonstrated that DNase I-treated DNA, but not intact DNA, increases the CpG motif- and TLR9-dependent cytokine production in murine macrophages. As shown in Fig. 3B, it was found that only the DNase I-treated DNA, but not DNase II-treated one, has an ability to increase CpG DNA-induced cytokine production. Both DNase I and DNase II are endonucleases and cleave the PO bond in DNA, which yields polynucleotides with a 5′-mono-phosphate and 3′-mono-phosphate, respectively 13, 25. Taking into Cell Cycle inhibitor consideration these results, an oligonucleotide

with a phosphate group PFT�� price at the 5′-terminal is required for increased cytokine production from macrophages. In addition, the results showing that dNMPs and dNTPs but not deoxynucleosides increased the TNF-α release by CpG DNA at the comparable level (Fig. 3A), support the importance of the presence of a phosphate at the 5′-end of DNA. Moreover, the results in Fig. 3A indicate that this activity of DNA with 5′-phosphate to increase CpG DNA-induced cytokine production is dependent on the type of base, because TMP and TTP were much less effective than other dNMP or dNTP. It is still unknown how short the DNA is when DNA is fully cleaved by DNase I. However, the present study has demonstrated that mononucleotides are sufficient to increase the CpG DNA-dependent cytokine release from macrophages. TNF-α production in RAW264.7 cells was not proportional to the concentration of ODN1668; a 3-fold increase in the concentration of ODN1668 resulted in a 18-fold increase in TNF-α

Masitinib (AB1010) production (Fig. 1A). ODN1668 at a concentration of 1 μM or lower was hardly effective for cytokine production (data not shown). In addition, CpG DNA was often used at the equal concentration to this study in multiple reports of immune responses to DNA 26, 27. Therefore, the concentrations of ODN1668 used in the present study (1 and 3 μM for RAW264.7 cells and splenic macrophages, respectively) were similar levels to those used in literatures 26, 27, even though they were higher than the concentration of DNA in the serum of active SLE patients (about 3.2±1.1 μg/mL) 28. It was excluded that increased cellular uptake or stabilization is involved in the DNase I-treated DNA-mediated increase in cytokine production (Fig. 5). Zwiorek et al.

The association of single-nucleotide polymorphisms (SNPs) in the

The association of single-nucleotide polymorphisms (SNPs) in the promoter region of

TNF-α (−308G/A), IL-2 (−330T/G), IL-4 (−589C/T) and in exon region of TGF-β1 (+869T/C) genes was assessed by ARMS & PCR-RFLP using specific primers in the above-mentioned subjects. The differences in allelic or genotypic frequencies of TNF-α (−308G/A) between patients, their HHC and HC were not statistically significant (P > 0.05). IL-2 (−330T/G) TG genotype was significantly different between patients, HHC compared to HC (P < 0.002, OR-1.997, 95%CI-1.260-3.168, P < 0.03, OR-1.602, 955CI-1.003-2.561).IL-4 (−589C/T) CC genotype was significantly different between patients and HC (P < 0.03, OR-1.791, 95%CI-1.009-3.189) as well as between HHC and Selumetinib price HC at P < 0.0001, OR-2.56, 95%CI-1.448-4.545. In addition, the TGF-β 1 (+869T/C) TC genotype was significantly associated with susceptibility to tuberculosis in patients when compared against HC(P < 0.0001, OR-3.416, 95%CI-2.063-5.670) and HHC (P < 0.0001, OR-2.357, 95%CI-1.439-3.868), respectively.MDR analysis indicated that TT genotype of TGF-β1 with TT and CT genotypes of IL-4 showed high risk

with GA, TT genotypes of TNF-α, IL-2, respectively. Our results suggest that IL-2 (-330T/G), IL-4 (-589 C/T) and TGF-β1 (+869T/C) gene polymorphisms may be associated with TB susceptibility. “
“We investigated the role of SIGNR1 in the recognition of Candida albicans and the subsequent cellular oxidative burst response. Soluble SIGNR1 (sSIGNR1) tetramer bound equally to zymosan and both heat-killed (HK) and live CHIR99021 C. albicans in an EDTA-sensitive manner, whereas sDectin-1 tetramer predominantly bound to zymosan and HK-microbes in an EDTA-independent manner. In cellular response, enhanced oxidative burst was observed in RAW264.7 cells expressing SIGNR1 (RAW-SIGNR1) compared with RAW-control cells upon stimulation with HK-C. albicans and zymosan. This

Dimethyl sulfoxide response was independent of TLR2 and the cytosolic portion of SIGNR1 but dependent on the recognition by SIGNR1 via carbohydrate recognition domain. Antagonistic laminarin and anti-Dectin-1 mAb cooperatively reduced the response with mannan and anti-SIGNR1 mAb, respectively, although they had no effect by themselves. Moreover, oxidative response and bactericidal activity largely relied on Syk-mediated signaling. RAW-SIGNR1 cells not only captured microbes more efficiently but also showed higher responses than RAW-control cells. Similar enhanced responses were observed in SIGNR-1-expressing resident peritoneal Mϕ. Interestingly, Dectin-1 was recruited to the phagosomal membrane upon the stimulation and physically associated with SIGNR1. These results suggest that SIGNR1 plays a significant role in inducing oxidative response to C. albicans by Syk-dependent signaling, possibly through Dectin-1.

Three enzymes involved in glycolysis were found to be more abunda

Three enzymes involved in glycolysis were found to be more abundant in the bradyzoite [glyceraldehydes-3-phosphate (GAPDH), fructose-1,6-bisphosphate and enolase], fitting with the belief that bradyzoites rely primarily on anaerobic glycolysis for energy metabolism (34). In the same vein, isocitrate dehydrogenase (Krebs cycle) exhibited higher abundance in tachyzoites. see more Additionally, two stress-related heat shock proteins (HSP70 and HSP90) were found to have higher expression in bradyzoites. Interestingly, both ROP9 and GRA9 were found to have greater expression in the bradyzoite stage,

although ROP9 has been previously shown as a tachyzoite-specific protein in Toxoplasma (65), and GRA9 has been associated with both stages (66). This preliminary study provides promising evidence that DIGE should be able to offer more clues as to the mechanisms behind tachyzoite–bradyzoite stage conversion in Toxoplasma, as well. DIGE has been used to examine how Toxoplasma infection modulates the host cell proteome. Nelson et al. (67) used 2DE and DIGE along with mass spectrometry to identify host cell proteins whose expression was modified by infection. Initial

proteomic comparisons were made between Small molecule library infected and noninfected human foreskin fibroblasts at time points ranging from 6 h post-infection (p.i.) to 24 h p.i., and protein samples were separated by 2DE. Spots of differentially expressed proteins were picked from the gels and identified via mass spectrometry. As 2DE studies are often plagued by inter-gel variations, DIGE analysis was performed to increase reproducibility and Isotretinoin sensitivity of the proteome analysis. A total of 157 protein changes were documented

with the combined dataset from the 2DE and DIGE studies. Intriguingly, approximately one-third of the modulated proteins were mitochondrial proteins based on the ontology predictions. This suggests the importance of that host organelle in parasite infection, an implication that is further supported by the extensive association that the PVM forms with the mitochondria (68). Significant changes occur in the levels of host cell proteins pertaining to amino acid metabolism, lipid metabolism and glycolysis. In fact, six of the ten glycolytic enzymes are modulated by infection, including up-regulation of GAPDH. The levels of numerous apoptosis-related proteins were altered upon infection, including voltage-dependent anion channel (a mitochondrial VDAC) and numerous heat shock proteins (HSP27, HSP70). To determine whether the proteome changes were specific to Toxoplasma or were common to other intracellular parasites, a preliminary DIGE study of host response to Leishmania major (a nonapicomplexan parasite) infection was performed. There were considerable differences between those seen in the Toxoplasma infection, suggesting that the host response to Toxoplasma may be specific. Nelson et al.

By definition, the ACR is dependent on albumin and creatinine exc

By definition, the ACR is dependent on albumin and creatinine excretion rates. The influence of age and sex on 24 h

urinary creatinine is well established. For example, one large population-based Belgian study of over 4000 people (26–60 years) demonstrated significantly lower creatinine excretion in females and significant negative correlation of 24 h urinary creatinine excretion with age.11 Therefore, increases in ACR with age can be explained Selleckchem GSK2118436 in part by the age related changes in AER and 24 h urinary creatinine excretion observed in both males and females. Normal ageing is characterized by a progressive decline in skeletal muscle mass and increase in body fat composition. Other age related factors that may influence ACR include the decline in skeletal muscle mass between the 20–80 years of age, which has been estimated to range from 22% to 40%,84,85 a decrease in the proportion of muscle in lean body mass85 and a lower meat intake in older subjects.81 Bakker71 has proposed the use of age-specific cut off values for ACR to help restrict the number of people selected for follow up with timed urine collections. In this large study (n > 2300) an increase in the ACR cut-off for each decade, from age group <50

to >70 years, was required to maintain equivalent sensitivities and specificities in each age subgroup. However, the use of both gender and age-specific cut off values for ACR may be confusing and impractical. The clinical importance of an age-related increase in ACR is an increased false positive rate in older patients (e.g. decreased specificity). Using the recommended cut off values, the age-related increase in false positive rates Palbociclib for spot ACR was approximately 30% for patients of either sex over 65 years limits.79 Table A4 presents a summary of studies (including those discussed above) that

provide evidence in relation to the use of AER and ACR ADAMTS5 for the screening and diagnosis of albuminuria. Included in the table is a summary of the key components of the cross sectional studies relevant to assessment of diagnostic accuracy. Where reported the sensitivity and specificity is shown along with the key conclusions made by the authors. It should be noted that only a few of the studies provided PPV and NPV values. Estimation of GFR (eGFR) based on serum creatinine is a pragmatic, clinically relevant approach to assessing kidney function in people with type 2 diabetes (Level III – Diagnostic Accuracy). The CG and the MDRD formulas for the estimation of GFR were developed predominantly in individuals without diabetes. Studies involving people with type 2 diabetes, are summarized in Table A5 and are generally consistent with the findings for the large number of studies in non diabetes populations.46 Nonetheless, the study by Walser86 questioned the acceptability of the CG and MDRD equations for monitoring kidney function in individuals with type 2 diabetes.

cruzi infected and LPS-treated mice in the absence of any adoptiv

cruzi infected and LPS-treated mice in the absence of any adoptive transfer procedure further confirm that this is a phenomenon that naturally occurs during acute Th1 inflammatory conditions and it does not represent an artifact induced after i.v. cell injections. It has been described that lymphopenic thymi are more permeable to peripheral leukocyte infiltration. For example, it has been reported that thymus lobes from aged or neonatal mice are much more leaky to peripheral T cells than are those from adult mice [4, 19]. Certain disease states have also been shown to promote thymic immigration by recirculating T cells;

for instance, mature resting T cells readily enter the atrophic thymus of T-cell deficient SCID mice and persist there for months [18]. Interestingly, our data show that after LPS treatment, C. albicans, or T. cruzi Selleck Navitoclax infection or simply after IL-12 + IL-18 systemic expression, thymi experience a great

loss of their cellularity, especially of DP cells [31]. However, data suggest that permeability to peripheral cells to the thymus is unlikely to be due solely to the sparse DP compartment found in the thymi, since dexamethasone treatment of a normal mice, known to deplete the DP compartment [26, 27], failed to promote the thymic immigration of adoptively transferred peripheral B and T cells from T. cruzi infected mice. These data make us believe that not only free space is necessary but also certain molecules involved in cell migration induced in these inflammatory models are needed in the migration of cells to the thymus. The first candidate

that we analyzed was the selectin CD62L, since it has been previously reported that cells that enter https://www.selleckchem.com/products/INCB18424.html the thymus are CD62Lhi [11]. Moreover, expression of CD62L on T cells has been demonstrated to mediate the interaction between peripheral node addressin on the thymic vasculature or stromal cells, thereby promoting T-cell immigration [28]. However, our data demonstrate that CD62L does not participate in this migratory effect. In a different experimental model, it has been reported that memory T cells that migrate to bone marrow express higher levels of CCR2 than memory T cells that reside in the spleen [38]. This fact led us to investigate if CCR2 is also involved in peripheral cell migration to the thymus. We found that when mice are treated with 12+18-cDNA or T. cruzi infection, CCR2 expression Coproporphyrinogen III oxidase in the thymus is increased. Moreover, B and T cells in the thymus of T. cruzi infected mice show positive expression of CCR2. MCP-1 is one of the C-C chemokines that has been reported to induces chemotaxis of B and memory T cells through its receptor CCR2 [39]. Moreover, MCP-1 has been reported to be important in mediating migration of CD8+ TCM cells to inflammatory sites [40] that is compatible with the TCM phenotype of T cells that enter the thymus in these three inflammatory/infectious conditions. Furthermore, MCP-1 is highly expressed in the thymus of LPS-treated, C.

8–4 g, given orally or as

suppositories One patient used

8–4 g, given orally or as

suppositories. One patient used antihypertensive medication (kandesartancileksetil; Atacand®ö, AstraZeneca, Södertälje, Sweden). In the patients with CD, one used sulphasalazine (Salazopyrin®, Pfizer, New York, NY, USA) (4 g) and one mesalazine (2 g) daily. A third patient with CD used nabumeton (Relifex®, Meda, Solna, Sweden) for arthrosis. All the included participants with UC (n = 10) and CD (n = 11) denied regular smoking. Prior to (day 0) and during (days 2 and 12) the intake of AndoSan™, heparinized blood collected from the included participants was, in one set of experiments, also immediately stimulated ex vivo with LPS (1 ng/ml) for 6 h at 37 °C in a 5% CO2 incubator. During this incubation, the tubes were shortly manually shaken each hour. Then, plasma was harvested and samples LY2157299 stored at −70 °C until analysis for levels of cytokines. The included UC and CD patients had median disease duration of 15 (2–29) and 10 (2–29) years, respectively. The patients with UC had pancolitis (n = 3), left-sided colitis (n = 3), proctosigmoiditis PD-0332991 molecular weight (n = 1) and proctitis (n = 3), of whom two had been treated in hospital for acute colitis. Disease location in CD was ileal (n = 1), ileocolic (n = 6) and colic (n = 4). Three patients had had ileocolic resections. To obtain baseline values of cytokine levels in healthy volunteers, equally treated plasma samples from unstimulated blood were

also analysed for this purpose. GABA Receptor The 15 healthy volunteers (eight men) had median age 36 (range 26–51) years and denied regular smoking and use of steady medication. Analyses.  Blood was harvested from the antecubital vein into glass tubes containing 15 IU heparin per ml or 10 mmol EDTA per ml. The EDTA blood was each time (days 0,

1, 2, 5, 8, 12) analysed for haemoglobin, haematocrite, mean cellular volume, mean cellular haemoglobin, reticulocytes, immature reticulocytes, leucocytes including a differential count of neutrophils, basophils, eosinophils, lymphocytes and monocytes, thrombocytes, C-reactive protein (CRP), urea, creatinine, bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, γ-glutamine transferase, alkaline phosphatase and pancreatic amylase. The harvested heparinized blood was immediately centrifuged (2300 g, 12 min) and plasma pipetted off and immediately stored at −70 °C till analysis for micro CRP (days 0, 2, 12) and cytokines (days 0, 2, 12). The CRP was analysed by both ordinary routine laboratory technique from EDTA blood and micro-CRP from plasma by the high sensitive Tina-quant CRP particle-enhanced immunoturbidimetric method performed using a COBAS INTEGRA 400 analyser (Roche Diagnostics, Indianapolis, IN, USA) [28]. This micro-CRP method is especially sensitive in concentrations ≤20 mg/l. Faecal calprotectin concentrations (mg/kg) (normal values <50 mg/kg) at days 0 and 12 were determined in duplicates as reported [18, 29].

, 2008) The data presented above suggest the participation of Sh

, 2008). The data presented above suggest the participation of ShET-2 in the invasive and/or pro-inflammatory processes that occur during Shigella infection. We evaluated the possible role of ShET-2 in the inflammatory and cellular stages of Shigella infection.

We constructed an S. flexneri sen mutant using the λ-red recombination system (Datsenko & Wanner, 2000); PCR and Western blot analyses confirmed the correct insertion and subsequent excision of the KmR cassette that was used to obtain the nonpolar selleck products sen null mutant, named 2457Tsen. 2457Tsen strain transformed with pSen plasmid secretes recombinant ShET-2 protein and IpaB protein in the presence of CR as well as wild-type 2457T strain (Fig. 1). The gentamicin protection assay selleckchem and the plaque assay showed no differences between the wild type and sen mutant, revealing no apparent role for this product in invasion, intracellular multiplication or spread from cell to cell (Table 2). Similarly, the guinea pig keratoconjunctivitis test revealed no significant difference

in the degree of inflammation between the wild type and sen mutant. These results are in agreement with previous observations (Ranallo et al., 2006). We did, however, observe a significant reduction in the amount of IL-8 secreted from epithelial HEp-2 cells infected with 2457T vs. 2457Tsen, when the cytokine was assayed 4 h after infection (Table 2). IL-8 secretion assayed 18 h after infection showed a significant reduction in the amount of this cytokine in T84 cell monolayers infected with 2457Tsen compared with wild-type 2457T (Fig. 4). Complementation of 2457Tsen with pSen and pJS26 [the latter encoding the sen gene cloned into pBluescript (Nataro et al., 1995)]

restored IL-8 secretion to wild-type levels (Fig. 4). Shigella type III effectors are classified into three categories, according Depsipeptide to the degree to which their expression is controlled by the T3SS activity (Parsot, 2009). Several studies have been proposed that ShET-2 belongs to the group of effectors that are positively controlled by T3SS activity. Here, we demonstrated that ShET-2 is in fact a type III effector, and is cotranscribed with ospC1, which is regulated by MxiE. These observations support the inclusion of ShET-2 with the group of Osp protein regulated by T3SS activity and MxiE protein. Recent studies have shown that Shigella type III effector proteins regulated by T3SS activity (OspF, OspB, OspG and IpaH9.8) interfere with the host signalling cascades at different level, mitigating intestinal inflammation (Kim et al., 2005; Okuda et al., 2005; Zurawski et al., 2006, 2009; Arbibe et al., 2007). In contrast to these observations, our data suggest that ShET-2 might have an additional function besides its previously reported enterotoxic activity: a contribution to the pro-inflammatory effect of S.