(2006). Three of nine auxiliary loci ETR-B, Mtub29 and Mtub34 (Supply et al., 2006) were not included in this study because they were previously shown to be monomorphic in ST125 strains

(Valcheva et al., 2008b). The amplicons were evaluated on 1.5% standard agarose gels using a 100-bp DNA ladder (GE Healthcare). The H37Rv strain was run as an additional control of the performance of the method. Size PI3K inhibitors in clinical trials analysis of the PCR fragments in 1.5% agarose gels and assignment of the VNTR alleles were carried out using totallab tl100 software (Nonlinear Dynamics Ltd, UK) and by comparison with correspondence tables kindly provided by Philip Supply. Some PCR reactions were repeated and allele scoring was performed by an independent analysis by two technicians. Analysis of the IS6110 element specific for the LAM genetic family was performed as described previously (Marais et al., 2006). In brief,

a 205-bp band indicates a LAM strain due to the presence of an IS6110 element Decitabine in a specific site in the genome, whereas a 141-bp band indicates a non-LAM strain lacking the IS6110 element in this site. To minimize the risk of laboratory cross-contamination during PCR amplification, each procedure (preparation of the PCR mixes, the addition of the DNA, the PCR amplification and the electrophoretic fractionation) was conducted in physically separated rooms. Negative controls (water) were included to control for reagent contamination. P-type ATPase NJ and UPGMA trees were built using the paup 4.0 package (Swofford, 2002) and minimum spanning tree – using the PARS program of the phylip 3.6 package (Felsenstein, 2004). At present, the mechanism of evolution of VNTR loci in M. tuberculosis

is not completely understood; for this reason, similar to other studies, VNTR alleles were treated as categorical variables, i.e. any change in a locus (gain or loss of any number of repeats) was considered equally probable. The search for historical links between the areas targeted in this work was carried out by searching Google (http://www.google.com/), Entrez Pubmed (http://www.ncbi.nlm.nih.gov/sites/entrez) and the History Cooperative (http://www.historycooperative.org/) search resources using the keywords ‘human migration,’‘tuberculosis,’‘history,’‘phylogeography,’‘coevolution’ as well as relevant geographic names used alone and in combination. This was followed by further sorting and mining of the large body of the retrieved information, and, if necessary, an additional search using more specific keywords covering bilateral relations between particular regions and countries. Although this method is neither exhaustive nor quite systematic, a quantitative approach to comprehensively study large events in human history still does not exist, to the best of our knowledge.

cruzi and L. major (15). The majority of species-specific genes – of which T. cruzi (32%) and T. brucei (26%) have a much greater proportion than L. major (12%) – occur at non-syntenic chromosome-internal

and subtelomeric regions and consist of members of large surface antigen families. These gene family expansions, along with structural RNAs and retroelements, are often associated with breaks in synteny. Gene divergence, acquisition and loss, and rearrangements within and between syntenic regions have shaped the genomes of the trypanosomatids (15). A remarkable click here feature of the T. brucei and T. cruzi genomes is the extensive expansion of species-specific genes, the large majority encoding surface proteins, such as Variant Surface Glycoproteins (VSGs) in T. brucei, trans-sialidase superfamily, mucin-associated surface proteins and mucins (TcMUC) among others in T. cruzi, all of them likely involved in important host-parasite interactions (15). These surface protein-encoding genes are often clustered into large arrays that can be as large as 600 kb and are/were subjected to intense rearrangements during the parasites’ evolution (15,20). It is likely therefore

that much of the striking polymorphism among the T. cruzi and T. brucei isolates that are reflected in several epidemiological and pathological aspects of Chagas disease and African sleeping sickness may be in part because of variability within these regions. Whole genome comparisons NVP-BKM120 clinical trial of distinct trypanosomatid lineages Org 27569 would allow further investigation of this. A wide range of pathologies is found within trypanosomatid parasite lineages. Thus, there remains a considerable evolutionary and pathological

space yet to be explored through additional comparative sequencing (we define pathogenomics as the genome analysis of pathogens). With the advent of massively parallel sequencing technologies, sequencing of additional trypanosomatid strains can now be performed at a fraction of the cost of the sequencing of the reference genomes. The Wellcome Trust Sanger Institute (WTSI) has initiated such efforts. The recent sequencing of the genomes of several Leishmania species, causative agents of cutaneous, mucocutaneous and visceral leishmaniasis, is beginning to unravel many features of potential relevance to parasite virulence and pathogenesis in the host. When compared to L. major, the genomes of Leishmania braziliensis and L. infantum displayed a highly conserved gene content and order. However, two hundred genes with a differential distribution between the three species were identified (21,22). Perhaps most unexpected was the discovery that L. braziliensis genome retained the components (Argonaute and Dicer) of a putative RNA interference pathway, which are absent in L. major and L. infantum. A subsequent functional study demonstrated the presence of a strong RNAi activity in L. braziliensis (23).

Grace et al. in a review of ANZDATA listed patients starting dialysis between 2000 and 2010 found only 7% of postcodes outside of major PI3K inhibitor cities were in the most advantaged quartile, compared with 54% of postcodes within major cities[9] Gray et al. in a similar review of non-indigenous patients on dialysis on found significant differences in disease burden between major capitals (MC), inner remote (IM), outer remote (OM) and very remote (VR) areas – Figure 2.[10] Patients want to be treated close to where they reside to avoid the cost of travel and dislocation involved in visiting metropolitan based clinics.

The implementation of renal palliative/supportive care services in rural areas requires a different model to metropolitan areas if these patients are to have the same standard of care as those in metropolitan areas. General practitioners and renal physicians tend to refer on the basis of previous personal exposure. Providing specialist renal palliative/supportive

care services will need to involve some on the ground outreach services to gain the trust and respect of the local physicians. Any model will need to enhance contact between palliative care services and local physicians. A ‘move aside while we show you how it is done in the city’ approach is unlikely to be successful. The knowledge base for renal palliative Rapamycin purchase care will need to be outsourced to the local physicians, GPs, and palliative care nurses to enhance patient care. Given that it is unlikely that rural units will have specialist renal palliative /supportive expertise on site the DNT committee supports the concept of a hub and spoke model of care to provide equity of service in all rural and remote areas.

This implies that metropolitan palliative care services will have a responsibility to provide outreach services and will need adequate resources. The same model is used to provide transplant services successfully in rural areas and not only allows rural patients to access these services locally but provides up skilling of the local workforce. Developments in information technology such as telemedicine are possible solutions to some of the problems associated with distance and isolation. The current Medicare click here rebate for consultations by videoconferencing should promote and compensate set up costs. This can be easily performed with currently available technology including Skype. There is a potential role for web based on going education for rural renal physicians and palliative care physicians in renal supportive care. This could potentially involve cased based scenarios in a chat room environment. A model currently working in the New England Area involves having a local supportive care nurse who is experienced in dialysis assess all patients referred to the service. Referrals can be from nursing colleagues, GPs, allied health workers and renal physicians.

Each sample was conducted in triplicate. The data were calculated by using 2−ΔΔCt method. The expression of TIPE2 protein in PBMC was assessed by Western blot. Proteins were extracted from PBMC using a modified TRIzol one-step extraction method. Protein concentration was determined by a Bradford kit (Bio-Rad, Hercules, CA, USA). Proteins (40 μg) were separated on SDS-polyacrylamide gel and then were electrotransferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 2% BSA in TBST containing 0.1% Tween-20 for 1 h at room temperature, the membrane was incubated overnight at 4 °C with 1:300 dilution of anti-TIPE2 antibodies (Boster Biological Technology Inc., Wuhan, China) and

then was washed three times and incubated with 1:2000 dilution of secondary antibody (goat anti-rabbit IgG) for 1 h at room selleck screening library temperature. After washing, the membrane was visualized by ECL Western blotting detection

system (Pierce Biotechnology, Rockford, IL, USA). β-actin was used as the loading control. Serum was obtained from peripheral blood of children with asthma and healthy controls. IL-4 and IFN-γ were both detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Briefly, the serum was added into 96-well plates coated with purified anti-human selleck compound IL-4 or IFN-γ antibody in duplicate and incubated at room temperature for 2 h. Human recombinant IL-4 or IFN-γ was included as standard. After five washes using wash buffer (1 × PBS, 0.05% Tween-20), biotin-conjugated anti-IL-4 or anti-IFN-γ antibody was added and incubated at room temperature for 1 h. Avidin-conjugated horseradish peroxidase (HRP) was added for an additional 1 h at room temperature, followed by the substrate working solution until colour development. Five washes were performed between the steps. The plates were detected at 450 nm using a multifunctional microplate reader (Bio-Rad). The level of serum total IgE in children with asthma and

healthy controls was determined by radioimmunosorbent test (IRST) using Germany’s Thiamine-diphosphate kinase Siemens fully automatic specific protein analyser (BNP, Marburg, Germany). Eosinophils (EO) were detected using BC-5800 Automatic Blood Cell Analyzer (Mairui, Shenzhen, China). All data were shown as mean ± SEM. Unpaired Student’s t-test was used to compare the difference between patients with asthma and healthy controls. Correlations were studied by Pearson’s correlation test. P < 0.05 was considered statistically significant. To determine the expression patterns of TIPE2 in childhood asthma, we firstly detected the expression of TIPE2 mRNA in PBMC from 42 children with asthma and 39 healthy controls using semi-quantitative RT-PCR. The results showed that the expression of TIPE2 mRNA was reduced in patients with asthma compared with normal controls (Fig. 1A).

Both eosinophils and neutrophils are protective against S. stercoralis larvae in primary infections, whereas neutrophils are more important during rechallenge infections (86). N. brasiliensis larvae are temporarily immobilized if co-incubated with eosinophil-rich leucocytes in the presence of normal mouse serum (87) (and Dent et al., unpublished), but by 24 h most have recovered motility. However, when injected into susceptible WT hosts most of these larvae fail to migrate to the lungs, suggesting that temporary immobilization may be accompanied by more serious damage and/or greater risk to multiple innate immune effector mechanisms in the recipient. From 1980 to 1990, whilst at the University of

Western Australia (Perth), David Grove and his colleagues including Hugh Dawkins, Graham Mitchell (Walter and Eliza Hall Institute, Palbociclib concentration Melbourne), Simon Carroll and Carolyn Northern published more than 20 articles on S. ratti and S. stercoralis infections in mice. Grove’s team were the first to establish convincingly S. ratti infections in mice, with substantial infections sustained in BALB/c, C57BL/6 and CBA inbred strains for at least 10 days (88), with all of the 12 strains tested being highly resistant to re-infection (88). S. ratti larvae entering the host via percutaneous infection rapidly transit to the underlying abdominal wall and then migrate to skeletal muscle, the selleck cerebrospinal

fluid and the lungs, though the route of migration is not clear (89,90). In contrast to primary infections with N. brasiliensis, where strong subcutaneous inflammatory infiltrates are detected within 2 h pi. (65,75), skin inflammation after exposure to S. ratti is initially modest at 2 h, increasing substantially

by 12 h pi. (91). Whereas in N. brasiliensis infections few inflammatory leucocytes are seen in the lungs in the first 24–48 h of either primary or secondary infections (65,69,76), strong early lung inflammatory responses are seen on re-exposure to S. ratti (91). Protection against primary S. ratti infections can be transferred with either immune serum or mesenteric lymph node cells harvested from mice infected 2–3 weeks previously (92). Mice can be immunized with soluble antigens prepared from filariform S. ratti larvae, but not by implantation of larvae within micropore chambers that are impervious oxyclozanide to leucocytes (93). Resistance to S. ratti is T cell-dependent, with higher intensity infections and persistence of intestinal worms for at least 6 weeks in athymic mice (94). More recently, Karen Ovington, Carol Behm and colleagues at the Australian National University have demonstrated that IL-5-deficient mice are more susceptible to S. ratti (95) and in collaboration with the Dent laboratory that IL-5 Tg mice are more resistant to this parasite (McKie, Ovington, Behm and Dent, unpublished). Groves and his colleagues linked their work with S. ratti to S.

Furthermore, metabolic gene changes seen in SALS, many of which were also evident in PLS fibroblasts, resulted in dysfunctional cellular respiration. The data demonstrate that fibroblasts can act as cellular models for ALS and PLS, by establishing the transcriptional changes in known pathogenic pathways that confer subsequent functional effects and potentially highlight targets for therapeutic intervention. “
“Magnetic Pifithrin-�� mouse resonance

imaging indicates diffuse white matter (WM) changes are associated with cognitive impairment in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). We examined whether the distribution of axonal abnormalities is related to microvascular pathology in the underlying WM. We used post-mortem brains from CADASIL subjects and similar age cognitively normal controls to examine WM axonal changes, microvascular pathology, and glial reaction in up to 16 different regions extending rostro-caudally through the cerebrum. Using unbiased stereological methods, we estimated length Selleckchem TSA HDAC densities of affected axons immunostained with neurofilament antibody SMI32. Standard immunohistochemistry was used to assess amyloid precursor protein immunoreactivity per WM area. To relate WM changes to microvascular pathology, we

also determined the sclerotic index (SI) in WM arterioles. The degree of WM pathology consistently scored higher across all brain regions in CADASIL subjects (P < 0.01) with the WM underlying the primary motor cortex

exhibiting the most severe change. SMI32 immunoreactive axons in CADASIL were invariably increased compared with controls (P < 0.01), with most prominent axonal abnormalities observed in the frontal WM (P < 0.05). The SIs of arterioles in CADASIL were increased by 25–45% throughout the regions assessed, with the highest change in the mid-frontal region (P = 0.000). Our results suggest disruption of either cortico-cortical or subcortical-cortical selleck compound networks in the WM of the frontal lobe that may explain motor deficits and executive dysfunction in CADASIL. Widespread WM axonal changes arise from differential stenosis and sclerosis of arterioles in the WM of CADASIL subjects, possibly affecting some axons of projection neurones connecting to targets in the subcortical structures. “
“Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and non-coding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may co-exist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function.

5% BSA and 0.05% Tween20). Blots were washed repeatedly in washing MAPK inhibitor buffer (15 mM NaCl, 50 mM Tris-HCl, 0.05% Tween20; pH 7.6) and incubated for 1 h at room temperature with 0.1 μg/mL peroxidase-conjugated donkey anti-mouse IgG in blocking buffer. Peroxidase activity was detected using chemiluminescence substrate (Pierce) and recorded with a chemiluminescence detector

(Vilber Lourmat). Mouse anti-MEK1/2 (phosphorylated and non-phosphorylated), mouse anti-JNK (phosphorylated and non-phosphorylated) and mouse anti-p38 (phosphorylated and non-phosphorylated) were obtained from Cell Signaling Technology, Danvers, MA, USA For TransAm analysis, primary human keratinocytes were stimulated for 2 h with recombinant cytokines. Nuclear

extracts were generated with the Nuclear Extract Kit (Active Motif) and analyzed for activated transcription factors using TransAm Kits (Active Motif) according to the manufacturer’s protocols. For dual luciferase assays, primary human keratinocytes were grown to 70% confluence and transfected with two plasmids, Alisertib mouse one containing the “Firefly Luciferase” under control of an AP-1-dependent promoter and a control plasmid expressing the “Renilla Luciferase” under the CMV promoter. The transfection was performed in presence of DMRIE-C (1, 2 -Dimyristyloxypropy l-3 – Dimethyl – Hydroxy – Ethyl–Ammoniumbromide plus Cholesterol) (Dual-Luciferase-Reporter Assay System, Promega). Eighteen hours after transfection, keratinocytes were stimulated for 48 h with recombinant cytokines. Concentration of CXCL-10, CXCL-11 and HBD-2 in cell-free supernatant of primary

human keratinocytes stimulated with 50 ng/mL IL-22, 50 ng/mL TNF-α or a combination of both were measured using commercially available sandwich ELISA kit according to the manufacturer’s instructions (CXCL-10, CXCL-11: R&D Systems, HBD-2: Phoenix Pharmaceuticals). C. albicans wild-type strain SC5314 was used for the infection of human oral keratinocytes (TR146, buccal carcinoma cell line) as described previously 33. C. albicans was grown on Sabouraud’s Janus kinase (JAK) dextrose agar (Difco) followed by two pre-cultures in 10 mL YPG (1% yeast extract, 2% peptone, 2% glucose) medium (Difco), first for 16 h at 25°C and then for 24 h at 37°C through orbital shaking. Human oral keratinocytes were cultured in DMEM medium supplemented with 10% FCS and 0.1% gentamicin solution (50 mg/mL) at 37°C and 5% CO2. For two-dimensional skin infection models, 30 000 human oral keratinocytes (TR146) were plated per well in 96-well plates in antibiotic and antimycotic free culture medium. Twenty-four hours after plating, cells were treated with 50 ng/mL TNF-α and IL-22 or Th22 supernatant. Each treatment was performed in triplicate. Keratinocytes were infected 30 min after treatment with a total amount of 3000 yeast cells (MOI 0.1).

STATISTICA® StatSoft, Inc. (StatSoft Scandinavia AB, Uppsala, Sweden) 9.0 software package was used selleck inhibitor for all statistical analyses. Positively skewed variables were logarithmically transformed prior to analysis. Values are presented as mean ± SD. The

study was approved by the Ethics Committee at Huddinge University Hospital, Stockholm, Sweden. The research was performed in accordance with institutional guidelines of the Karolinska Institute and in accordance with the Declaration of Helsinki. All subjects gave their informed consent. As shown in Table 1, the level of ascorbate in plasma increased significantly after treatment with ascorbate. Likewise, the level of α-tocopherol in plasma increased after treatment with vitamin E, whereas measured levels of retinol remained unchanged. As shown in Table 2, inhalation of cigarette smoke induced a significant reduction in capillary blood flow velocity. This effect of smoking was very prompt both before (p < 0.0007) and after treatment with ascorbate (p < 0.0004). However, there was no significant difference in terms of relative reduction in CBV before or after intervention by either of the antioxidants. The reduction was 65% before ascorbate and 60% after ascorbate (ns). At baseline, TtP was significantly prolonged after inhalation of cigarette smoke, an increase in TtP from 7.3 to 10.6 seconds (p < 0.05). When comparing

the response to provocation by PRH before and after two weeks of treatment with ascorbate, there was a highly significant shortening of TtP DAPT purchase as compared with baseline: 7.3 seconds vs 5.2 seconds (p < 0.002). Furthermore, the TtP in response to smoking after treatment with ascorbate was prolonged from 5.2 to 7.4 seconds (p < 0.002). The relative change in response to smoking did not differ between subjects treated or not treated with ascorbate (ns). The same experimental protocol was repeated in volunteers using vitamin E. Again, there was an effect on resting CBV with a similar effect of acute smoke inhalation on CBV as for ascorbate. The reduction in CBV after smoking was highly significant: from 0.72 ± 0.24 to 0.40 ± 0.22 mm/sec (p < 0.000008). Concordant with the results of treatment

with ascorbate, there was no difference Histamine H2 receptor in the response of CBV to the effects of smoke inhalation before and after treatment with vitamin E, i.e., it was not possible to demonstrate any significant effect on the reduction in CBV in response to smoking before or after the two-week treatment with vitamin E. The baseline TtP before treatment with vitamin E was similar to before ascorbate, 7.0 ± 3.0 seconds compared to 7.3 seconds (ns). However, there was no difference in TtP before or after the two-week treatment with vitamin E, 7.0 ± 3.0 seconds vs 6.8 ± 2.6 seconds (ns). Baseline CBV before either treatment did not differ (ns). In contrast to baseline measurements, the CBV increased significantly after treatment with ascorbate, from 0.64 ± 0.33 to 1.00 ± 0.53 mm/sec (p < 0.

Especially in ICU patients frequently showing single- or multi-organ failure and receiving a multitude of drugs with complex interactions, echinocandins have become the treatment of first choice for candidemia.

“
“Women suffering from buy BAY 80-6946 recurrent vulvo-vaginal candidosis (RVC) often follow medical and non-medical advices to diminish the severity and frequency of the recurrences, but the impact of such interventions is unclear. The aim of this study was to identify differences in life style habits of women with RVC compared with normal women and to define which changes have influenced the frequency of recurrences in these women. Fifty-one women with RVC and 51 age-matched control women without a history of RVC were sent a questionnaire. History of allergic disease (OR 2.8) and use of corticoids (OR 5) were more frequent in patients with RVC than controls. When interrogated about beneficial changes introduced in their life style habits, lowering the intake of sugars, preventing perineum humidity and stopping contraceptive pills were factors offering substantial improvement. Apart MK-8669 from an increased risk of having an allergic constitution, no differences in the medical history or life style habits were evident between women with RVC and healthy women. However, women with RVC have introduced several changes in life style habits that proved beneficial to them. Among these changes, lowering

intake of sugars, preventing perineum humidity and stopping oral contraceptives were the most important. “
“Evidence-based clinical pathways to direct antifungal treatment options in patients with breakthrough fungal infections during current systemic antifungal therapy are not available. Nonetheless, for defined settings of such breakthrough infections approaches to management can be recommended based on clinical, epidemiological, pharmacological and in vitro susceptibility

data. “
“Invasive aspergillosis (IA) has a wide spectrum of clinical presentations and is associated with high mortality rates. Early initiation of systemic antimould therapy remains the most important measure to reduce mortality. Surgical debridement is an important additional therapeutic option mainly in cases of extrapulmonary IA. The main intention for surgical intervention in IA is to obtain material for Casein kinase 1 diagnosis and antifungal susceptibility testing. There are, however, also therapeutic implications for surgical interventions in rare manifestation of IA such as endocarditis or mycotic aneurysm. Here, we will review the role of surgical interventions in the treatment of different clinical manifestations of IA. Aspergillus spores are ubiquitous, and – once aerosolised and inhaled – may colonise the airways and cause invasive aspergillosis (IA). Host factors such as severe and prolonged neutropenia, allogeneic stem cell transplantation, prolonged use of corticosteroids or receipt of recognised T-cell immunosuppressants may predispose patients for developing IA.

Briefly, freshly isolated cells were incubated for 15 min at RT in the dark with 5 μm CFSE in PBS/0,1% BSA. Afterwards, excess dye was washed by centrifugation and cells were re-suspended in fresh complete medium and plated at a concentration of 1 × 106 cells per well in 96-well flat-bottom plates (Costar, Acton, MA, USA). Cells were stimulated with 10 μg/mL of complete somatic antigen (AgS) or with 5 μg/mL of antigenic fractions

(F9, F13, F17). The response was also measured when cells were costimulated with anti-CD3/CD28 antibodies; 0.5 μg/ml anti-CD3 (plate bound) and selleck 0.5 μg/mL anti-CD28 antibodies (BD Biosciences, Pharmingen, San Diego, CA, USA) were used in culture with antigen and fractions. After 72 h of culture cells were collected, washed with PBS, stained with anti-CD4-PerCP and anti-CD8-APC

antibodies (BD Biosciences, Pharmingen, San Diego, CA, USA) as described above and analysed in FACS. Cell proliferation, tracked by dye dilution, was monitored in CD4+ and CD8+ cells, and division index was calculated (DI = number of all cells/number of parent cells). The MLN cells at a concentration of 5 × 105 per well were plated onto 96-well flat-bottom plates (Costar) and treated simultaneously with 10 μg/mL of somatic antigen or with 5 μg/mL of separate antigenic fractions and 10 ng/mL of recombinant tumour necrosis factor-α (rTNF-α) or 100 nm synthetic glucocorticoid, dexamethasone, DEX (Sigma-Aldrich, Steinheim, Germany) 5-Fluoracil purchase in complete medium. Cells were cultured for 72 h, then collected and harvested by centrifugation at 800 g for 10 min. Cell pellets were resuspended in 100 μL of PBS (pH 7.4) with 2% BSA (Sigma-Aldrich). The effect of a stimulant dose on cell apoptosis was determined in a preliminary study using ssDNA ELISA. The method is based on the selective denaturation of DNA in apoptotic cells by formamide and detection of denatured DNA with a monoclonal antibody to single-stranded DNA (ssDNA).

The assay was performed following the manufacturer’s protocol using an ssDNA Apoptosis ELISA kit (Chemicon International, Inc., Temacula, CA, USA). Apoptosis Phenylethanolamine N-methyltransferase of specific populations of T cells was measured by four-colour flow cytometry. Cells were phenotyped for surface markers: 1 × 106 cells were incubated with 10 μL rat Alexa anti-mouse CD3, Allophycocyanin (APC) anti-mouse CD25, phycoeritrin (PE) anti-mouse CD4 (L3T4) and peridinin–chlorophyll–protein complex (PercP) anti-mouse CD8 monoclonal antibodies (BD Biosciences, Pharmingen) for 30 min at 4°C. The CD25 molecule (IL-2R-α chain p55) is widely used, but it is not a unique marker for Treg as it is also expressed on activated effector T cells. For this reason, we analysed CD4+CD25+ cells with a high expression of the IL-2Rα chain (CD4+CD25hi) to distinguish the natural from adaptive Treg subsets.