The tail vein is usually used, although other veins (e.g. penile vein,14 femoral vein15 and retro-orbital plexus16) have been used. A great deal of technical expertise is required to perform this, particularly in mice (due to the small size of their veins and their predisposition not to lie still despite restraint, particularly in the case of the C57BL/6 strain). The main complication of tail vein injection is skin necrosis in the event of tissue extravazation. Gefitinib nmr Due to the narrow therapeutic index of Adriamycin, a small difference

in dose administered can potentially lead to a large variation in disease severity. Another route of administration is the substernal intra-cardiac (∼7 mg/kg in male Wistar rats) approach,17 which requires general SB203580 anaesthesia. The intra-renal route, whereby Adriamycin is injected directly into the kidney (pre- and post-contralateral nephrectomy) is associated with induction of renal injury within 4 weeks. Direct injection

of the renal artery has not been used except in pharmacodynamic studies in dogs.18 Despite their reported safety, the invasiveness of the intra-cardiac and intra-renal routes of administration has precluded widespread application. Intraperitoneal administration has been favoured for its ease of use, particularly in mice19 but due to variable absorption through the peritoneal membrane, inconsistency in induction of renal injury compared with the intravenous route has made this method less favoured. A variety of conditions can affect the delivery of Adriamycin to the target organ. Temporary clipping of one renal artery during the intravenous administration of Adriamycin partially protects the clipped kidney from proteinuric renal injury.14,20 In addition, inhibition of renal blood flow by nitric

oxide inhibition protects against glomerulosclerosis. These studies provide substantial proof that Adriamycin acts directly on the kidney to induce tissue injury.21 Male rats are more Phosphatidylinositol diacylglycerol-lyase susceptible than female rats to Adriamycin-induced nephropathy. Castration renders male rats less susceptible compared with sham-operated rats, indicating that sex hormones may contribute to the pathogenesis of Adriamycin-induced renal injury.22 Because of the difference in severity of renal injury, choice of sex is a major factor in designing an experiment using AN as a model of renal injury. In this animal model, the histological changes resemble those of human focal glomerulosclerosis, with podocyte fusion, focal segmental and global glomerular sclerosis and tubulointerstitial inflammation and fibrosis (Fig. 1).23 Adriamycin induces thinning of the glomerular endothelium and podocyte effacement associated with loss of size- and charge-specific barrier to filtration of plasma proteins.11 These changes are seen as early as 1–2 weeks after Adriamycin injection, and are severe by 4 weeks (Fig. 2).

To our knowledge, this is the first study to report association of these genotypes in household contacts. Based on MDR analysis, high-risk combination between IL-1β and IL-10 genes suggests that these SNPs interact synergistically affecting signalling impairment, and hence, effector mechanisms significantly leading to pathogenesis of tuberculosis. Our study illustrates that IL-1β CC and IL-10 GG genotypes may be useful for early detection of the disease

in high-risk www.selleckchem.com/products/jq1.html individuals, that is, household contacts. However, there is a need to evaluate the data in large sample size. We thank Bhagwan Mahavir Trust and staff of the free chest clinic Mahavir PPMDOTS, Tuberculosis Unit (TU). Financial support was provided by DBT-RGYI (Sanction no: 102/IFD/PR/2029/2007-2008 dated 18/01/2008) and COE (Sanction No: BT/01/COE/07/02, dated 30/12/08). “
“Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed

that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis PRKACG or pulsed with M. tuberculosis selleck culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen-specific IFN-γ and IL-2-expressing CD4+ and CD8+ T cells. In exosome-vaccinated mice, there was a similar TH1 immune response but a more limited TH2 response compared to BCG-vaccinated mice.

Using a low-dose M. tuberculosis mouse aerosol infection model, exosomes from CFP-treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell-free vaccine against an M. tuberculosis infection. Currently, more than 2 billion individuals have been infected with Mycobacterium tuberculosis and about 5–10% those infected will develop active tuberculosis (TB) disease during their lifetime. In 2011, there was an estimated 8.7 million new cases of TB (13% co-infected with HIV) and among the approximate 1.5 million individuals who died from TB, 430 000 were HIV positive [1]. In 1921, the vaccine Mycobacterium bovis BCG, developed by Albert Calmette and Camille Guérin, was first used in humans [2, 3]. Currently, M. bovis BCG has been administrated to over 4 billion people and remains the only licensed anti-TB vaccine worldwide [4].

Medium was changed on days 3 and 5 and usually 7-day-old cultures were used for experiments. For cytokine measurement, sorted lung DC or BMDC were pulsed with OVA or OVA-IC (see below) for 45 min or 48 h, respectively. Then supernatant was harvested and IL-6 and TNF-α determined using a commercial available Cytometric Bead Array (BD Biosciences, Germany) according to the manufacturer’s instructions. For stimulation of OT-II or DO11.10 cells, single cell suspensions from the LN were treated with monoclonal antibodies against MAC-1, F4/80, erythroid cells, Gr-1, MHC

class II and CD8α. The antibody-coated cells were incubated with anti-rat IgG-coupled magnetic beads (Biomag® Quiagen, Germany) following the manufacturer’s protocols. Finally, enriched T cells were labeled with CFSE as described elsewhere 6. For www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html the experiments using soluble OVA (Grade VI, Sigma or EndoGrade

OVA, endotoxin click here conc.<1 EU/mg, Hyglos, Germany) or OVA-IC, DC were plated in U-bottom 96-well plates (1×104 cells/well in RPMI 1640 supplemented with 10% FBS and 25 mM HEPES) with 25 μg/mL OVA or OVA-IC (made by mixing a 1:4 ratio of 25 μg/mL OVA and anti-OVA IgG) for 45 min at 37°C in complete medium. A Limulus Amebocyte Lysate revealed that OVA grade VI (Sigma) had a non-significant endotoxin content of <0.1 EU at a concentration of 100 μg/mL, that the EndoGrade™ OVA (100 μg/mL) was endotoxin free with no signal in the Limulus Amebocyte Lysate assay, and that the anti-OVA IgG at a concentration used in our experiments had an endotoxin level of 0.24 EU. The DC were washed three times and co-cultured in 200 μL of complete medium containing 5×104 CFSE-labeled OT-II or DO11.10 cells. For experiments using OVA in

combination with serum from sensitized (OVA or BSA) or non-sensitized mice, 1×104 lung DC were incubated with OVA (25 μg/mL) and serum (100 μL) for 1 h at 37°C. Afterwards, OVA and serum were removed by washing the cells with PBS and DC were used to stimulate 5×104 CFSE-labeled OT-II cells in 200 μL MG-132 research buy of complete medium. For proliferation analysis after 60 h of culture, OT-II or DO11.10 cells were stained with CD4-APC (BD Biosciences, Germany), and samples were analyzed by flow cytometry. The total number of dividing (CD4+PI−CFSElow) cells was determined in duplicate. For some experiments with OVA-IC, the data are presented as fold increase above T-cell proliferation obtained with OVA-pulsed DC in order to normalize for variability among experiments. Twenty-four hours after the last allergen challenge, airway hyperresponsiveness to inhaled methacholine (MCh) was assessed. Invasive but repetitive technique was performed to measure lung function in orotracheally intubated mice, using a body plethysmograph (HSE-Harvard Apparatus, March-Hugstetten, Germany) and an inhalation unit, which has been designed specifically for this mouse model 39.

GDM seems associated with low l-arginine transport (Figure 4), but higher expression of hCAT-1 in hPMEC, and insulin reverses these effects of the disease selleck chemicals to values in cells from normal pregnancies [65]. Thus, we hypothesize that insulin could be a key factor mediating reversal of the GDM deleterious effect in hPMEC to a phenotype resembling that in cells from normal pregnancies. Adenosine uptake is reduced in hPMEC primary cultures from GDM pregnancies, a phenomenon that has been proposed

as an explanation, at least in part, of the increased vein and whole plasma adenosine concentration detected in this disease [71]. Adenosine uptake in hPMEC is mediated via hENT1 and hENT2 in a similar proportion [30, 71] suggesting that under normal

conditions these two transport mechanisms could share a role in controlling the extracellular levels of adenosine in the human placenta microcirculation. Interestingly, reduced hENT1 and hENT2 expression and activity in hPMEC from GMD pregnancies compared with cells from normal pregnancies is reported [71]. This effect of GDM was most likely due to reduced expression of SLC29A1 and SLC29A2 (for hENT2) in this cell type. Since SLC29A2 promoter transcriptional activity is reduced in hPMEC from GDM pregnancies and the p42/p44mapk/Akt activity Selleck Deforolimus ratio was <1 instead of a predominant mitogenic signaling pathway (i.e., p42/p44mapk/Akt activity ratio >1), a potential metabolic phenotype will predominate in hPMEC from GDM pregnancies [71]. There are no studies addressing the potential modulatory action

of adenosine on the l-arginine/NO pathway in the microcirculation of the human placenta in normal or GDM pregnancies [39, 81]. Preliminary studies suggest that adenosine could acts BCKDHB as modulator of l-arginine transport in hPMEC from normal pregnancies, a phenomenon that seems to require A2AAR and A2BAR activation in this cell type (E Guzmán-Gutiérrez and L Sobrevia, unpublished observations). However, in cells from GDM pregnancies l-arginine transport was lower compared with cells from normal pregnancies, a phenomenon that was further reduced by the use of A2AAR, but not A2BAR antagonists. Thus, GDM is a condition potentially associated with reduced activity of the microvascular endothelial l-arginine/NO pathway due to tonic activation of A2BAR. However, we have recently reported that adenosine also causes vasodilation of human chorionic stem villi vein rings via a mechanism that require endothelium-derived NO [85]. Thus, adenosine is a vasodilator at the microcirculation of the human placenta from normal pregnancies (Figure 5). Since NO synthesis in human fetoplacental endothelium seems to require l-arginine uptake, it is likely that adenosine vasodilation also involved a likely increase in the l-arginine/NO pathway in cells from normal pregnancies.

R. Bellomo has received consultancy fees from Gambro Pty Ltd & Baxter Pty Ltd, for consultation regarding acute dialysis and fluid market. An Honorarium has been provided by B. Braun Pty Ltd for consultation regarding fluid management, Gambro Pty Ltd additionally paid for R. Bellomo travel to a dialysis meeting. V. D’Intini received financial support from Servier to attend the DNT Workshop in Alice Springs in March 2013. Z. Endre has received an Honorarium from Novartis Transplant Advisory Board (2012, 2013), financial support for travel from Alere (2010) and Novartis (2011) and Accommodation

Amgen (2013). Research funding has been provided by Alere, Argutis, Abbot (2007–2010) for provision of assay kits. M.P. Gallagher received an honorarium from Amgen and Abbvie, Amgen also sponsor a research fellowship at The George Institute. S. McGuinness received financial support from Fresenius for CHEST MK-2206 mouse study and financial support from Baxter for the SPLIT and Supplement PN studies. B.B. Hickey has no financial affiliations that would cause a conflict of interest according to the conflict of interest statement set down by KHA-CARI. R.K.S. Phoon has received consultancy fees from Baxter (2011, 2012), Janssen (2012), Novartis (2011, 2012, 2013), and Sanofi (2012). Financial support has been provided for R Phoon for travel and conference registration from Novartis (2013), Amgen (2013) and Roche (2012).

Research funding has been provided to R Phoon by Amgen in 2012. K. Salamon has no financial affiliations that would cause a conflict of interest according to the conflict of interest statement set down by KHA-CARI. J. PI3K inhibitor Woods has no financial affiliations that would cause a conflict of interest according to the conflict of interest statement set

down by KHA-CARI. The evidence and recommendations in this KHA-CARI guideline have been evaluated and graded following the approach detailed by the GRADE working group (http://www.gradeworkinggroup.org). A description of the grades and levels assigned to recommendations is provided in Tables A1 Liothyronine Sodium and A2. For a full text version of the guideline, readers need to go to the KHA-CARI website (http://www.cari.org.au). “
“We aimed to examine the association between preoperative use of statins and postoperative acute kidney injury (AKI) in patients undergoing major surgery by performing a systemic review and meta-analysis. MEDLINE and EMBASE, from inception to April 2013, and the reference lists of related articles were searched for relevant studies. Trials comparing preoperative statin therapy with no preoperative statin in patients undergoing major surgery were included. Outcome measures of interest were the risk of cumulative postoperative AKI and postoperative AKI requiring renal replacement therapy (RRT). Fixed or random effect meta-analysis was performed to derive summary effect estimates.

We suggest that the individual’s wishes and comorbidities when considering referral, be taken into account (2D). *It is important to note that intra-individual variation in eGFR readings can be as high as 15–20% between consecutive eGFR measurements, such that a number of readings are required before one can be confident that a decrease in eGFR of >5 ml/min per 1.73 m2 in 6 months is real. Chronic kidney disease is associated with considerable morbidity and increased mortality risk. Biochemical evidence of CKD (reduced estimated GFR, elevated serum creatinine) usually indicates the presence of tubulointerstitial fibrosis within BGJ398 the kidney. Such pathology is irreversible, therefore the aim of

treatment in many patients with CKD is to delay progression of disease rather than achieve a cure. In light of this it is clear that implementation of primary prevention measures to avoid development of CKD is a preferable strategy. While much information is available about risk factors for development of CKD (refer to Early CKD CARI Guideline Part I) it is less clear whether risk factor modification

prevents development of CKD. In addition to primary prevention strategies, the needs of patients and their families to access MAPK Inhibitor Library purchase CKD education and information tailored to the stage and cause of CKD, has been highlighted by some studies. White et al.[25] conducted a cross sectional survey of participants of the AusDiab study to assess the level of awareness of the causes of kidney disease. The results indicated an overall low level of awareness of risk factors for kidney Alectinib concentration disease and low level of recall of kidney function testing even among subgroups of the

cohort who were at greatest risk of CKD.[25] A study by Ormandy et al.[26] found that CKD patients had clear information needs, which changed according to their CKD stage. Moreover, Nunes et al.[27] reported disparity between perceived knowledge and objective knowledge in patients with CKD. Although information is crucial to knowledgeable decision-making by patients, how it is provided is also very important. Successful contemporary educational interventions for people with a chronic disease typically incorporate psychological methods to empower patients and change behaviour.[28] The aim of this guideline was to evaluate currently available clinical evidence of interventions relevant to lifestyle modification, patient education, elevated blood pressure, diabetes mellitus, referral to multidisciplinary care and the effect of pregnancy in the primary prevention of CKD. In this guideline prevention of CKD is defined as a normal serum creatinine, eGFR above 60 mL/min and absence of urinary albumin, protein or haematuria. a. We suggest the maintenance of a stable (within 5%), healthy weight as it is associated with a lower risk of developing CKD (2C) c.

3c), suggesting that lymphoid cells are involved in the increase in this population during infection with P. yoelii. Because lymphoid cells were required for the accumulation of MHC II+CD11c−CD3−CD19−IgM− cells during infection with P. yoelii, the following two possibilities

Selleckchem Ibrutinib were considered: (1) these cells were derived from the lymphoid lineage; or (2) they were of myeloid lineage and became MHC II+CD11c−IgM− cells under the influence of lymphocytes during infection. To examine these possibilities, Rag-2−/− mice (CD45.2+) were adoptively infused with splenocytes, which contain lymphoid cells, from B6.Ly5.1 (CD45.1+) mice. These mice were maintained for 3 weeks to allow homeostatic proliferation of the donor cells and were then infected with P. yoelii [24]. Eight days post-infection, accumulation of MHC II+CD11c−CD3−CD19−IgM− cells was

separately examined in CD45.1+ and CD45.1− populations (Fig. 4). The number of MHC II+CD11c−CD3−CD19−IgM− cells did not significantly increase in the donor CD45.1+ population; however, the number in the host CD45.2+ population did significantly increase, suggesting that the majority of MHC II+CD11c−CD3−CD19−IgM− cells that are derived from the myeloid lineage accumulate in the spleens of P. yoelii-infected mice mainly have a non-lymphoid lineage. Thus, it was concluded that MHC II+CD11c−CD3−CD19−IgM− cells that are derived from the myeloid SCH772984 mw lineage accumulate in the spleens of P. yoelii-infected mice under the influence FER of lymphocytes. The functional capacities of MHC-II+CD11c− non-lymphoid cells that accumulate in the spleen as a defense mechanism against P. yoelii infection were examined. First, purified populations of MHC II+CD11c−CD3−CD19−IgM− cells

were incubated with iRBCs and production of TNF-α, IL-6 and IL-12 evaluated (Fig. 5). Conventional DCs from uninfected mice were used as positive controls. In response to iRBC, MHC II+CD11c−CD3−CD19−IgM− cells from infected mice produced TNF-α and IL-6, but not IL-12. Production of IL-10 was undetectable (data not shown). Second, the ability of these cells to present antigens to CD4+ T cells was evaluated by using OT-II OVA-specific TCR transgenic mice (Fig. 6). OT-II mice were immunized with OVA to enrich memory/effector type OT-II cells that are sensitive to the antigen presentation of OVA. MHC II+ subpopulations isolated from the spleens of infected and uninfected mice were pulsed with OVA323–339 or OVA and cocultured with OT-II cells. OT-II cell proliferation was assessed on the basis of diminution in CFSE and the amount of IL-2 production, which was determined by ELISA. MHC II+CD11chi DCs from both uninfected and infected mice efficiently stimulated proliferation of, and IL-2 production by, OT-II cells.

This is not what we observed. In contrast, the absence of the proximal promoter did not decrease circulating sST2 concentrations, either in naïve or allergen-challenged mice. Although the cellular source of sST2 in the blood is still not known, these findings suggest fibroblasts are not a major source under the conditions tested. It remains possible, however, that fibroblasts and/or the proximal promoter and enhancer are important for sST2 induction in

other physiological settings; this is something future studies with these mice may help reveal. In the course of these experiments we also found that fibroblasts use the proximal promoter to express ST2L and are functionally responsive to IL-33, as demonstrated by the Tanespimycin mw gene induction of the neutrophil-attracting CXCL1 and other chemokines. Examination of these mice in models of fibrosis could therefore be informative due to the central role of fibroblasts and recent evidence implicating IL-33 in fibrotic disease [21]. Finally, we hypothesize that there are other nonimmune cell types that require the proximal promoter for ST2L expression and that these mice may thus be useful for examining tissue-specific IL-33 responses in vivo. A targeting vector was

constructed to delete a region in the ST2 locus beginning 4490 bp upstream of the +1 initiation site (ACGTGGGT) in exon 1b and ending at the 3′ end of exon 1b (83 bp downstream from the +1 site), as illustrated in Fig. 1A. The Dabrafenib nmr targeting construct was electroporated into 129×C57Bl/6 F1 hybrid ES cells and clones were then transfected with a CRE recombinase-expressing plasmid to delete the Neo cassette prior to injecting for germline transmission in C57Bl/6 mice using standard conditions. For splenocytes, spleens were minced

and single cell suspensions were collected through a nylon mesh. RBCs were lysed and cells were cultured for 3 h in RPMI with 10% FBS prior to RNA isolation. For mast cells, bone marrow cells were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, IL-3 (5 ng/mL, Amgen), and SCF (100 ng/mL, ADAM7 Amgen) at approximately 2–5 × 105 cells/mL. Every 3–4 days nonadherent cells were transferred to new flasks. Flow cytometry was performed after 5 weeks using antibodies to ST2 (MD Bioproducts, clone DJ8) and c-kit (CD117, BD Pharmingen, clone 2B8). BMMCs were cultured overnight at 105 cells/well with or without IL-33 (Amgen) and IL-6 was measured in the supernatant by ELISA (R&D Systems). For fibroblasts, deboned tails from 12-week-old euthanized mice were minced in HBSS followed by digestion in a 1:1 solution of collagenase (Type XI-S Sigma in HBSS; 2000 U/mL) at 37°C for 30 min, and then 0.05% trypsin at 37°C for 20 min, followed by quenching (DMEM + 15% heat-inactivated calf serum). Cells were cultured in 10 cm plates for 5–7 days.

Both types of monocytes are F4/80+ Cabozantinib supplier and CD86− 6. Data are accumulating on the presence of local tissue precursors for DCs and macrophages and the contribution of these precursors to DC and macrophage accumulation under pathological conditions. In organs, such as the skin and brain, local precursors for macrophages and Langerhans cells have been detected 9–11. We earlier described the presence of local precursors for macrophages in the fetal pancreas

of C57BL/6 mice 12. However, little is known about the origin of the DCs that accumulate in the pre-diabetic NOD pancreas and the factors driving this accumulation. It is generally assumed that these cells are inflammatory in nature and infiltrate from the circulation. However, previous studies from our group suggest that the early accumulation of DCs in the pre-diabetic NOD pancreas cannot only be explained by a massive influx of DCs and DC precursors from the blood. First, pro-inflammatory chemokines that normally attract monocytic cells (CCL2 and find more CCL3) could not be detected in the pancreas at the time of DC accumulation 13. Second, DCs and monocytes of NOD mice have an impaired migration towards pro-inflammatory chemokines in vivo and in vitro 13, although the contribution of other chemokines cannot be excluded. Finally, the depletion of phagocytic

cells with clodronate resulted in a late re-appearance of DCs in the NOD pancreas (28 days after depletion), while monocytes and DCs had already re-appeared in the blood and spleen 4 days after depletion. This late re-appearance suggests that pancreatic

DCs are not only replenished from the circulation 14. We therefore hypothesized that local precursors for DCs are present in the pancreas and that an enhanced proliferation and differentiation of these cells is responsible for the enhanced accumulation of pancreatic DCs initiating the islet autoimmune reaction. In this study, the presence of local pancreatic precursors for DCs, their proliferative capacity and the actual generation of DCs from these pancreatic precursors was investigated in the fetal pancreas and the pre-diabetic pancreas of NOD and control mice. The presence of precursors for DCs in the fetal pancreas was studied using the myeloid progenitor marker ID-8 ER-MP58. ER-MP58 has previously been described by our laboratory as a marker for all myeloid progenitor cells in BM 15. A double staining with ER-MP58 and insulin was performed on the E15.5 pancreas of C57BL/6 and NOD/LTj mice using immunofluorescence (Fig. 1). The results showed that ER-MP58+ cells were present in and around the insulin positive islets of Langerhans in the E15.5 pancreas. To investigate the phenotype of this myeloid precursor in the pancreas a FACS staining was performed on fetal pancreas cells and compared with blood monocytes (4 weeks) from C57BL/6 and NOD/LTj mice.

However, it is also being shown that the recovered immune function in these natural revertants might be very variable, suggesting that the effects of ERT might be unique to each patient. In this report, we describe the molecular and immunologic abnormalities associated with ADA deficiency in a child Galunisertib mw diagnosed at the age of 1 month with T-B- SCID, in whom low numbers of PB T lymphocytes were found later at the age of 23 months and became normal by 50 months of age. This was associated initially

with homozygosity for a mutation that later resulted in a mosaic because of a monoallelic reversion of this mutation documented in his T cells. As this child was not eligible for HSCT or GT, he was placed on ERT, and we describe the molecular and immunologic changes due to partial immune reconstitution and the clinical outcome after 17 months of ERT. Patient and control subjects.  Our patient was a boy diagnosed with ADA-SCID at the Primary Immunodeficiencies Clinic in the University of Antioquia in Medellin (Colombia), that we followed until the age 67 months. https://www.selleckchem.com/products/Lapatinib-Ditosylate.html Written informed consent approved by the IRB at the University of Antioquia was obtained from both parents and healthy age- and sex-matched controls. Immunophenotyping of peripheral blood lymphocytes.  Peripheral blood lymphocytes (PBL) from EDTA

whole blood were stained with different combinations of fluorochrome-conjugated monoclonal antibodies against CD3, CD4, CD8, CD19, CD21, CD27, IgD, CD16, CD56, TCRαβ, TCRγδ, CD45RA and CD45RO (eBioscience

Inc, San Diego, CA, USA and BD Biosciences, San Jose, CA, USA) for 30 min at room temperature, followed by treatment with lysing solution (BD FACS Lysing Solution®; BD Biosciences) for 10 min to remove RBC. After this, the cells were washed twice in PBS (Dulbecco’s phosphate-buffered saline; Sigma Aldrich, Saint Louis, MO, USA), fixed in 200 μl of 2% formaldehyde and read on a FACScan Flow Cytometer equipped with a 388-nm laser (Becton Dickinson, San Jose, CA, USA). Files were analysed using the software FlowJo v8.2 (TreeStar Inc, Ashland, OR, USA), and the results were compared with the controls as indicated [15]. Mutation analysis.  Genomic DNA from the patient HSP90 and controls was extracted from whole blood, PBL and buccal epithelial cells as well as from negatively enriched CD3+ T cells using a DNA Purification Kit (Puregene, Gentra Systems, Minneapolis, MN, USA). Primers and PCR conditions used for the amplification of all ADA exons have been described previously [5, 16]. The nucleotide sequences were determined using the genetic analyzer ABI-PRISM 3100 (AB Applied Biosystems, Foster City, CA, USA) and analysed using the Sequencher software v. 4.8 (Gene Codes Corporation, MI, USA). ADA activity and adenine nucleotide content in RBC.