Recently, OXA-48-producing E. coli identified in France from patients transferred from Egypt were described [16]. Our findings thus confirm the hypotheses about a likely endemic Wnt activity circulation of OXA-48 in Egypt and other north African countries [16]. Of special interest, the carbapenem-resistant isolate of phylogroup B1 containing blaCMY-2, blaOXA-48 and blaVIM-29 was attributed with ST101. This supports the concerning evidence of a previous study by Mushtaq et al. who reported that 9/18 isolates of NDM-producing

E. coli from England, Pakistan and India were B1-ST101 [17]. Finally, ciprofloxacin resistance was associated with the presence of qnrS in only two phylogroup A isolates, whereas in all the remaining strains aac(6′)-Ib-cr was detected (Table 1). Twenty of 27 ciprofloxacin resistant E. coli isolates showed an association with blaCTX-M-15 and aac(6′)-Ib-cr genes. Thus, the genetic makeup which has driven the success of the ST131 pandemic clone appears to be diffuse among E. coli strains of different lineages and habitats. Acquisition of multidrug resistance gene traits by a widely disseminated human commensal organism on a global scale may seriously affect human health JNK inhibitor chemical structure and healthcare resources by causing difficult-to-treat infections in both community and healthcare settings, thus increasingly fueling the antibiotic crisis [1, 2]. The impact may be devastating in limited resource countries

and immunocompromised hosts, such as cancer patients. A previous report from Egypt described rates of resistance to third generation cephalosporins of approximately 60%in bloodstream isolates of E. coli from five hospitals in Cairo, Egypt in 1999–2000 [18]. Our findings confirm an alarming picture of multidrug resistance in E. coli and highlight acquisition of a variety of resistance genetic determinants in association with PMQR genes and the emergence of resistance to carbapenems. This work was financially supported by Institutional funds of the Department of Sciences for Health Promotion and Mother-Child Care “G. D’Alessandro. The authors declare no potential conflicts of interest with respect to the research, authorship,

and/or publication of this article. “
“The reports on fish parasite Anisakis simplex allergy have increased in countries with high fish consumption in the last decade. of In Norway, a high consumption country, the prevalence of immunoglobulin E (IgE) sensitisation to A. simplex was still unknown. Thus, our objective was to investigate the sensitisation prevalence in this country. At the Haukeland University Hospital, Bergen, Norway, two main groups of surplus serum samples were collected; one from newly recruited blood donors, and one from the Allergy laboratory after analysing IgE and IgE antibodies. The latter was divided into three series, one containing unsorted sera, and two sorted either by Phadiatop®≥ 0.35 kUA/L or total IgE ≥ 1000 kU/L. The sera were analysed for total IgE and IgE antibodies against A.

2). In contrast, when the above target mRNAs were correlated with 16S rRNA or rpoD, their expression was unaltered (Fig. 2). Expression of two other T3SS mRNAs (cpn0186 and cdsJ) appeared unaltered by the addition of INP0010 if expression was correlated with rpoA or gyrA (Fig. 2). On the other hand, cpn0186 and cdsJ show a reduced level in the presence of INP0010 when correlated with 16S rRNA or rpoD (Fig. 2). We conclude that when different control RNAs are used, a large variation of target mRNA expression can be observed. Previously,

selleck compound a method involving combined control transcripts has been used (Maurer et al., 2007). We tested this method by relating each target mRNA to a combination of the control transcripts (16S rRNA, rpoA, rpoD, and gyrA). Our results indicate that the expression of most target mRNAs were slightly stimulated, or unaltered by the addition of INP0010 (Fig. 2). The amount of any transcript at a given time point is directly GSK3235025 mw correlated with its synthesis and subsequent decay. It is plausible that the transcript stability of different control RNAs varies, which would explain the diverse target gene expression seen in Fig. 2, and it is also possible that the transcript stability can be affected

by the presence of INP0010. To investigate this, de novo synthesis of RNA was inhibited by the antibiotic rifampicin, which binds and inactivates the RNA polymerase. Such blockage allowed us to measure transcript decay of specific mRNAs. To test the stability of both virulence-associated mRNAs and control RNAs, we added rifampicin to infected cells in the presence or the absence of INP0010 at 14 h p.i. Samples were collected 0, 1, and 2 h after adding the antibiotic. As shown in Fig. 3 and Table 2, the stability of the various transcripts differed considerably.

The 16S rRNA transcript was stable in both the presence and the absence of INP0010 (mRNA half-life>2 h). In Liothyronine Sodium contrast, several transcripts (rpoD, cpn0186, cdsS, and cdsN) could be detected at the time rifampicin was added, but they were undetectable 1 h after antibiotic treatment (data not shown). This suggests a quick turnover of these transcripts during the transition from the metabolically inactive to the metabolically active state. The remaining transcripts (rpoA, gyrA, groEL_1, incB, and cdsJ) had mRNA half-lives ranging from 8 to 23 min (Fig. 3, Table 2). Although not statistically proven, these transcripts seemed to be somewhat stabilized by the addition of INP0010 (Fig. 3, Table 2). In conclusion, the transcripts used as internal expression controls in our experiments (16S rRNA, rpoA, rpoD, and gyrA) displayed varying stability. Hence, the read-out of an experiment will be complex if an added drug affects transcription, and the control and target mRNAs differ with regard to stability. Many C.

Cells were collected by centrifugation, fixed in 1·5% paraformaldehyde in PBS for 10 min at room temperature and treated with ice-cold methanol (500 µl/106 cells) for 10 min at 4°C. Cells were washed twice in PBS containing 1% BSA and stained with polyclonal antibodies to p-JNK (1:200), p-p38 (1:100) or p-c-Jun (1:20) in PBS 1% BSA for 30 min at room temperature. After incubation, the cells were washed twice with PBS 1% BSA, stained with FITC-conjugated goat polyclonal anti-rabbit IgG (1:200) or Cy3-conjugated see more rabbit polyclonal anti-goat IgG (1:100), washed twice more in PBS 1%

BSA, then 5000 events were analysed by FACScan (BD Biosciences). Autofluorescence was assessed using untreated cells. MonoMac6 (1 × 106/ml) cells were incubated alone or with JNK inhibitor SP 600125 (0·5 µM) or p38 inhibitor SB

203580 (1 µM) for 30 min at 37°C, or with antibody to FcγRIIB or irrelevant goat polyclonal IgG (0·1 µg/ml) for 30 min at 4°C. After culture, the cells were incubated alone or with GXM (100 µg/ml) in RPMI-1640 for 2 h at 37°C with 5% CO2. After incubation, the cells were washed and lysed with M-PER in the presence of protease inhibitors (BioVision, Mountain View, CA, USA) and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay reagent kit (Pierce). The lysates (100 µg of each sample) were separated by sodium dodecyl sulphate-10% polyacrylamide gel electrophoresis (PAGE), and transferred to a nitrocellulose membrane (Pierce) for 1 h at 100 V in a blotting system selleck chemicals llc (Bio-Rad) for Western blot analysis. Membranes Phosphatidylinositol diacylglycerol-lyase were then placed in blocking buffer, and incubated overnight at 4°C with rabbit polyclonal antibody to phospho-JNK (Thr183/Tyr185, Thr221/Tyr223) (1:1000). Membranes were stripped, blocked and incubated with rabbit polyclonal antibody to phospho-p38 MAPK (Thr180/Tyr182) (1:1000)

in blocking buffer, stripped, blocked and incubated with rabbit polyclonal antibody to phospho-c-Jun (Ser 63/73) (1:1000) in blocking buffer, stripped again and incubated with rabbit polyclonal antibody to FasL (1:1000). Immunoblotting with the rabbit polyclonal anti-actin antibody (H-300) (1:200) was performed in the same membrane and was used as an internal loading control to ensure equivalent amounts of protein in each lane. Detection was achieved using appropriate HRP-linked anti-rabbit IgG, followed by Immun-Star™ HRP chemiluminescent kit (Bio-Rad). Immunoreactive bands were visualized and quantified by Chemidoc Instruments (Bio-Rad). Heparinized venous blood was obtained from healthy donors. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia), as described previously [23]. For lymphocyte purification, PBMC were plated on culture flasks for 1 h in RPMI-1640 plus 5% FCS at 37°C and 5% CO2.

001). The viral loads of all of these discordant samples were low copy numbers. Indeed, complete concordance was observed in the quantitative results for the samples with ≥36 copies/ml in the prototype assay. Comparison of the prototype assay and each home-brew assay for all positive samples according to both assays had a high degree of correlation (Fig. 3). Longitudinal monitoring of five representative Pritelivir individual transplant recipients is demonstrated in Figure 4. The dynamics of the CMV load in all patients were similar, although some discrepancies were observed within the follow-up period.

Standardized calibration materials and commercially available assays have been developed for standardized quantification for specific viruses, such as HIV and hepatitis C virus (12–14). Standardization is necessary for consensus guidelines in patient management. Hayden et al. (7) reported a multicenter comparison of different real-time PCR assays for EBV. This study was carried out at eight sites using three panels consisting PD98059 of serial dilutions of commercially available EBV DNA and extracts from 19 whole blood specimens. Strong concordance among laboratories was observed with respect to the qualitative results, whereas quantitative discordance was seen at a maximum of 4 log-units. This discrepancy decreased when a common reference standard was used to obtain quantitative results. Preiksaitis et al.

(15) reported an international comparison of EBV DNA quantitative assays. They distributed a panel of samples to 28 laboratories. The panel of samples consisted

of seven constructs using EBV-positive cell lines and three clinical plasma samples. Half of the quantitative results were within ±0.5 log-units, whereas the maximum variation was approximately 4 log-units. With regard to CMV quantification, Pang et al. (16) recently reported an international comparison of CMV viral load assays. They distributed a panel of samples to 33 laboratories. The panel of samples consisted of seven constructs using purified CMV stock and three Orotidine 5′-phosphate decarboxylase clinical plasma samples. Fifty-eight percent of the quantitative results were within ±0.5 log-units whereas the maximum variation was approximately 4 log-units. In the present study, five independent laboratories were involved in comparing the quantitative values for EBV and CMV from each home-brew assay and the prototype assay. The maximum variations were 4.15 for EBV and 3.03 for CMV, which is acceptable in comparison with previous reports (7, 15, 16). Additionally, the dynamics of the EBV load in 12 patients and the CMV load in five patients were found to be similar, and this comparison may be unique. Even the inter-laboratory variation appears to be small; however, it is uncertain whether this variation is a problem for treating patients. The development of a prototype assay may help eliminate concern related to variability.

The expressed EdIII, not the NusA -Tag protein, was detected by antibodies that detect the E proteins of the tick-borne flavivirus by Western blot. These

results indicated that EdIII can be useful as the antigen in the diagnosis ELISA. One hundred and twenty serum samples from wild rodents captured in Kamiiso, Hokkaido, were tested for TBE virus-specific antibodies by EdIII-ELISA, SP-ELISA and the neutralization test. The detection accuracy of each ELISA was evaluated by comparing the results between the neutralization test and the ELISAs. Figure 2 shows the sensitivity and specificity of the EdIII-ELISA by comparison with the neutralization test, using the corresponding cut-off values. The sensitivity of the EdIII-ELISA decreased with increasing cut-off values, while the specificity increased. The difference between the sensitivity and MK-2206 chemical structure specificity was a minimum selleck screening library value when a cut-off value of 0.61 was used. Then at a cut-off value of 0.64, a higher specificity (80.0%, 68/85) and equal sensitivity (77.1%, 27/35) were obtained, compared to the cut-off value of 0.61 (Table 1). The SPs were expressed by the transfection of the plasmid pCAGprME into 293T cells

and precipitated using PEG solution as described previously (15). Anti-E protein rabbit IgG was prepared by immunization of a rabbit with the EdIII in order to use it as the capture antibody in the SP-ELISA (23). The anti-E protein rabbit IgG was confirmed to be reactive to both the E protein from the authentic

TBE virus antigen and the SPs (Fig. 3). These results indicated that the anti-E protein rabbit IgG can be useful for the capture antibody of the diagnostic SP-ELISA. Figure 4 shows the sensitivity and specificity of the SP-ELISA by comparison with the neutralization test, using the corresponding cut-off values. The sensitivity of the SP-ELISA decreased with increasing cut-off values, while the specificity increased. The difference between the sensitivity and specificity was at a minimum value when a cut-off value of 0.042 was used. Then at a cut-off value of 0.089, a higher specificity (100%, 85/85) and equal sensitivity (91.4%, 32/35) were obtained, compared to the cut-off value of 0.042 (Table 2). To investigate Bumetanide whether our ELISAs using recombinant antigens can be applied to the epizootiological survey, wild rodent samples were collected in Khavarovsk, Russia, an area in which many TBE patients were reported (24), and examined for anti-TBE virus antibodies by the ELISAs. Twenty-nine serum samples from wild rodents were tested by the EdIII-ELISA and the SP-ELISA, and the same three samples were diagnosed as positive by both ELISAs (Table 3). The three samples were also positive for the neutralization test and the other 25 samples, which were negative for the ELISAs, were also negative for the neutralization test.

Mechanisms that control normal T-cell homeostasis are not well understood. In this study, we demonstrate

that TSC1 plays a critical role for in maintenance of peripheral T-cell numbers by promoting T-cell survival through the maintenance of normal mitochondrial homeostasis. We have shown that TSC1 inhibits mTORC1 activity, but promotes mTORC2 signaling in T cells. TSC1 may affect mTORC2 signaling through several potential mechanisms. In cell line models, the TSC1/TSC2 complex can associate with mTORC2 to promote mTORC2 signaling 33. In HEK293 cells, it has been demonstrated that Rictor, a component of mTORC2, is directly phosphorylated at Thr1135 by S6K1 after growth AG-014699 clinical trial factor stimulation, and that this phosphorylation is sensitive to rapamycin. In cells that express a Rictor T1135A mutant, which cannot be phosphorylated by S6K1, mTORC2-dependent Akt phosphorylation was markedly increased, strongly suggesting that mTORC1 activation can directly suppress mTORC2 activity 34. In our model,

TSC1-deficient T cells exhibit highly phosphorylated S6K1 and decreased phosphorylation of Akt and its downstream targets. Whether or not the regulation of mTORC2 by mTORC1, through Rictor, is true during the activation of primary T cells remains to be determined. It has also Selleck PF2341066 been reported that elevated S6K1 activity can trigger a negative feedback mechanism to inhibit growth factor induced mTOR activation. For example, S6K1 can phosphorylate IRS-1 to inhibit insulin receptor signaling 35. Elevated S6K1 activity in TSC1 T cells may elicit similar negative feedback inhibition on mTOR-dependent signaling. The exact mechanism as to how TSC1-deficiency leads to mTORC2 inhibition in T cells will require further examination. Our studies indicate that the TSC1/TSC2 complex is paramount for mature T-cell Isoconazole survival. mTORC2 and Akt activities are decreased in TSC1KO T cells. Since only expression of Akt-DD but not Akt-S473D can rescue

these cells from death, it suggests that the increased death of TSC1KO T cells could not be solely due to decreased mTORC2 activity. The lack of survival defects in Rictor-deficient T cells also supports the idea that mTORC2 is not essential for T-cell survival 10. Increased mTORC1 signaling has been reported to promote cell death. In hepatocyte cell lines, S6K1-deficiency led to down-regulation of caspase-8, caspase-3 activation, cytochrome c release, and protected against the onset of apoptosis 36. S6K1 may promote cell death by inhibiting BAD phosphorylation 37. Since rapamycin cannot rescue TSC1KO T cells from death, enhanced mTORC1 may not directly cause death of these cells. However, this result does not completely rule out a role of increased mTORC1 activity in the death of TSC1KO T cells.

The activation of NK cells observed in response to LASV- and MOPV-infected MΦs is particularly interesting in that it is almost as robust as for positive controls, regarding the expression of NKp30 for instance, but nevertheless presents a different phenotype. We show here that LASV and MOPV do not infect NK cells. This result was expected and consistent

with previous studies showing that α-dystroglycan, the LASV and MOPV entry receptor, is expressed preferentially Small Molecule Compound Library on DCs and poorly on lymphocytes and that lymphocytic choriomeningitis virus, the prototypic Arenavirus that is closely related to LASV and MOPV, can infect only a few types of lymphocyte. Moreover, after direct contact with the viruses, NK cells were not activated and displayed no change in their effector functions. A slight downregulation of NKp30 expression and an increase in the expression of CXCR3 on the cell surface was even

observed in the presence of LASV or MOPV. Interestingly, TLR7 stimulation induced NKp30 downregulation as well. These results suggested that NK cells can detect both viruses, possibly through TLR7 stimulation requiring further investigation. NK cells display a rapid decrease in surface CXCR3 when cocultured learn more with LASV- or MOPV-infected MΦs. However, the significance of this downregulation is unclear. It is unlikely to be accounted for by the modulation of CXCR3 mRNA synthesis, as analysis of the mRNAs revealed no change during LASV or MOPV infection (data not shown). Aspartate CXCR3 is the receptor for the inflammatory chemokines CXCL9, 10, and 11. These chemokines, initially described as attracting activated T lymphocytes, are secreted in large amounts during the infection of MΦs with LASV and MOPV in vitro (Pannetier et al., manuscript in preparation). Moreover, the transcripts for CXCL10 and CXCL11 are found in PBMCs and lymph nodes from infected Cynomolgus monkeys [18]

and we show here that CXCL11 is detected in LASV- and MOPV-infected NK/MΦ cocultures. CXC chemokines, such as CXCL11, have been reported to induce the rapid desensitization and internalization of their receptor, CXCR3 [20]. Thus, the downregulation of surface CXCR3 expression could partly be accounted for by receptor internalization. This hypothesis is consistent with our observations, showing that CXCR3 surface expression is also downregulated when cell contact is prevented, implying that soluble factors are involved. Moreover, it is also consistent with our results with neutralizing Ab directed against CXC chemokines that abolish or reduce the downregulation of CXCR3 at the surface on NK cells in the presence of LASV- or MOPV-infected MΦs respectively.

The currently available commercial PCV2 vaccines include two subunit vaccines based on the PCV2 capsid protein expressed in the baculovirus system and an inactivated vaccine based on a PCV2 virus (9). All of these vaccines are based on the PCV2a CP-673451 cost subtype,

which several studies have shown to be cross-protective against PCV2b challenge (35, 36). An experimental live chimeric vaccine was generated with the idea that it might provide more broad cross protection and better immunity, and could be adapted for use by the oral route. The experimental chimeric PCV2 vaccine was developed by replacing the ORF2 of PCV1 with the ORF2 of PCV2a in the genomic backbone of the non-pathogenic PCV1 (37). An inactivated version of the chimeric PCV2 vaccine, which was known under JQ1 chemical structure the trade name Suvaxyn PCV2 (Fort Dodge Animal Health, Overland Park, KS, USA) and developed and licensed for pigs 3 weeks of age and older, became commercially available in 2006 (9). It was later voluntarily removed from the market but was then reintroduced in August 2011 in a reformulated version under a new name: Fostera PCV (Pfizer Animal Health, Madison, NJ, USA). Previous studies using the experimental live attenuated PCV2 vaccine demonstrated no evidence of reversion of

the live attenuated PCV1-2 to its parental wild-type viruses (PCV1 or PCV2) after 11 serial passages in PK-15 cells and the PCV1-2 was found

to be genetically stable during three serial passages in pigs (38). In addition, the experimental live chimeric PCV2 vaccine was shown to be attenuated in pigs and to induce strong protective immunity in the PCV2a HSP90 challenge model (39) and in a triple challenge model (40). Recently, the vaccine efficacy of IM administration of the live-attenuated chimeric PCV2 experimental vaccine based on subtype PCV2a was tested in a triple challenge model using PCV2b, PPV and PRRSV (41). In conventional pigs with variable amounts of anti-PCV2 antibodies and degrees of PCV2 viremia at the time of vaccination, the live-attenuated chimeric PCV2 vaccine was found to reduce the amount of PCV2 DNA in serum compared to non-vaccinated challenged pigs (41). In addition to the chimeric PCV2 vaccine based on PCV2a, a novel chimeric PCV2 virus with the PCV2b capsid gene cloned into the backbone of PCV1 was recently described (42). In a single challenge model in SPF pigs using a PCV2a or PCV2b challenge, IM administered attenuated live chimeric PCV2b vaccine was found to decrease lymphoid lesions and to prevent detectable PCV2 viremia (42). The efficacy of the live-attenuated chimeric PCV2b vaccine administered by combined IM and intranasal routes was also evaluated in a PCV2b-PRRSV-PPV triple challenge model and found to induce protective immunity in SPF pigs (40).

Contrasting with this result, we found a statistical trend for a lower prevalence KIR2DS1 in patients. Pellet et al.[11] also reported

that the presence of at least one of the two activating KIR (KIR2DS1 and/or 2DS2) was increased BIBW2992 significantly in patients (80%) when compared with controls (62%). We were also unable to reproduce this finding, observing 60·0% of KIR2DS1 and/or 2DS2 in cases and 69·6% in controls. The main finding from our study was that the inhibitory KIR2DL2 is a strong protective factor for SSc (OR = 0·22). Furthermore, we observed that the presence of the activating KIR2DS2 (the corresponding activating counterpart of KIR2DL2) is a significant risk for the disease, but only in the absence of KIR2DL2 (Tables 3 and 4). When KIR2DS2 was present concomitantly with KIR2DL2, protection from disease was observed (Table 3), suggesting that KIR2DL2 has a dominant protective effect over KIR2DS2. This can probably be explained by the interaction between KIR and HLA molecules. The most important ligands for inhibitory KIR are HLA-C molecules

[5]. The HLA binding domains of the corresponding activating KIR are almost identical to the inhibitory KIR binding domains, but have a lower affinity for HLA-Cw Palbociclib research buy [24]. This may be a possible explanation for the preponderance of KIR2DL2 over KIR2DS2 that was observed in our data and also shown by Momot et al.[10]. Considering the results of Momot et al.[10] and ours, it is possible that KIR2DS2 and KIR2DL2 (activating and inhibitory KIRs, respectively) are antagonistic molecules involved in regulation of

the activity of 4-Aminobutyrate aminotransferase NK cells and T cell activation in systemic sclerosis [6]. This combination of genes has also been implicated in the pathogenesis of other rheumatic diseases. In rheumatoid arthritis, the presence of KIR2DS2 was related to vasculitis [25]. Another study observed an association of KIR2DS2 in the absence of ligands of KIR2DL2 with increased risk of psoriatic arthritis [26]. Recent evidence suggests involvement of the combination KIR2DS2+/KIR2DL2- in the pathogenesis of Sjögren’s syndrome [27]. In our study, patients and controls presented a statistically significant difference in mean age. However, SSc is relatively rare. The prevalence of SSc is reported to be between 242–286 and 86–233 per million in North America and Australia, respectively, while the incidence is estimated to be around 20 per million per year [28]. Therefore, it is extremely unlikely that a significant number of control individuals will develop SSc in the future. Considering the high complexity of this gene system, with a great variety of possible genotype profiles, we believe that these observations are physiologically relevant. Despite the differences observed in studies from distinct ethnic groups, they all point to susceptibility and protective roles of certain activating and inhibitory KIR genes in SSc.

RNA isolation, cDNA synthesis and quantitative PCR.  The samples frozen in liquid nitrogen were homogenized in Tri Reagent (Applied Biosystems, Foster City, CA, USA), using the Ultra-Turrax apparatus (Janke&Kunkel, Staufen, Germany) or VWR Pellet Mixer (VWR, West Chester, PA, USA) for the solid organs. Total RNA was isolated after chloroform phase separation using the RNeasy kit (Qiagen, Düsseldorf, Germany), as instructed by the manufacturer.

First-strand PD98059 supplier cDNA was synthesized using oligo-dT primer (Sigma Aldrich, St. Louis, MO, USA) and avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland). Real-time quantitative PCR (qPCR) was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the appropriate primer-probe sets, and the samples were analysed using the iCycler iQ instrument (Bio-Rad, Hercules, CA, USA). All samples were run in duplicate and unless otherwise stated, were normalized against Hprt expression

levels. The primer-probe sets for Hprt, Foxp3 and CD19 were commercially available assays-by-demand (Applied Biosystems). The primer-probe set for T cell receptor (TCR) Cα was an assay-by-design (Applied Biosystems), consisting of the primers 5′-CAA AGA GAC CAA CGC CAC CTA and 5′- CGG TCA ACG TGG CAT CAC, and probe 5′-6FAM- CCA GTT CAG ACG TTC CC-quencher. All assays were intron spanning. Statistical analysis.  The data are shown as mean ± SD. Comparison of means was carried out by Student’s two-tailed t-test, with P < 0.05 as the limit for statistical significance. To test how cells from Aire−/− selleckchem mice behave in a situation strongly biased in favour of autoreactivity, but with normally functioning Aire protein, we transferred

lymph node cells from Aire−/− and control Aire+/+ mice into lymphopenic Aire+/+ recipients, thus triggering LIP. In the first Adenosine triphosphate group of recipients (hereafter Aire-group), the proliferating cells have developed in the absence of Aire, but the proliferation takes place in an Aire-sufficient environment. The second group of recipients will hereafter be denoted the control group. In this way, we can separate the central effects of Aire from its peripheral functions, and all the differences we note between the Aire and control group will be consequences of the defective thymic development in Aire−/− donors. The major lymphocyte populations in the donors were analysed prior to the adoptive transfer, and no significant differences were found in the frequency of T or B cells, CD4+ cells, CD8+ cells, Foxp3+ Treg cells, or in the fraction of Ki-67+ cells within these subsets (data not shown). Each lymphopenic recipient was injected with 1 million mononuclear cells to tail vein. After the transfer, we allowed the lymphocytes to proliferate for 2 months in order to reach the plateau phase of LIP.