DNMT3A and DMMT3B are responsible for de-novo methylation and modification of unmethylated DNA, whereas DNMT1 is required to maintain DNA methylation [9,10]. Previous studies have shown that mRNA levels of DNMT1 and DNMT3A are reduced in patients with atopic dermatitis [11],

and that DNMT1 mRNA levels were also decreased in patients with systemic lupus erythematosus (SLE) [12]. There are several noteworthy polymorphisms in the genes encoding enzymes. It has been reported that the A-allele of the DNMT1+14395A/G polymorphism (rs16999593) is present more frequently in patients with infiltrating ductal breast carcinoma than among controls [13]. The DNMT1+32204A/G (rs2228612) polymorphism A-769662 cost is a non-synonymous substitution in which the frequency of the minor allele is 5% in the Japanese population, according to the National Center for Biotechnology Information (NCBI)-SNP (http://www.ncbi.nlm.nih.gov/snp/) and Japanese (J)-SNP databases (http://snp.ims.u-tokyo.ac.jp/). The A-allele of the DNMT3A−448A/G (rs1550157) polymorphism showed significantly

higher promoter activity (>twofold) compared to the G-allele [14]. Carriers of the T-allele of the DNMT3B−283T/C polymorphism (rs6087990) showed significantly lower promoter activity compared to carriers of Roscovitine order the C-allele [15]. However, unambiguous genotyping of the DNMT3B −283T/C polymorphism by restriction fragment length polymorphism (RFLP) analysis is complex. Therefore, we examined The DNMT3B−579G/T polymorphism (rs1569686), which is in linkage

disequilibrium Orotidine 5′-phosphate decarboxylase (LD) with the −283T/C polymorphism. The two common haplotypes formed by these SNPs, −283T/−579G and −283C/−579T, account for 98% of the chromosome [15]. Methylenetetrahydrofolate reductase (MTHFR), which is involved in the supply of the methylation group, is an enzyme necessary for the folate metabolic pathway (Fig. 1) and is considered to result in hypermethylation of genomic DNA [16,17]. The MTHFR+677C/T polymorphism (rs1801133) results in an alanine (C)-to-valine (T) substitution and renders the enzyme less active [18,19]. The MTHFR+1298A/C polymorphism (rs1801131) results in a glutamic acid (A)-to-alanine (C) substitution and the CC genotype of the SNP results in a significant decrease of MTHFR activity [20]. Methionine synthase reductase (MTRR) plays a crucial role in maintaining the active state of methionine synthase (MTR), which is associated with an increase in the DNA methylation level [21,22]. Because the minor allele frequency of a functional polymorphism in the MTR gene (rs1805087) [23] was less than 5% in the Japanese population, we focused on another polymorphism in the MTRR gene as an alternative candidate. The most common polymorphism in the MTRR gene is the +66A/G polymorphism (rs1801394), which results in an isoleucine (A)-to-methionine (G) substitution at position 22; its minor allele frequency in the Japanese population is 30%.

Trophoblast and endothelial co-expression of Slit/Robo implies an autocrine/paracrine regulatory system for the regulation of placental trophoblast and endothelial cell function.

It is likely Palbociclib mw that the other neuronal guidance systems may also have a role in placental angiogenesis although whether they are expressed in the placenta is not known. Global and placenta-specific gene “knock-out” animal studies have provided informative evidence as to the relative significance of a large number of genes (reviewed in [118, 103]) in placental development and function based on embryonic lethality owing to the severity of the placental defects in the homozygous mutant mice. Surprisingly, reduced vasculature in the labyrinth generally occurs in mouse mutants of only a few genes, including the extracellular matrix protein Cyr61 [85] and the Notch-signaling components Dll4 [30], Notch1/4 [65], Hey1/2 [38], and Rbpsuh [64]. Of note, these genes are expressed in the vasculature itself and their mutations lead to a poorly vascularized allantois where the placental vasculature stems from during mouse embryogenesis Lorlatinib [25]. Nonetheless, these studies implicate

that these genes, especially these encoding the Notch-signaling components, are of significant importance for placental vasculogenesis. Genetic studies also have provided convincing data showing that disruption of several transcription factors results in impaired placental angiogenesis although the downstream target genes are incompletely understood. For example, targeted inactivation of Fra1 (a member of the activator protein-1 transcription factors) [57] results in fetal death between E10.0 and E10.5 owing to defects in extra-embryonic

tissues in mouse. The placental labyrinthine layer is reduced in size and largely avascular, owing to a marked decrease in the number of VEGFR1-positive vascular endothelial cells, without affecting the spongiotrophoblast layer. The mutant fetuses are severely growth restricted possibly due to yolk-sac defects. Importantly, when the placental defect is rescued by injection of Fra1−/− embryonic stem cells into tetraploid wild-type blastocysts, the pups obtained are no longer growth retarded and survived up to two days after birth without apparent phenotypic defects. These Tolmetin results suggest that Fra1 plays a crucial role in establishing normal vascularization of the placenta, which is crucial for fetal development and survival [105]. PPARγ is another critical transcription factor that regulates placental vascular development. PPARγ belongs to a family of ligand-activated transcription factors of the nuclear hormone receptor superfamily, which mainly regulate the expression of genes involved in lipid and energy metabolism [116]. It is highly expressed in the trophoblast cells of the rodent labyrinth and in the cytotrophoblasts and syncytiotrophoblasts in human placentas [42], which is increased at late gestation [89].

(reviewed in ref. 35) It is therefore possible that IL-10, produced by a small number of skin-resident Treg cells, mediates potent anti-inflammatory effects by serving to limit the amplification of inflammatory networks. With this in mind it is

therefore tempting to speculate that in our model, IL-10 produced by skin-resident Treg cells, acts to suppress the accumulation and survival of neutrophils at the site of antigenic challenge thereby reducing the overall immunogenicity of the antigen. These findings have implications for vaccine efficacy because they indicate that even partial removal of Treg cells will alter vaccine immunogenicity through limiting the influence of the cells on both innate and adaptive immune responses. This work was supported by an MRC non-clinical

selleck compound senior fellowship (G117/488), an MRC collaboration grant (G0500617) and project grants from the AICR (05-028) and the Wellcome Trust (067046). The authors declare that there are no conflicts of interest. “
“Membrane microdomains play an important role in the regulation of natural killer (NK) cell activities. These cholesterol-rich membrane domains are enriched at the activating immunological synapse and several activating NK-cell receptors are known to localize to membrane microdomains upon Selleckchem GPCR Compound Library receptor engagement. In contrast, inhibitory receptors do not localize in these specialized membrane domains. In addition, the functional competence of educated NK cells correlates with a confinement of activating receptors in membrane microdomains. However, the molecular basis for this confinement is unknown. Here we investigate the structural requirements for the recruitment of the human activating NK-cell receptors NKG2D and 2B4 to detergent-resistant membrane fractions in the murine BA/F3 cell line an in the human NK-cell line NKL. This stimulation-dependent recruitment occurred

independently of the intracellular domains of the receptors. However, either interfering with the association between NKG2D and DAP10, or mutating the transmembrane region of 2B4 impacted the recruitment of the receptors to detergent-resistant Nutlin-3 in vitro membrane fractions and modulated the function of 2B4 in NK cells. Our data suggest a potential interaction between the transmembrane region of NK-cell receptors and membrane lipids as a molecular mechanism involved in determining the membrane confinement of activating NK-cell receptors. This article is protected by copyright. All rights reserved “
“Immunoinflammatory-mediated demyelination, the main pathological feature of multiple sclerosis (MS), is regularly accompanied by neurodegenerative processes, mostly in the form of axonal degeneration, which could be initiated by glutamate excitotoxicity. In the current study, the relationship between Th17-mediated inflammatory and excitotoxic events was investigated during an active phase of MS.

Conversely, two Syk ligands were approximately twofold enriched with the S297A mutant, i.e. Igβ and ubiquitin. Hence, our “reverse proteome approach” directly confirmed the critical role of the major Syk phosphorylation site for 14-3-3 binding and indicated that this complex inhibits BCR recruitment and ubiquitinylation of Syk. Reduced BCR recruitment is likely to attenuate Syk function while ubiquitinylation of Syk

has been associated with its increased degradation 8, Selleck ABT263 9. We tested the functional impact of 14-3-3γ for Syk-mediated activation of the Ca2+ mobilization pathway. Importantly, all subsequently described studies were conducted with batches Selleckchem LDK378 of retrovirally transduced B cells expressing identical amounts of WT or mutant Syk (Fig.

4A, right panel). Hence, we could exclude that conclusions are based on individual responses of single cell clones produced and selected by conventional transfection methods. We immunoprecipitated the proximal Syk substrate SLP65 from resting and BCR-activated B cells expressing either WT Syk or its S297A variant, and subjected the obtained proteins to anti-phosphotyrosine immunoblot analysis (Fig. 4A, upper left panel). SLP65 purified from S297A-expressing cells showed strongly enhanced and prolonged phosphorylation compared to SLP65 obtained from cells expressing WT Syk. Similarly, PLC-γ2 that was co-immunoprecipitated with SLP65 and also acts as important Syk substrate exhibited increased and sustained tyrosine phosphorylation in the absence Protein kinase N1 of the Syk/14-3-3γ complex (Fig. 4B, upper left panel). The latter finding was directly demonstrated by anti-phosphotyrosine immunoblotting of anti-PLC-γ2 precipitates (Fig. 4B). Equal loading of purified proteins was confirmed by reprobing the blots with antibodies to SLP65 or PLC-γ2, respectively (Fig. 4A and B, lower panels). Hence, loss of 14-3-3γ binding promotes phosphorylation of Syk substrates. Flow cytometric recording

of BCR-induced Ca2+ responses demonstrated that this effect translated into dramatically prolonged Ca2+ fluxing (Fig. 4C). Interestingly, the maximal Ca2+ peaks of WT and mutant B cells were almost identical. We conclude that 14-3-3γ binding to phospho-S297 of Syk serves as negative feedback regulation that limits the activation of BCR-proximal signaling events. Next, we assessed how 14-3-3γ inhibits Syk function. Two main mechanisms control Syk activation and interaction of Syk with downstream targets. Doubly phosphorylated ITAMs in Igα and Igβ recruit Syk to the plasma membrane and concomitantly provide an allosteric trigger for its catalytic activity. The latter is further amplified by auto- and trans-phosphorylation on activatory tyrosine residues 6.

32 The majority of studies reviewed use this method to determine vitamin B6 status, with the exception Dabrafenib price of Mydlik and Descombes who use erythrocyte activity. This method has been criticized by some because of the shortened life span of red cells in chronic renal failure and the higher activities of some enzymes in younger erythrocytes.33 Some data, however, suggest that erythrocyte glutamic-oxaloacetic transaminase levels are more reliable than plasma or serum.9 Other information suggests pyridoxal may be a more reliable indicator of vitamin B6 metabolism as inorganic phosphate and alkaline phosphatase may interfere with plasma PLP measurements.34 While there is conflict, plasma

PLP is probably more readily available as a therapeutic guide.3 Differences in reference ranges for the classification of vitamin B6 status can, however, further cloud the picture of deficiency. While this review focuses on measures of vitamin status, dietary intake of vitamins has previously been shown to be low in the haemodialysis population.35 This is especially true of vitamin B6. While nutrient reference DNA Damage inhibitor values (NRV) have been determined from depletion/repletion studies, and are set for the Australian population at 1.5–1.7 mg/day,36 a recent US population-based study showed that vitamin B6 intakes between 3 and 4.9 mg/day would leave at risk

groups with inadequate vitamin status.32 US nutrition intake information in the haemodialysis population has shown that the mean intake is far less than these lower end recommendations, at 1.21 ± 0.39 mg/day.37 Australian data for the same population indicates intake levels are less again; 1.0 ± 0.3 mg/day in men and 0.6 ± 0.3 mg/day in women.38 More recent data show vitamin B6 intakes of 0.9 ± 0.37 mg/day in 67 haemodialysis patients.39 These data show suboptimal intake in this population, which is well below the NRV. In addition, foods high in vitamin B6, such as wheat bran, avocado, banana,

lentils, walnuts, soybean, potatoes, eggs, meat, fish, cheese and milk, are often limited in the haemodialysis population owing to their potassium and phosphate contents. As it is water soluble, Smoothened vitamin B6 is affected by the cooking process, which further diminishes availability.40 More recent nutrient intake data along side accurate dialysate PLP measures would provide further insight into current vitamin B6 status of the haemodialysis population. What does a deficiency in vitamin B6 mean for the haemodialysis population? Vitamin B6 is involved in many vital metabolic functions, and is important for the normal function of multiple organ systems. It is a cofactor for enzymes involved in the synthesis and catabolism of neurotransmitters, homocysteine trans-sulfuration and the metabolism of other amino acids, fats and glycogen. It also modulates the action of hormones and affects immune competence.

Although alternatively activated microglia exert a beneficial role in early disease phase, continuous activation has been implicated as a contributor to neurodegeneration; indeed, microglial activation has been shown to correlate with neuronal degeneration in several neurodegenerative diseases, as demonstrated by positron emission tomography (PET) imaging,[35] which enables monitoring of microglial activation in vivo,[36] and classical Selleck Ganetespib activation of microglia through chronic local infusion of LPS was shown to trigger neurodegeneration

in animal models.[37] In primarily non-inflammatory neurodegenerative diseases, such as Alzheimer’s disease, ALS and Parkinson’s disease among others, misfolded proteins play a crucial role in the pathogenic process[38] and their involvement in microglial activation has been demonstrated in several neurodegenerative diseases. Early activation of microglia was observed in mice transgenic for wild-type α-synuclein, an animal model of Parkinson’s disease[39, 40] and in vitro and in vivo studies have suggested that transgenic expression of mutant superoxide dismutase 1 in models of ALS results in activated microglial phenotypes that are inherently

neurotoxic.[26] The importance of the role of glial cells in ALS www.selleckchem.com/products/Dasatinib.html was demonstrated in the animal model whereby conditional transgenic mice with simultaneous over-expression of mutant superoxide dismutase 1 in both neurons and microglia developed motor neuron degeneration,[41] whereas selective motoneuronal expression was not pathogenic.[42] Release of misfolded protein Casein kinase 1 from damaged neurons is a possible trigger for microglia activation. Among non-mutually exclusive mechanisms that implicate release of misfolded protein by neurons in microglial activation in neurodegenerative diseases, a possible common mode of action has been postulated in Alzheimer’s disease and Parkinson’s

disease whereby binding to the scavenger receptor CD36 mediates microglial inflammatory response to fibrillar amyloid β[43] and α-synuclein,[39, 44] respectively. Other studies suggest another pathway triggering microglial inflammatory response to α-synuclein through binding to Mac-1 receptors, thereby signalling to activate reactive oxygen species production by NADPH oxidase.[45] Signalling through TLR4 might also represent a common pathway for microglia activation to neurotoxic phenotype in Alzheimer’s disease and ALS. Mutant superoxide dismutase 1, which is released from neurons and astrocytes through interaction with the neurosecretory proteins, chromogranin A and B,[46] binds to the microglial pattern recognition receptor, CD14, signalling in conjunction with TLR2 and TLR4 to induce in vitro morphological and functional activation changes in microglia that lead to neurotoxicity through release of nitric oxide and superoxide.

We investigated Galunisertib the effect of parameters of classical indication for CRRT on mortality in patients on continuous renal replacement (CRRT) therapy. Methods: We prospectively and consecutively enrolled a total of 519 patients who stared renal replacement therapy. Results: Mean age was 63.4 ± 14.5 years old, and men were 59.5%

in all enrolled patients. Causes of acute kidney injury (AKI) were septic (46.4%), ischemic (19.5%), post-operation (9.1%), and nephrotoxic (6.2%) AKI. Level of pH (hazard ratio (HR) 1.403, 95% confidence interval (CI) 1.181–4.774, 7.20 < pH ≤ 7.25; OR 3.520, 95% CI 1.330–9.316, 7.15 < pH ≤ 7.20; HR 4.315, 95% CI 1.649–11.286, pH ≤ 7.15; P-for-trend 0.001, reference pH > 7.3), weight gain over 2 kg (HR 2.501, 95% CI 1.552–4.032), urine output (HR 2.190, 95% CI 1.408–3.406, urine output ≤ 0.3 ml/min/kg), and phosphorus level (HR 2.136, 95% CI 1.199–3.805, 5.5 < P ≤ 6.5; HR 4.737, 95% CI 2.613–8.590; P-for-trend < 0.001, reference P < 5.5). However, serum creatinine level (HR 0.892, 95% CI 0.824–0.966)

and increased amount of serum creatinine level (HR 1.083, 95% CI 0.930–1.260) were not associated with in-hospital mortality. Diagnostic values of composite of these factors (pH, weight gain, urine output, and phosphorus levels) (area under Lapatinib price the curve (AUC) 0.7145, 95% CI 0.656–0.771) was higher than serum creatinine level (AUC 0.449, 95% CI 0.382–0.517), GFR (AUC 0.553, 95% CI 0.485–0.62), and AKIN stage (AUC 0.589, 95% CI 0.521–0.657). Conclusion: These data may suggest that classical indication should be considered for the optimal timing for initiation of CRRT in critically ill patients. HATTORI YUKA1, KIM HANGSOO2, TSUBOI NAOTAKE2, YAMAMOTO AKIHITO1, UEDA MINORU1, MATSUO SEIICHI2, MARUYAMA SHOICHI2 1Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine; 2Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine Introduction: Acute kidney injury (AKI) is a critical condition which is

associated with high mortality rates of 30 to 50%. Ischemia-reperfusion injury (IRI) is a major cause of AKI. However, available treatments for AKI are limited. Preclinical studies indicate that administered MSCs ameliorate HSP90 renal injury and accelerate kidney repair. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are medical waste, have received attention as a novel stem cell source. The purpose of this study is to clarify whether SHED have therapeutic effect on AKI induced by IRI. Methods: SHED were isolated from human exfoliated deciduous teeth as described previously. For all experiments 7- 8-wk-old male C57BL/6 mice weighing 18–22 g were used. Under anesthesia mice were subjected to right heminephrectomy.

Mutations within a viral genome often confer advantages in vivo, the evolution of which is driven strongly by immune selection pressures. Immune control of the virus before it is able

to mutate is therefore crucial in determining long-term outcome to infection (see Fig. 5). Selleckchem Fludarabine In HIV and simian immunodeficiency virus (SIV), viral escape mutations within immunodominant epitopes play a critical role in early and late loss of immune control [50–52] and this is also shown to influence long-term outcome in acute HCV infection [53,54]. There is a variation in the degree of escape between different epitopes within the viral genome of such persistent viral infections, where some epitopes are observed to escape while others are often conserved. One explanation which has been proposed for this is that more sensitive T cells are associated with escape (‘driver’ responses), while see more less sensitive cells may be simply ‘passengers’ which have little impact on viral evolution or disease outcome [55]. More sensitive populations are observed to drive viral escape, whereas less sensitive CTLs are associated with epitope stability in both HCV [56] and SIV [57]. In HIV, CTL responses

to the promiscuous epitope TL9-Gag were compared between HLA types within the B7 supertype. B*8101-restricted TL9-Gag responses were found to be of significantly higher functional sensitivity than those restricted by B*4201. Higher TL9-Gag sequence variation is observed in B*8101 compared to B*4201-positive

patients [58]. There is a clear conflict of interest in the outcome of better-quality CTL responses. The immune advantages of improved clearance of the more sensitive responses would appear to be balanced against the disadvantage of driving evolution of the virus in its ability to escape the host immune response. However, viral fitness costs associated with the acquisition of escape mutations may contribute to the protective nature of some HLA class I alleles, such as B57 [3]. CTL dysfunction is seen in a number Sodium butyrate of chronic viral infections in humans [59,60] and animal models [61,62]. The genesis of such dysfunction is not well understood, but is thought to be related to repetitive triggering through the TCR. One possible outcome is that more sensitive cells might become preferentially over-stimulated and anergic in the presence of high antigen load. This is supported by in vivo studies showing the persistence of anergic CTLs with high functional sensitivity under such conditions [63,64]. The distinct sensitivities observed in cells of the acute and chronic phase of HIV-1 appears to be a consequence of deletion of the more sensitive cells, as determined by clonotypic analysis of TCR VB chains by polymerase chain reaction (PCR).

Apart from recognition

of triphosphate group of ATP, arginine fingers may be responsible for displacement of water out from the binding site. Such a role of arginine fingers was recently demonstrated for the Ras–RasGAP complex in the QM/MM calculations (Heesen et al., 2007). A more detailed analysis of JEV NS3 helicase/NTPase structure may lead to the conclusion that to function as a catalytic base, the pKa of Glu286 would need to be much higher than that of a typical glutamic acid residue in a protein, as PF-01367338 concentration suggested for HCV helicase (Frick, 2007). It was thus proposed that the neighboring aspartic acid residue (Asp285 in JEV NS3 helicase/NTPase) may serve as a catalytic base instead. Docking of known JEV NS3 helicase/NTPase inhibitors 1–2 revealed engagement of crucial binding pocket residues in the interactions

with ligands. In particular, the role of Glu286 and Arg464 was clearly depicted. Moreover, docking of 1–2 allowed the identification of Arg202 as an additional important residue of the binding pocket, making this arginine a straightforward candidate for mutational studies. The analysis of ATP–enzyme complex allowed speculation about the role of conserved threonine Thr201. Most probably, it directs the ligand properly toward interactions with Lys200 and the conserved arginine residues. A similar role may be assigned to the branched side chains of apolar amino acids (especially Val227 and Ile411), which was demonstrated in the case of 2 and was suggested www.selleckchem.com/products/Liproxstatin-1.html earlier for ionotropic glutamate receptors (Kaczor et al., 2008). Docking of 1–2 indicated Asn417 as an additional

anchoring point, whereas docking of identified hits 8–22 also indicated Glu231 as a potentially important residue for interactions with inhibitors. Virtual screening procedure made it possible to identify 15 potential inhibitors of JEV NS3 helicase/NTPase. Only one of them, namely the one containing pentose moiety 14, may be treated as a far analog of nucleosides. This structural diversity may prove beneficial because it increases the likelihood that the new inhibitors will be selective toward human ATPases. This is a significant problem: CYTH4 it is worth emphasizing that ring-expanded nucleosides 1 and 2 also have high affinity to human Suv3 mitochondrial helicase (routinely used to test the selectivity of novel inhibitors of viral helicase/NTPase), which excludes them as drug candidates (Zhang et al., 2003). On the other hand, compounds 1 and 2 were also active toward all the tested viral helicase/NTPases: WNV and HCV. This seems promising, as the research on specific anti-JEV compounds may lead to the development of a drug with broad antiviral spectrum of activity.

OS is invariably fatal within the first months of life unless immune restoration is performed by haematopoietic stem cell transplantation (HSCT). Abnormal autoreactive T cells may infiltrate and expand BI 6727 order into different organs (e.g. skin, gut, liver and spleen) and cause significant tissue damage [3]. Poor clinical status before the HSCT results in high transplantation-related mortality [4]. In the past, interferon (IFN) gamma was used to counteract the predominance of T cell activation and proliferation,

to down-regulate interleukin (IL)-4 and IL-5 production, to modulate the inflammatory reaction by enhancing phagocytic functions and to improve clinical status [5]. Today, topical/systemic steroids or cyclosporin A (CsA) are the widely used medications to control the skin manifestations [6]. CsA, a known calcineurin inhibitor, seems to act on the IL-2 by inhibiting its production and

repressing the activity of various transcription factors, thus leading to a decrease in the proliferation of the activated lymphocyte [7,8]. Moreover, it may interfere with specific signal transduction pathways which are important to the hypertrophic response [9]. Little is known about the immune modifications induced by CsA in OS patients. Such information will further improve our understanding the pathophysiology underlying OS and mechanisms of potential treatment modalities. Here we describe two OS patients selleck kinase inhibitor and their clinical and immune response to CsA. Two patients with recombinase activating gene (RAG)2 deficiency SCID and clinical and immunological features suggestive of the diagnosis of OS phenotype were reported. Significant transplacentally acquired maternal T lymphocyte was excluded in both patients by fluorescence in-situ hybridization (FISH). The study was approved by the Institutional Review Board and informed consent was obtained from all participants’ Calpain parents. Cell surface markers of peripheral blood mononuclear cells (PBMCs) and lymphocyte proliferative

responses to mitogens were performed as described previously. The amount of signal joint (sj) T cell receptor excision circles (Trecs) were determined by quantitative real-time reverse transcriptase – polymerase chain reaction (qRT–PCR). Reactions were performed using 0·25–0·5 µg genomic DNA extracted from the patients’ PBMCs. The standard curve was constructed by using serial dilutions of a known Trec plasmid (generously provided by Dr Daniel Douek, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA). The number of Trecs in a given sample was calculated automatically by comparing the obtained Ct value of a patient’s sample to the standard curve using an absolute quantification algorithm.