There may be other possible factors that promote the proliferation of DN Treg cells in combination with IL-15, possibly other cytokines or co-stimulatory molecules that deliver signals to DN Treg cells. This is the subject of ongoing investigations. The function of Treg cells has been described, see more both in vitro and in vivo. It has been proposed that Treg cells function as modulators of autoimmune responses because of their suppressive effect on autoreactive lymphocytes. Furthermore, this suppressive function can be transferred by injecting

Treg cells into autoimmune animal model systems.7 The Treg cells have also been shown to function in many non-autoimmune models such as graft-versus-host disease and allergy.48–51 In contrast, Treg cells can interrupt the activation of effector T cells responding to tumour cells and infectious pathogens.46 However, clinical applications using Treg cell suppressive function have been limited GPCR Compound Library because of the hypoproliferative property and polyclonal nature of Treg cells. In vitro studies using cTreg cells show that only a relatively high ratio of Treg : effector cells can suppress the effector cells (i.e. 5 : 1 to 1 : 1). As a result

of this inefficient in vitro suppression, the therapeutic potential of Treg cells has been critically limited. However, HBeAg-specific DN Treg cells demonstrate superior suppressive effects on effector cells at effector cell : Treg Fossariinae cell ratios as low as 32 : 1 (see Fig. 5). The multiple mechanisms of suppression used by Treg cells is an ongoing subject of research and remains somewhat controversial. The suppressive effects of cTreg cells in vitro have been reported mostly on CD4+ and CD8+ effector cells, but have also been found to act directly on APCs and natural killer cells.52–56 Inhibitory cytokines, IL-10 and transforming growth factor(TGF)-β are

known to be produced by cTreg cells and thought to be a part of the mechanism of Treg cells.57,58 According to our preliminary data in a transwell system, IL-10 and TGF-β are not candidates as the primary mediators of suppression demonstrated by HBeAg-specific DN Treg cells (data not shown). Another report showed that the regulatory function of Treg cells is serine protease granzyme-B (GZ-B)-dependent using GZ-B−/− mice.59 Other suppressive mechanisms have been suggested to function via cell–cell contact. CTLA-4, FAS–FASL, GITR and CD103 have also been suggested to play a role in the function of Treg cells. Recently, the inhibitory function of Treg cells has been demonstrated to be mediated through the exoenzymes CD73/CD39.60–62 Interestingly, a high frequency of HBeAg-specific DN Treg cells are CD73+/CD39+ after activation (Fig. 11). We are investigating whether this pathway may explain the efficient immunoregulation mediated by HBeAg-specific DN Treg cells.

microsporus and L. ramosa revealed growth at 45 °C. Furthermore, both the species of Apophysomyces showed sporulation on 2% water agar plates incubated

at 28 °C after 5–7 days. AFLP profiles of 33 strains of Rhizopus species, comprising R. arrhizus var. delemar (n = 16), R. arrhizus var. arrhizus (n = 12), R microsporus (n = 5) and four reference strains viz., R. microsporus var. chinensis CBS 294.31T, R. microsporus var. tuberosus CBS 113206, R. azygosporus CBS 357.93T and R. arrhizus var. arrhizus CBS 112.07T, revealed bands in a 40–400 bp range. The ALK inhibitor dendrogram derived from the AFLP banding pattern was generated using Pearson algorithm and single linkage cluster analysis (Fig. 3). AFLP analysis of R. arrhizus revealed heterogeneity among the isolates comprising five distinct genotypes including Genotype III and IV, solely representing variety delemar and Genotype V variety arrhizus. On the other hand Genotype I and II showed overlapping of both the varieties. The different genotypes of R. arrhizus were well separated from R. microsporus. Results of in vitro antifungal susceptibility profiles are summarised in Table 4. Over all, AMB was found to be the most potent antifungal agent for all the mucorales tested, showing MICs of ≤1 μg ml−1, with geometric mean MIC of 0.06 μg ml−1. Among the azoles, POS exhibited highest activity (GM MIC, 0.4 μg ml−1). Interestingly, a new azole, ISA (GM MIC, 1.27 μg ml−1),

had less in vitro activity than POS but better activity as VRC. Although POS was the second most potent antifungal against mucorales, 46% isolates had MICs of ≥0.5 μg ml−1 and 7.5% isolates exhibited GW-572016 concentration MICs above ≥2 μg ml−1, which included 2 isolates of R. arrhizus var. delemar, 2 of R. arrhizus var. arrhizus, one isolate each of R. microsporus and Mucor circinelloides. ISA showed limited in vitro activity in 36% (29/80) isolates with MICs >1 μg ml−1. Notably, highest activity was observed for Rhizopus

species of which 62% (37/60) of the isolates had ISA MICs ≤1 μg ml−1. Overall 15% (12/80) of isolates revealed very high MICs of ISA ranging from 8 to 16 μg ml−1 which included four isolates of R. arrhizus var. delemar, 3 of S. racemosum, 2 of L. ramosa Alanine-glyoxylate transaminase and one each of R. microsporus, M. circinelloides and Apophysomyces variabilis. Similarly, ITC also exhibited limited activity with MIC of ≤0.5 μg ml−1 in 45% (36/80) of all the Mucorales tested. FLU, VRC and echinocandins demonstrated no or poor activity. Notably, TERB was active against all the species tested except R. arrhizus (MIC90, 32 μg ml−1). Etest MICs of AMB, revealed a high categorical agreement of 87% with CLSI method (Table 5). On the other hand Etest MICs of POS revealed a low agreement (67%) with CLSI MICs. Etest MICs of POS were observed to be statistically higher than CLSI MICs (P = 0.003). Also, the MICs of POS obtained by Etest showed varied values against all the Mucorales tested.

Obesity may be a greater risk factor for loss of GFR in patients who already have impaired kidney function. This is analogous to the greater impact of hypertension in causing progressive

disease in patients with CKD when compared with those with normal kidney function. There are some data (n = 162) to suggest that obesity promotes more rapid loss of renal function in patients with IgA nephropathy.46 Patients who were overweight had heavier proteinuria at time of biopsy, were more likely to be hypertensive, have more severe tubulointerstitial changes on biopsy and to subsequently develop hypertension and renal impairment. Gestational diabetes: a systematic review47 demonstrated that gestational diabetes is associated with a 17–63% increase in risk of Type 2 diabetes within 5–16 years of pregnancy. The highest risk occurs in the first 5 years after pregnancy and then appears to plateau. BMI > 30 kg/m2 MLN8237 concentration Doxorubicin was identified to further increase risk associated with gestational diabetes in most but not all studies. Renal cell carcinoma (RCC): although RCC only accounts for 2.8% of cancers in Australia (Cancer in

Australia, 2001), it is of particular relevance to potential donors. A systematic review48 of 22 small studies demonstrated an increase in the relative risk of RCC of 1.07 (95% CI: 1.05–1.09) per unit increase in BMI and the risk was equivalent in men and women. Therefore, the relative risk for patients with a BMI of 30 kg/m2 is 1.35. Subsequent large cohort studies have been consistent with this finding49,50 although others have failed to find an Rucaparib association between obesity and RCC in men.51,52 There is a biologically plausible link between obesity and RCC as increasing BMI is associated with elevated levels of fasting serum insulin-like growth factor,53 which has been shown

to increase cellular proliferation in RCC in animal models. Kidney stones: analysis of data from the Nurse’s Health Study I and II and the Health Professionals Follow-up Study54,55 demonstrated that prevalence and incidence of new stone disease was directly associated with BMI, with a stronger relationship evident in women. The age-adjusted prevalence OR for women with a BMI ≥ 32 kg/m2 compared with 21–22.9 kg/m2 was 1.76 (95% CI: 1.50–2.07), and 1.38 (1.51–2.36) for the same analysis in men. For incident stone formation in women, the OR was 1.89 (1.51–2.36) in women, but not significantly different in men. Increases in rates of donor obesity have occurred over the past decade and demonstrate regional variation. In a survey of UK transplant centres published in 1999,56 only one centre was identified as accepting patients with a BMI greater than 30 kg/m2 or a weight greater than 20% above ideal. Results of a survey of US centres, published in 1995, reported that only 16% of centres would exclude a donor with moderate obesity.

The aim of the present study was to evaluate the relationship between LV mass and mild-to-moderate renal dysfunction in a group of non-diabetic hypertensives, free of CV diseases, participating

in the Renal Dysfunction in Hypertension (REDHY) study. Methods:  Patients with diabetes, a body mass index (BMI) of more than 35 kg/m2, secondary hypertension, CV diseases and a glomerular filtration rate (GFR) of Protein Tyrosine Kinase inhibitor less than 30 mL/min per 1.73 m2 were excluded. The final sample included 455 patients, who underwent echocardiographic examination and ambulatory blood pressure monitoring. Results:  There was a significant trend for a stepwise increase in LV mass, indexed by both body surface area (LVMI) and height elevated to 2.7 (LVMH2.7), with the declining renal function, that remained statistically significant after correction for potential confounders. The prevalence of LVH, defined either as LVMI of 125 g/m2 Selleckchem Carfilzomib or more or as LVMH2.7 of 51 g/m2.7 or more, was higher in subjects with lower values of GFR than in those with normal renal function (P < 0.001 in both cases). The multiple regression analysis confirmed that the inverse association between

GFR and LVM was independent of confounding factors. Conclusion:  The present study confirms the high prevalence of LVH in patients with mild or moderate renal dysfunction. In the patients studied (all with a GFR of 30 mL/min per 1.73 m2), the association between LVM and GFR was independent of potential confounders, including 24 h blood pressure load. Taking into account the negative prognostic impact of LVH, further studies focusing on a deeper comprehension of the mechanisms underlying the development of LVH in chronic kidney disease patients are needed. “
“Aim:  To investigate whether urinary angiotensinogen (UAGT) levels are correlated with renal involvement of Henoch-Schonlein purpura (HSP) in children, and to explore whether UAGT has any relation to the severity of HSP. Methods:  The

study sample consisted of 107 patients (50 boys and 57 girls, 6.68 ± 2.41 years) with clinical diagnosis of HSP. A 24 h urine sample was collected before treatment. Demeclocycline UAGT levels were measured in patients with HSP in the acute and convalescent phases by enzyme linked immunosorbent assay. Results:  Urinary angiotensinogen/urinary concentration of creatinine levels were significantly higher in proteinuric HSP in the acute phase and the convalescent phase (32.02 ± 3.95 and 25.31 ± 4.11 µg/g) compared with those with HSP without renal involvement (17.26 ± 2.60 and 15.14 ± 3.81 µg/g) and those with hematuric HSP (19.70 ± 2.21 and 17.28 ± 3.62 µg/g) (P < 0.0001 and P < 0.01, respectively). Using matched urine samples from the same patients, UAGT/urinary concentration of creatinine (UCr) levels of proteinuric HSP patients were significantly lower in the convalescent phase (25.31 ± 4.11 µg/g, P < 0.01) than in the acute phase (32.02 ± 3.95 µg/g).

A total of 46 responses were diagnostic between at least some of the species and are listed in Table 3. Results for identification of P. minutispora and Petriellopsis africana from which only single strains were analysed are only included in the Table if they were remarkable and therefore usable as specific identification markers. Scedosporium prolificans was clearly GSI-IX price distinguishable from remaining species by nine compounds (l-valine, p-aminohippuric acid, adonitol, dulcitol, sedoheptulose, β-d-glucosamine, glycine-tryptophan-βNA and d-alanine-para-naphthylamide

(pNA), bolded values in Table 3). The single P. minutispora isolate was the only strain positive for γ-hydroxybutyrate. l-Asparagine and l-glutamine distinguished P. minutispora and Petriellopsis africana. Additional species-specific reactions were acid production PLX3397 price from sucrose for the differentiation of S. aurantiacum (–) from all other species of the P. boydii complex (+), assimilation of glycine-glycine-βNA, sucrose-phenylanaline-glycine-leucine-βNA, and praline-pNA for differentiation of S. aurantiacum (+) and S. dehoogii (–) as well as assimilation of p-nitrophenyl-β-d-maltoside (pH 7.5) and p-nitrophenyl-β-d-glucopyranoside (pH 5.5) for separation of P. boydii (–) from S. aurantiacum (+). Pseudallescheria apiosperma could be distinguished from the P. boydii complex only by a combination of characters obtained with p-nitrophenyl-α-l-rhamnopyranoside

(pH 7.5), p-nitrophenyl-β-d-maltoside (pH 7.5) and p-nitrophenyl-β-d-glucopyranoside (pH 5.5). Intraspecific variability was present in all species for which more than one strain was analysed. In Table 4, the numbers of species-specific positive,

negative and variable results of each species from which more than one isolate was available for study are listed. The lowest degree of variation regarding all reactions was found in S. aurantiacum (22.5%) and in S. prolificans (27.2%). Variabilities of P. boydii (53.5%), P. see more apiosperma (49.2%) and S. dehoogii (48.4%) were in the same range. Especially in P. apiospermua, large differences were found in the variability of the different Taxa Profile microtitre platforms: 61.3% in Profile A (amino derivates), 25.6% in Profile C (carbohydrates) and 59.8% in Profile E (aminopeptidases, glucosidases, phosphatases) respectively. The environmental strain CBS 467.76 of S. prolificans differed from clinical isolates of this species by positive results for catechol, 3-aminobenzamide, gum xanthan, pectin and negative results for protocatechuate, asparagine-βNA, hypoxanthine-βNA-HCl, glutaminic acid-glutaminic acid-βNA, glutaminic acid-histidine-βNA and histidine-leucine-histidine-βNA. Both algorithms for cluster analysis (SSM and SJ) generated seven robust clusters, with S. prolificans in a remote position. The dendrogram constructed from SSM analysis is presented in Fig. 1.

Monolayers of Madin-Darby canine kidney cells in 12-well plates were incubated with 0.1 mL of the dilutions for 1 h, and the cells were overlaid with 1.5 mL of agar medium. The plates were maintained

in a humidified atmosphere containing 5% CO2 for 2 days, and the plaques in wells were counted. The virus titers of the lungs were expressed as the number of pfu per unit weight of lung. The left lobes of lungs were fixed in 10% neutral buffered formalin solution, sectioned, and stained with hematoxylin and eosin. Histopathological Paclitaxel ic50 scores were established on the basis of the extent of the histopathological findings including hypertrophy, hyperplasia, abruption and necrosis of bronchial epithelium, infiltration of inflammatory cells in bronchial submucosa

and alveolar septa, exudation of inflammatory cells in alveolus, atelectasis, edema, and hemorrhage in the alveolus. Each histopathological finding was scored as follows: 0, normal; 1, mild; 2, moderate; and 3, severe. Histopathological scores were estimated from the average of the extent of these findings. Data are expressed as mean ± SD, and P < 0.05 indicated significant differences as PLX4032 cell line determined by Student’s t-test for comparisons between groups. A total of 85 strains consisting of 57 strains from 16 species of Lactobacillus, 14 strains from 5 species of Bifidobacterium, 8 strains from 2 species of Lactococcus, 4 strains from 2 species of Enterococcus, and 2 strains from 1 species of Streptococcus were examined for their ability to induce IL-12. Murine splenocytes were cultured with heat-killed

bacteria (1 μg mL−1) for 2 days and the levels of IL-12p70 in supernatants were determined Idoxuridine (Fig. 1). Lactobacillus paracasei MoLac-1 most strongly induced IL-12. Heat-killed MoLac-1 induced IL-12p70 and IFN-γ production in a dose-dependent manner between 0.1 and 1 μg mL−1 (Fig. 2). To examine the cell types exhibiting MoLac-1-induced IL-12 production, the IL-12 production by splenocytes depleted of various cell populations was compared with that of complete splenocytes. We prepared splenocytes depleted of CD90.2+ cells (mainly T cells), B220+ cells (mainly B cells), CD11b+ cells, CD11c+ cells (mainly dendritic cells), and DX5+ cells (mainly NK cells and NKT cells). Splenocytes and the depleted splenocytes were cultured with heat-killed MoLac-1 (1 μg mL−1) for 2 days. The secretion levels of IL-12 induced by MoLac-1 were diminished in CD11b− cells but maintained in the other subsets of splenocytes depleted of CD90.2+ cells, B220+ cells, CD11c+ cells, or DX5+ cells (Fig. 3a). CD11b is expressed on macrophages/monocytes, granulocytes, NK cells and subsets of dendritic cells. Using Ly-6G, a marker expressed on granulocytes, we found that Ly-6G− cells produced IL-12 induced by MoLac-1 (Fig. 3b).

40 CDK4 and CDK6 were both induced upon CD3/CD28 costimulation. nIL-2 abrogated the up-regulation of CDK6, and partly inhibited CDK4 induction, while BMS-345541 and PS-1145 suppressed the induction of both kinases. Taken together, these results emphasize that an important effect of IKK activation on CDK4 and CDK6 expression relies on IL-2/IL-2R LY294002 solubility dmso signalling. However, as full CDK4 up-regulation requires the activation of IKK and IL-2 signalling, these data add new information about the mechanisms that govern CDK4 expression in human T cells. CDK2–cyclin E/A complexes are implicated in the

regulation of major processes governing the G1/S transition.5 In our experiments, CDK2 induction was detected in 24-hr costimulated cells, and was preserved in the presence of nIL-2, but abolished by BMS-345541 and PS-1145. We thus Selleck Poziotinib conclude that, in activated T cells, CDK2

induction is independent of IL-2 signalling, and relies instead on IKK activation, which is a novel finding. To acquire catalytic activity, CDK2 must bind to cyclin E (G1/S phase transition) or cyclin A (S phase).5 We found that T-cell stimulation caused a significant increase in cyclin E and cyclin A gene expression. nIL-2 prevented cyclin A up-regulation but did not affect cyclin E, a clear indication that in activated human naïve CD4+ T cells only cyclin A expression is dependent on the IL-2/IL-2R signalling pathway, consistent with previous reports.3 Interestingly, BMS-345541 and PS-1145 prevented the expression not only of cyclin A, but also of cyclin E, providing compelling evidence for involvement of IKK in the regulation of cyclin E expression in human naïve CD4+ T cells. In light of the essential role played by the CDK2/cyclin E complex in initiating DNA replication,5 this finding underscores a critical function of IKK in the regulation of T-cell entry into S phase. Degradation of p27KIP1 by the ubiquitin–proteasome

pathway at the Janus kinase (JAK) G0/G1 transition results in activation of the cyclin E/CDK2 complex, and commitment of cells to S phase.41 In our results, stimulation of human naïve CD4+ T cells resulted in a considerable decrease in p27KIP1 that was prevented by nIL-2, or BMS-345541 or PS-1145. The degradation of p27KIP1 is a complex process that requires the formation of a ternary complex with cyclin D/CDK4, followed by p27KIP1 phosphorylation on Thr187 by cyclin E/CDK2.4 The RING finger-type ubiquitin ligase complex SCFSKP2-CKS1B recognizes phosphorylated p27KIP1 through the C-terminus of two of its subunits, SKP2 and CKS1B, resulting in targeting of p27KIP1 for ubiquitination and degradation.42 SKP2 and CKS1B levels periodically oscillate during the cell cycle: they are low or absent during G0 and early G1 phases, increase in late G1 phase, and peak in S phase, dropping as cells proceed through M and early G1 phases.

Satisfying this requirement would necessitate

a clarification of the relationship between educated APCs and the several Signal 3s (i.e. one APC-one Signal 3 or all Signal 3s), and of what tells them which Signal 3 to transmit. Under the Alarm Model, the role of specificity for the Eliminon is lost. The response must rid the Eliminon, not the host. To argue as an illustrative example of tissue-based control of effector class that privileged sites are protected by tissue-selected effector mechanisms that are ineffective in attacking host components but effective against pathogens (assuming that such a discrimination is possible for an effector mechanism coupled https://www.selleckchem.com/products/PD-0332991.html to an unsorted repertoire) is equivalent to saying that privileged sites are susceptible targets for all categories of pathogen against which the unexpressed effector mechanisms would normally protect. If the privileged site does not provide a physical barrier that excludes the immune system, then its components (epitopes),

in one way or another, must have participated in the sorting of the repertoire (Module 2/Decision 1). In fact, there exists a clear experimental example of this, namely, autoimmunity to an eye protein in the absence of its ectopic expression in thymus in Aire-defective mutants ([51]). This shows that click here the wild-type animal is normally tolerant of a protein said to be in a privileged site. The question of the relationship between Pyruvate dehydrogenase lipoamide kinase isozyme 1 ‘healthy tissue’ and the immune system needs consideration. Whatever evidence we have tells us that the immune effector mechanisms are as lethal for the ‘healthy tissues’ of the host as they are for pathogens. This conclusion derives not only from a major evolutionary selective pressure to provide mechanisms that protect healthy sensitive tissues from immunopathology but also from all of the

studies of autoimmunity in the Aire-defective mutants ([52, 53], discussed in ref. [49]). This being the case, if trauma signals are required for the expression of the G, A or E ecosystems responsible for an autoimmune situation, then they must be endogenously provided by an M-ecosystem attack/insult. This tells us why the M-ecosystem is so dangerous and, in general, is kept as ephemeral in expression as possible. The Matzinger and Kamala Alarm Model might be reduced to the following picture that accords best with their above-cited admonition. The insulted tissue triggers an alarm signal that, in the end, is interpreted by a master organizing T cell (a chef d’ orchestre, probably of the helper category). This cell selects, directly or indirectly, from a pool of cellular elements, a compatible family of components that would comprise an ecosystem that is optimal (or appropriate) in ridding a given Eliminon.

This transient deficiency in IFN-I benefits the host as it does not lower resistance to common secondary bacterial infections (Fig. 1). In support of this hypothesis, IFN-I exhaustion is most likely to be evolutionarily as it

appears to be a consequence of all primary viral infections. We and others have shown this to be the case for adenoviruses, alphaviruses, orthomyxoviruses, murine cytomegalovirus and lymphocytic choriomeningitis virus [16, 21]. From an evolutionary perspective, there must have been a strong selective advantage to transiently exhaust IFN-I responses after primary viral infections signaling pathway to occur. Thus, it is reasonable to speculate the evolutionary advantage of negative feedback regulation to suppress virus-induced immune responses that are detrimental against secondary bacterial infections. It has been shown previously, exploring influenza virus/S. pneumoniae co-infection models, that secondary challenges, with either virus or bacteria, at the peak or during the IFN-I response, are highly lethal and the increased lethality is attributable to IFN-I [34-36]. It would be interesting

to find out whether the outcome of such co-infection experiments would differ if mice undergoing a primary virus infection were challenged with bacterial pathogens at the time of IFN-I exhaustion, 5–9 days post-infection. Thus, to provide evidence for the above-outlined hypothesis, all that https://www.selleckchem.com/products/AG-014699.html would be required is to establish correlates of strength of IFN-I response and exhaustion with severity of secondary bacterial challenges. A time course of bacterial infections after primary virus infection and/or poly I:C treatment would provide an answer to this question. Poly I:C, a synthetic analogue of double-stranded RNA, mimics RNA viral infections, but would eliminate potential unrelated viral-induced pathologies affecting secondary bacterial pathologies. It has been shown that poly I:C-treated mice mount IFN-I responses that render the host transiently more susceptible to bacterial infections [41, 46]. Evaluation of the severity of bacterial growth, morbidity and mortality should establish whether IFN-I exhaustion ameliorates secondary bacterial pathology.

Poly I:C-treated experimental groups will eliminate potential unknown viral-induced complications. It is somewhat surprising that the by now widely known phenomenon, that of an Tideglusib IFN-I refractory period after a viral infection, has as yet not been investigated as to its consequences for the host’s susceptibility to bacterial infections, given its potential clinical implications. The known detrimental consequences of the refractory period to secondary viral infections, namely heightened susceptibility, are somewhat hard to understand in evolutionary terms unless there exists an overriding host–benefit rationale. This may well turn out to be protection from potentially lethal bacterial infection, which can be controlled in the absence of IFN-I.

Multiple clinical parameters were obtained for the long-term stable patients within the GenHomme project, including donor and recipient demographic characteristics, clinical history of renal graft failure, transplantation

monitoring, full blood counts and medications biochemical screening. Non-transplanted patients with “non-immune” RFA (n=8) had a creatinemia 654±193 μmol/L and proteinuria >1 g 24 h−1. The causes of RFA were polycystic kidney (4/8 patients), renal dysplasia (2/8 patients), interstitial nephropathy (1/8 patients) and malformative uropathy (1/8 patients). Finally, healthy individuals (HEI, non-transplanted individuals, n=14) with normal renal function and no known infectious pathology for at least 6 months prior to the study were enrolled. MLN0128 PBMC from HLA-A2 CMV+ patients were stained with PE-labeled anti-human CD8 mAb, Alexa700-labeled anti-human IWR-1 CD3 mAb, Alexa 647-labeled anti-human CD4 mAb and pp65-HLA-A2 APC-labeled multimer. DAPI was used to exclude dead cells. pp65-HLA-A2 APC-labeled multimer was prepared by incubating for 1h APC-streptavidin with biotinylated pp65-HLA-A2 monomer. All mAb were purchased from BD Biosciences and biotinylated pp65-HLA-A2 monomer was produced by INSERM core facility (Nantes, France). DAPI−CD3+CD4−CD8+, DAPI−CD3+CD4−CD8+pp65-HLA-A2 multimer− and DAPI−CD3+CD4−CD8+pp65-HLA-A2 multimer+

were separated from PBMC using a high-speed cell sorter (FACSAria, BD Biosciences). Purity was greater than 98%. Blood, collected in EDTA tubes, was obtained Vasopressin Receptor from a peripheral vein or arteriovenous fistula. PBMC were separated

on an MSL layer (Eurobio) and frozen in TRIzol® reagent (Invitrogen) for RNA extraction. Total RNA was reverse-transcribed using a classical MMLV cDNA synthesis (Invitrogen). Complementary DNA was amplified by PCR using pairs of primers specific of each Vβ gene 10, elongated and electrophorezed using a gel sequencer (ABI Prism 377 DNA sequencer – Applied Biosystems) 35. The CDR3 profiles obtained were transformed into mathematical distributions and normalized so that the total area was equal to one. In parallel, the level of Vβ family transcripts was measured by real-time quantitative PCR and normalized by a housekeeping gene (HPRT). The CDR3-LD was then combined with each normalized Vβ transcript amounts to obtain the TcL data as described previously 15, 36, 37. Several parameters or metrics can be used to describe, and summarize with one value, the shape of the Vβ CDR3-LD. Indeed, the distribution of 13 lengths of Vβ CDR3 reflects different immunological situations which can be analyzed 12. Kurtosis, a mathematical index, has been chosen to quantify the CDR3-LD diversity 17. The Kurtosis reflects the degree of “peakedness” of a distribution 38 and is perfectly suitable for describing CDR3-LD with expansions.