“
“T cell recognition of gliadin from dietary gluten is essential for the pathogenesis of coeliac disease (CD). The aim of the present study was to analyse whether gliadin-specific T cells are detectable in the circulation of children with newly diagnosed coeliac disease by using a sensitive carboxfluorescein diacetate succinimidyl ester (CFSE)

dilution method. Peripheral blood CD4+ T cell responses were analysed in 20 children at diagnosis of CD and compared to those in 64 healthy control children carrying the CD-associated Ulixertinib clinical trial human leucocyte antigen (HLA)-DQ2 or -DQ8 alleles. Deamidated gliadin (gTG)-specific T cells were detectable in the peripheral blood of more than half the children with CD (11 of 20, 55%) compared to 15 of 64 (23·4%) of the control children (P = 0·008). Proliferative responses to gTG were also significantly stronger in children with CD than in controls (P = 0·01). In contrast, T cells specific to

native gliadin were detectable at comparable frequencies in children with CD (two of 19, 10·5%) and controls (13 of 64, 20·3%). gTG-specific T cells had a memory phenotype more Sirolimus in vitro often than those specific to native gliadin in children with CD (P = 0·02), whereas controls had similar percentages of memory cells in both stimulations. Finally, gTG-specific CD4+ T cells had a higher expression of the gut-homing molecule β7 integrin than those specific to the control antigen tetanus toxoid. Collectively, our current

results demonstrate that the frequency of circulating memory CD4+ T cells specific to gTG but not native gliadin is increased in children with newly diagnosed CD. Coeliac disease (CD) is a T cell-mediated chronic inflammatory disorder of the small intestine, and is mediated by intestinal T cells that recognize peptide epitopes of gluten in the context of disease-associated human leucocyte antigen PRKACG (HLA)-DQ molecules [1,2]. The highest risk for CD is associated with the presence of the DQ2 molecule, encoded by the DQA1*05 and DQB1*02 alleles [3]. More than 90% of patients are positive for HLA-DQ2, and most of those without DQ2 express the DQ8 molecule, encoded by the DQA1*03 and DQB1*0302 alleles [3]. In the gut mucosa, ingested oral gluten is deamidated by tissue transglutaminase (TTG). In turn, this deamidation enhances the immunogenicity of gluten by increasing the affinity between deamidated gliadin (gTG) epitopes and DQ2 and DQ8 molecules [4–6]. Gluten-specific T cell responses have been studied mainly on lymphocytes from small intestine biopsy samples [1,5,7–9], but they can also be detected in the peripheral blood. The frequency of these specific T cells in the circulation of CD patients is low, and studies have been performed mainly after oral gluten challenge in order to increase the number of circulating gliadin-specific cells in vivo[10–12].

For each patient, see more demographic and anthropometric data, laboratory data, electrocardiographic findings, ultrasound results, etiology of AKI and short-term outcomes were recorded. Results: The male to

female ratio was 1.57 to 1. Mean age was 5.28 ± 6.3 (SD) years and the median was 1.8 years. The more frequent age group was children less than 2 years. The mortality rate was 22.2% (40 patients). The mortality was not correlated with age (p= 0.74). Renal replacement therapy was recommended for 62 patients (34.4%). Mean of the first and last glomerular filtration rate (GFR) were 18.33 ± 1.12 ml/min/1.73 m2 and 52.53 ± 2.98 ml/min/1.73 m2, respectively. The most common urinary sediment finding in approximately 70% of the patients was either renal epithelial cell or renal cell cast. Increased kidney echogenicity was the most common ultrasound finding (48%). Using ANOVA regression analysis, the etiology of disease was the only predictor of mortality (p = 0.0001). Conclusion: Conclusions: We concluded that the mortality is still high in AKI. Furthermore, the poor outcome (defined as low

GFR) are higher among patients with low levels of first GFR and higher RIFLE score. SUBUN CHANTIDA, SRISUWAN KONGGRAPUN, CHULAMOKHA YUPAPIN, THIRAKHUPT PRAPAIPIM, LAMPAOPONG ADISORN Division of Pediatric Nephrology, Department of Pediatrics, Phramongkutklao find protocol hospital, Bangkok, Thailand Introduction: Peritonitis is one of the most important complications of peritoneal dialysis (PD) and often leads to membrane failure or even changing dialysis modality in children. The most common organisms responsible for PD-related peritonitis are gram-positive

bacteria such as Staphylococcus spp.and Streptococcus spp., gram-negative bacteria such as E. coli, Klebsiella spp. and Pseudomonas spp., and fungus. Micrococcus spp. is rarely found as a pathogen in a healthy individual. It is generally thought to be RG7420 research buy a commensal organism. However, several reports showed that Micrococcus could be an opportunistic pathogen, particularly in immunocompromised hosts, with one published report on Micrococcus PD peritonitis. Case report: A 17-year-old Thai boy with end-stage renal disease secondary to Immunoglobulin A nephropathy, who has been on chronic ambulatory peritoneal dialysis (CAPD), presented with a fever, abdominal pain and cloudy effluent. A complete blood count (CBC) showed leukocytosis with neutrophil predomination. The effluent cell count revealed white blood cells 530 cells/cu.mm with 70% polymorphonuclear cells. The effluent gram-stain revealed numerous polymorphonuclear white blood cells although no organisms were noted. A PD-related peritonitis was diagnosed, so, the patient was empirically treated with intraperitonealcefazolin and ceftazidime.

Results: To date 20 patients have been recruited (female 75%), mean age 69 years. Rapamycin molecular weight Reasons for referrals included decline in renal function (50%), uncontrolled hypertension (30%), albuminuria (15%) and haematuria (5%). 6 patients were at HR. Referrals have included specialist opinion for symptoms,

palliative care support and diabetes management. Time to review averaged 1 week and 3 weeks if a second specialist was consulted. Conclusions: The program allowed safe, quick and efficient consultation from multiple specialists online. It expedited time to nephrologist review and reduced face to face referral. To date it has proved to be safe and secure. Ongoing evaluation will occur and feasibility to a larger study is planned. 209 BIOLOGICAL VARIATION AND ANALYTICAL STABILITY OF SERUM SOLUBLE α-KLOTHO IN HEALTHY VOLUNTEERS SJ TAN1,2, ER SMITH1,3, SG HOLT1,2, ND TOUSSAINT1,2 1Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria; 2Department of Medicine (RMH), The University of Melbourne, Parkville, Victoria; 3Monash University, Clayton, Victoria, Australia Aim: To investigate the biological variability and analytical stability of soluble α-klotho in serum. Background: Recent evidence suggests that the cleaved extracellular domain of the α-klotho receptor, soluble α-klotho (sKl), has effects on phosphate homeostasis, ion channel

regulation and anti-fibrotic/anti-oxidant pathways. However, measurements of serum sKl in healthy individuals and in cohorts of patients Selleckchem XAV 939 with renal disease have yielded inconsistent results with respect to their relationship with renal function, other markers of mineral Ixazomib metabolism and patient outcome. Pre-analytical factors such as biological variation and analyte

stability may affect the interpretation of sKl results but have yet to be formally assessed. Methods: For assessment of biological variability, serum samples were collected from four healthy volunteers at three time-points during the day (morning, midday and afternoon). For assessment of analytical stability, separate aliquots from morning samples were allowed to stand at room temperature for 30, 60 and 120 minutes, prior to centrifugation and processing. All samples were stored at −80°C until batched analysis. sKl was measured using a commercial ELISA kit (Immuno-Biological Laboratories Co., Gunma, Japan) according to the manufacturer’s protocol. Biological and analytical stability was assessed using repeated-measures ANOVA. Results: Delayed separation of samples yielded mean (± SD) sKl levels of 222 (± 69) pg/mL, 208 (± 99) pg/mL and 193 (± 72) pg/mL, at 30, 60 and 120 minutes, respectively, revealing a small but non-significant trend towards analyte degradation over time. Mean (± SD) sKl levels were 222 (± 69) pg/mL, 220 (± 51) pg/mL and 207 (± 69) pg/mL at morning, midday and afternoon time-points, respectively showing no evidence of significant diurnal change.

1) according to site of injury along the pedicle path, each yielding different surgical strategies as follows. Type A includes injuries within 2 cm from its origin; type B includes injuries from 2 cm below Selleck Autophagy Compound Library its origin up to TD gives off the serratus branch; type C includes injuries between the level of the serratus branch and the neurovascular hilus, while type D injuries are intramuscular damages involving both horizontal and descending

branches. Whenever possible, primary anastomosis is the treatment of choice. This method is suited for type A without vessel tissue loss and B injuries that involve sharp lacerations of the pedicle with minimal (about 1 cm) or without vessel tissue loss. After locating proximal parts of artery and vein, the stumps are dissected free of surrounding tissue to provide for a tension-free coaptation. Whenever vessel tissue loss involves proximal segment up to the branch of the serratus or whether TD pedicle is cauterized about 1 cm in its length or not, direct anastomosis is not recommended. The need to refresh vessels’ edges and the shortening of proximal stump can

lead to excessive size discrepancy with unsafe anastomosis and limit the useful arch of rotation on the chest. The flap should be click here revascularized by end-to-end anastomoses to circumflex scapular (CS) vessels so to compensate vessel shortness and diameter difference. In type C that involves sharp lacerations with minimal (about 1 cm) or without vessel tissue loss direct microsurgical anastomosis can be still performed between proximal and distal stumps. This method is not appropriate whether the proximal segment of neurovascular hilus is Edoxaban involved or TD pedicle is cauterized about 1 cm in its length. In that case, the axillary vessels should not be used because of insufficient length of CS vessels and calibre

discrepancy to allow proper flap insetting and safe anastomosis. The flap can be revascularized by end-to-end anastomosis with serratus branch if intact. Whenever it is damaged as well as in all cases of LD pedicle’s avulsion and in type D lesions, a pedicled-to-free flap conversion to IMV recipient vessels at the third/fourth intercostal junction would be required. In attempt to spare IMV for possible future life-saving procedures, the wiser option is to dissect the ascending/descending branch of TD pedicle and prepare either IMV-perforators or the pectoral branch of the thoracocromial artery as recipient vessels. Since the ascending branch parallels the upper/medial LD border, anastomoses to IMV-perforators are suggested providing a free-tension horizontal insetting of the flap. For the same reason, since the descending branch parallels the lateral border of the muscle anastomoses to the pectoral branch should be performed with oblique insetting of the flap. The implant positioning under the muscle is not recommended because it can strain the anastomosis and consequently lead to arterial or vein impairment or both.

1), B220 (clone RA3-6B2). Intracellular AIRE staining was performed using the BD Cytofix/Cytoperm kit according to the manufacturer’s instructions 9. Cell sorting and analysis were performed on FACS (DakoCytomation MoFlo®, DakoCytomation MoFlo® XDP, BD FACSAria™, BD FACSCanto™, BD FACSCalibur™). Normal and transduced cells were plated on chamber slides (ICN Biomedicals) and permeabilised using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit. For AIRE staining, cells were incubated with monoclonal rat anti-AIRE Ab (Clone 5H12) buy Rucaparib followed by Alexa

568 nm goat anti-rat IgG (H+L) (Invitrogen). For the detection of MOG protein, cells were stained with monoclonal mouse anti-MOG Ab (Clone 8-18C5; gift from Prof. C Bernard, MISCL, Monash University, Victoria, Australia) followed by secondary Ab (Alexa 594 nm goat anti-mouse IgG). Slides were mounted using Dako Fluorescence mounting medium (Dako Cytomation) and images acquired with an Olympus IX71 Inverted Research Microscope. For confocal microscopy, transduced cells were cultured on glass coverslips, fixed with 4% PFA in PBS and permeabilised with 1% Triton X-100 in PBS prior to staining. Cells were stained with FITC-conjugated

anti-AIRE 5H12 9 and nuclear stain Hoechst 33342 (Sigma), mounted using fluorescent mounting media (Dako) and images acquired on a confocal microscope (Leica TCS SP2, Leica Microsystems). Statistical significance was evaluated using two-tailed Student’s t test for 2 groups. p values less than or equal to 0.05 were considered significant (*p≤0.05, learn more **p≤0.01, ***p≤0.001). Significant difference between two curves was evaluated via a permutation test offered by the Walter and Eliza Hall Institute for Medical Research (Melbourne, Australia) (http://bioinf.wehi.edu.au). We thank K. Webster for help with mTEC isolation and

P. Crewther for animal and laboratory management. We thank AMREP and WEHI Animal Services for animal care and management. This work was supported by fellowships from La Fondation pour la Recherche Medicale (FRM) and the this website 6th FP of the EU, Marie Curie, contract 040998 (to F.-X.H.), by Australian Postgraduate Awards (to S. A. K), NHMRC fellowships (171601 and 461204), NHMRC program grants (257501, 264573, 406700), Eurothymaide and EURAPS, 6th FP of the EU, and the Nossal Leadership Award from the Walter & Eliza Hall Institute of Medical Research to H. S. S., and NHMRC project grant (491004), to F. A., H. S. S. and F. X. H. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“This unit describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell migration and polarization.

Such a finding may enhance the negative and/or the positive predictive value of a chemical biomarker. Previous study demonstrated that sonographic measurement of fetal membrane thickness could be helpful in the prediction of preterm delivery.[16] Using the amniotic fluid and cervical length data from the randomized trials noted above, we examined the relationship

between cervical length and levels of inflammatory mediators in amniotic fluid.[17] Selleckchem Metabolism inhibitor Spearman correlations were used to determine which cytokines correlate with cervical length. Stepwise regression identified the most significant cytokine predictive of early delivery, and a ROC curve determined the cervical length cutoff predictive of intra-amniotic inflammation. Our results indicate that cervical length ≤5 mm is associated with significant

increases in amniotic fluid inflammatory cytokines, even in the absence of infection or labor. A cervical length of ≤5 mm was associated with significant increases in inflammatory mediators (Interleukin (IL)-1β, IL-2, IL-6, IL-8, and MCP-1), which have been previously shown to be associated with preterm labor.[18, 19] Unfortunately, the dataset was too small to allow a multivariable analysis including both cervical length and mediator levels in predicting outcome. While a very short cervical length is a good indicator of intrauterine inflammation, it represents the final common pathway of multiple inciting events that can result in preterm labor. As https://www.selleckchem.com/products/Romidepsin-FK228.html such, it is not an ideal biomarker when utilized alone. Many of these patients will go on to delivery prematurely despite intervention. It is likely that markers which identify earlier in-utero events will allow more effective therapies Molecular motor to be designed to stop the preterm labor cascade before the cervix becomes shortened. It appears that the intrauterine compartments are mostly immunologically distinct, and the expression of inflammatory markers in various maternal-fetal compartments will

be differentially expressed in non-invasive sampling sites. Because the etiology of preterm labor is multifactorial, using multiple biomarkers from distinct biologic pathways will better predict the risk of preterm labor. Furthermore, combining non-invasive tools such as a physical or ultrasound finding physical finding may improve the ability of specific biomarker in predicting outcome. Platforms to measure for example the levels of inflammatory mediator are commercially available and can easily be incorporated into ongoing trials looking at interventions to treat preterm labor. Initially, data can be collected in an observational manner and correlated with outcomes.

The tests were done in duplicate. Briefly, a microtiter plate (Costar, Cambridge, MA, USA) was coated with 100 μL/well of 5 μg/mL monoclonal mouse anti-human learn more granulysin (clone RB1) (MBL International, Nagoya, Japan) in 0.05 M carbonate-bicarbonate buffer (pH 9.5) overnight at 4°C. The plates were washed with PBS containing 0.05% Tween 20 and blocked with buffered protein solution with ProClin-150 at room temperature for 1 hr. After being washed, the undiluted plasma was added and incubated for 2 hr at room temperature. The bound antigens were detected

with 0.1 μg/mL of monoclonal mouse anti-human granulysin biotin (RC8) (MBL International) and avidin-horseradish peroxidase (Av-HRP) conjugate (BD Biosciences Pharmingen) diluted to 1:1000. After incubation for 1 hr, the reactions were developed by coloring with TMB substrate (BD Biosciences Pharmingen) for 20 min in the dark. The reaction was stopped by 2N H2SO4 solution (BD Biosciences Pharmingen). Optical densities were measured at 450 nm wavelength by an ELISA reader (ELx808 IU ultra microplate reader, Bio-Tek instruments, Winooski, VT, USA). Granulysin

concentrations were Palbociclib in vitro calculated from a standard curve using granulysin containing culture supernatant obtaining from Cos7 cell transfected with gene encoding 15K granulysin. The lower detection limit for granulysin was 0.047 ng/mL. Interferon-γ concentrations in plasma and stimulated PBMC supernatant were determined by ELISA according to the manufacturer’s instruction (BD Biosciences Pharmingen). The tests were done in duplicate. Briefly, a microplate (Costar) was coated with 100 μL/well of anti-human IFN-γ (diluted to 1:250 in 0.1 M sodium carbonate) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% Tween 20, blocked with 200 μL/well of buffered protein solution

with ProClin-150 and incubated at room temperature for 1 hr. After being washed, 100 μL of undiluted sample was added and incubated for 2 hr at room temperature. The bound antigen were detected with biotinylated anti-human IFN-γ monoclonal antibody and streptavidin-horseradish peroxidase conjugate (diluted to 1:250 with 10% FBS in PBS) and incubated for 1 hr at room temperature. Then, 100 μL of TMB substrate solution was added and incubated for 30 min at room temperature in the ADAMTS5 dark. The reaction was stopped by 2N H2SO4 solution. Samples were analyzed at 450/550 nm wavelength with a microplate ELISA reader (ELx808 IU ultra microplate reader) and IFN-γ concentrations were calculated from a standard curve using recombinant human IFN-γ. The lower detection limit was 4.7 pg/mL. Statistical analyses were performed by SPSS software version 17.0. IFN-γ and granulysin concentrations in different independent subject groups were compared by Mann-Whitney U test. A P value < 0.05 was considered statistically significant.

8,9 However, the long-term effects (over 10 years of therapy) of ARB or ACEi on kidney function in type 2 diabetes

are less clear. In addition, assessment of the effects of ARB or ACEi in normotensive, microalbuminuric people with type 2 diabetes need to take into account the potential cardiovascular benefits. The review by Boersma et al.10 focused on the pharmacoeconomics of ARB and ACEi treatment of people with type 2 diabetes and nephropathy. The conclusion with respect to ARBs was considered unequivocal in that the trials show both health gains and net cost savings compared with conventional treatment therapy, largely because of the high cost of dialysis and transplantation. The outcome with respect to the use of ACEi INCB018424 supplier was concluded to be less clear due to the limited head-to-head trials comparing ACEi to ARB. It has been demonstrated that aggressive BP reduction in hypertensive, normoalbuminuric people with type 2 diabetes reduces the incidence of microalbuminuria.11

Taken together with the progressive lowering of recommended BP thresholds for initiating treatment of elevated BP,12 it is possible that transition rates between stages of diabetic kidney disease will be substantially lower in the future than suggested by previous studies.13,14 It is important to note the assumptions inherent in cost-effectiveness analyses. A major concern about cost-effectiveness analysis is the validity of find more extrapolating to different populations in which costs, risk of diabetic kidney disease and effects of treatment on progression to renal failure may differ from the study population. why Socio-economic differentials in health are widely recognized with individuals of lower socioeconomic status (SES) having a higher risk for mortality and morbidity compared with those of higher SES.15,16 These guidelines consider evidence for socioeconomic influences as they relate to outcomes relevant to the prevention and management of CKD in people

with type 2 diabetes. The increasing prevalence of type 2 diabetes has been identified as the prime cause for the increasing prevalence of ESKD in Australia.2,17 The duration of diabetes, age, BP control and blood glucose control have been identified in the Australian population as independent risk factors for the development of albuminuria.18 Thus the consideration of the impact of socioeconomic factors on the diagnosis, prevention and management of CKD in people with type 2 diabetes, needs to be cognisant of factors that influence the development and treatment of type 2 diabetes, or that influence the likelihood of having undiagnosed diabetes and poorly treated hypertension and blood glucose. It is reasonable to assume that socioeconomic factors that influence the diagnosis and management of type 2 diabetes will also be important factors relevant to the progression of CKD.

After removing the template RNA, double-strand cDNA was generated using DNA polymerase I (Promega) and RVuni13: 5′-CGTGGTACCATGGTCTAGAGTAGT AGAAACAAGG-3′. PCR was performed using AccuPrime Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA), FWuni12 and RVuni13. The amplification products were separated by electrophoresis in agarose gels and the 1.8 kb fragments corresponding to the HA genes were excised from the gels to be purified. The amplicons were directly sequenced with BigDye Terminator ver1.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). The sequences were analyzed with an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Phylogenetic analysis

was carried out based Selleck MK 2206 on the 1,032 bp sequence corresponding to the HA1 region of the HA gene. Sequence data of each sample, together with those from GenBank, were analyzed by the clustalW program. A phylogenetic tree was constructed with FigTree software (http://tree.bio.ed.ac.uk/software/figtree). From the 71 nasal swab specimens collected between September and December 2009, we obtained 70 cytopathogenic agents using MDCK cells as described above. We confirmed that all of the agents were influenza A virus by RT-PCR (9) and designated them T1-T70. We purified and directly sequenced the amplification products corresponding

to the HA and NA genes. All of the nucleotide sequences found in both ends of the genes showed more than 99% homology to those of A(H1N1)pdm09 (accession: GQ165814 and GQ166204). These results indicate that only A(H1N1)pdm09 was isolated check details from the students during the study period. We analyzed the nucleotide sequences of the HA1 region of 4��8C the gene from the 70 isolates by the neighbor-joining method. The phylogenetic tree indicates that the 70 isolates are clustered into three groups (Fig. 2). The first group is composed of isolates from two (3%) sporadic cases, T1 on 3 September and T23 on 21 October 2009, which are related to A/Mexico/4115/09 (H1N1) (Mexico)

isolated on 7 April and A/Narita/1/09 (H1N1) (Narita) isolated on 8 May, Narita virus being detected as A(H1N1)pdm09 for the first time in Japan. The second group, consisting of 16 (23%) isolates from 13 October to 17 November, is related to A/Sapporo/1/09 (H1N1) (Sapporo) isolated on 11 June, which was the first A(H1N1)pdm09 isolated in Hokkaido, and A/Shanghai/1/09 (H1N1) isolated on 23 May. The last group is composed of 52 (74%) isolates obtained from 30 September to 15 December. These isolates are genetically related to A/Texas/42102708/09 (H1N1) (Texas) isolated on 10 June in the USA and A/Australia/15/09 (H1N1) isolated on 20 July. Based on the sequence of Narita, we observed a fixed amino acid change, Q293H, among the first group isolates and additionally found that T23 possessed R45G mutation.

CMV+ donors carry a high precursor frequency of CMV-specific Cabozantinib molecular weight T cells, and CMV-reactive T cells lines are

already in use to treat infection in stem cell transplant patients [5]. Here we stimulated PBMC with CMV antigen, isolated the antigen-specific cells using IFN-γ secretion and expanded the T cells into T cell lines CMV-specific cells isolated.  Human PBMC from CMV+ donors were stimulated with CMV lysate antigen (Dade Behring) for 16 h. For some HLA-A2+ donors, pp65 NLV(495–503) peptide was added during the last 3 h of the protein stimulus. IFN-γ selection isolated a mean of 7·7 × 104 CMV-reactive CD4+ T cells and 2·9 × 104 CD8+ T cells per 1 × 108 starting PBMC; adding the pp65 NLV peptide boosted the mean number of CD8+ T cells to a mean of 3·7 × 104 (Fig. 5a). Culturing these isolated T cells as described previously [9] for one round of expansion (2 weeks) led to a 2-log overall expansion selleck screening library rate, with slightly better proliferation of CD4 T cells (CD4 cells mean 2·3 log expansion versus CD8 cells 1·8-log expansion n = 20 Fig. 5b); also see [9]. Thus an average of 1 × 105 total CMV-reactive T cells isolated from 1 × 108 PBMC can be expanded to more than 1 × 107 total specific cells in 2 weeks – this is already similar to the total doses of cells currently given therapeutically [5]. The specificity of CD8+ cells can be checked easily by major histocompatibility

complex (MHC)-tetramer staining, but can be influenced heavily by the HLA-type of the donor – here we illustrate two HLA-A2+ T cell lines made following pp65 stimulus, but one donor is also HLA-B7+. In the HLA-B7- donor the cells produced are >99% positive for the dominant NLV(495–503) antigen (Fig. 5ci), but are almost completely absent in the HLA-B7+ donor, where most cells are specific for the B7-restricted TPR(417–426) peptide (Fig. 5cii). Thus care must be taken in understanding the immunodominance of different antigens in different

HLA-types. CD4+ T cells are best assayed by antigen-specific cytokine production – here we illustrate CD4+ T cells restimulated with autologous dendritic cells and CMV-lysate – the effector memory phenotype for these cells is illustrated graphically, as 88% of the cells make IFN-γ in response to restimulation but only 2% from make IL-2 (Fig. 5d). This section describes the protocol for cytokine detection and enrichment in detail. In this protocol, there are a number of critical steps, and failure to follow these will render results impossible to interpret. Critical steps and common areas that require troubleshooting are highlighted Prepare human PBMC or mouse spleen/lymph node (LN) cells. Critical step – foreign protein such as fetal calf serum (FCS) leads to higher background cytokine production in the non-stimulated control – use human AB serum or mouse serum. Resuspend cells in culture medium at 1 × 107 cells/ml and 5 × 106 cells/cm2 (e.g.