In mice, the CXCL10-binding chemokine receptor CXCR3 was shown to play a crucial role in the recruitment of autoaggressive T cells to pancreatic islets [24,25]. CXCL10 is produced by β cells [24] and increasingly detectable in serum of newly diagnosed or prediabetic subjects [26]. Inhibition of CXCL10 homing to islets prevents autoimmune diabetes in experimental models [25,27]. CXCL10 production by islets of type 2 diabetes patients has been described and claimed to impair β cell function [28]. Our observed CXCL10 and CXCR3 expression in pancreatic islets of a new-onset type 1 diabetes patient with enterovirus infection in β cells is strikingly similar to a very recent

report on fulminant diabetes and enterovirus infection [29]. A role for CXCL10 and CXCR3 was proposed in which enterovirus infection of the pancreas initiated co-expression of CXCL10 in β cells, attracting autoreactive T cells and macrophages GW-572016 solubility dmso to the islets via CXCR3. We described the expression of this particular chemokine receptor on human autoreactive T cell clones obtained from peripheral blood samples of (pre)diabetic individuals and demonstrated their capacity to home to pancreatic tissue of NOD/SCID mice after adoptive transfer [9]. In addition, recruited T cells

were found to express CXCR3 in situ, suggesting that peripheral blood T cells display the proper homing receptors, which is an important check-point for participation in the Stem Cell Compound Library process of insulitis and also perhaps in β cell destruction. Indeed, a type 1 diabetes patient-derived autoreactive CD8 T cell clone against preproinsulin, which was shown

to kill human pancreatic β cells, selectively expressed CXCR3 [30]. The CXCL10–CXCR3 pathway facilitating leucocyte migration to pancreatic islets is active in all donors, but not in non-diabetic controls. This may provide the basis for the development of a novel therapeutic target in type 1 diabetes [24,25,27]. Our report underscores the value of extensive studies on human insulitis [31–34]. Indeed, the Juvenile Diabetes Research Foundation has launched IKBKE an initiative to collect pancreatic tissue from diabetic donors to facilitate and drive such studies that are likely to bridge the gap in knowledge on immune as well as environmental factors contributing to β cell destruction in human type 1 diabetes (http://www.jdrfnpod.org/). These studies were supported by the Juvenile Diabetes Research Foundation, the Dutch Diabetes Research Foundation, the Italian Ministries of Health, University and Research and the Italian Diabetes Society Research Foundation (FORISID). Printing of the colour graphs was supported by a donation from the Lugtenburg family. The authors declare no conflict of interest. “
“The identification of soluble factors involved in stem cell renewal is a major goal in the assessment of the BM niche.

In order to complement

the null mhuA allele, the full-length mhuA gene was amplified by PCR with the primer pair A1 and A4, and the resulting amplicon was ligated into the XbaI site of a broad-host-range plasmid, pRK415. The resulting hybrid plasmid, pRK415-mhuA, was transformed into E. coliβ2155, and crossed with the ΔiucDΔmhuA strain. Tetracycline-resistant colonies were selected, and plasmid transfer confirmed by PCR and restriction enzyme analysis of the extracted plasmid. Two kinds of DNA fragments containing upstream regions of the mhuA gene were amplified by PCR with primer pairs Xba-I-P (5′-ctagtctagaACGGAACCGCAGACATGGTGTTG-3′), or Xba-I-P2 (5′-ctagtctagaTTTGATAACTCAAGGAGCTAGGAGC-3′) and Sph-I-P (5′-acatgcatgcTACAACAATTGCACTAGCGAGC-3′) Acalabrutinib selleck kinase inhibitor (the small italic letter sequences in Xba-I-P and Xba-I-P2, and Sph-I-P are polylinkers with XbaI and SphI sites, respectively). PCR fragments digested with

SphI-XbaI were ligated into the same enzyme sites of pAA224 (17), and the resulting promoter-lacZ reporter plasmids, termed pVMB2 and pVMB3, were then individually transformed into E. coli WAM131 (15). The degree of expression of the promoter-fused lacZ gene in E. coli WAM131 cells harboring the respective reporter plasmids was estimated by β-galactosidase activity measured by the method of Miller (22), after they had been grown at 37oC in +Fe or −Fe (with DPD) medium for 20 hr and 12 hr, respectively. Nucleotide sequence data for the V. mimicus mhuA and mhuB genes have been deposited in the EMBL/GenBank/DDBJ databases under the accession number AB048382. In order to eliminate background growth resulting from aerobactin-mediated iron uptake in the −Fe medium, a ΔiucD mutant incapable of synthesizing aerobactin was first constructed from V. mimicus 7PT and then www.selleck.co.jp/products/Fludarabine(Fludara).html used to assess whether this species can utilize heme and hemoglobin as iron sources. The deletion

in the iucD gene was confirmed by PCR analysis of the ΔiucD chromosomal DNA with the primer pair D5 and D6, which revealed an amplicon of the expected size (ca. 2.8-kb) (Fig. 1a). In the growth assay, the ΔiucD strain showed no growth in the −Fe medium, while the addition of hemin at 10 μM or hemoglobin at 2.5 μM to the same medium restored growth to a degree comparable to that found in the +Fe medium (Fig. 1b) These data clearly indicate that V. mimicus can utilize heme and hemoglobin as iron sources. The ORF in the 5121-bp cloned region together with relevant inserts in the constructed plasmids are shown in Figure 2. Fur-box-containing gene fragments from V. mimicus 7PT (10, 21) were isolated through application of FURTA system (14). One of the FURTA-positive clones, termed pVM3, possessed two partial ORF, whose deduced amino acid sequences were significantly homologous to the V. cholerae HutA and VCA0575 proteins involved in heme and hemoglobin utilization (11, 23). To clone surrounding regions of these partial ORF, V.

rubrum. Seventeen nail samples were positive for fungal elements in the KOH-mounts only and were negative in cultures and T. rubrum PCR. In scales (Fig. 2) as well as in nails (Fig. 3), the sensitivity of the T. rubrum PCR was clearly higher than the culture method with regard to detection of T. rubrum. This superiority was higher for nail probes than for scale samples. The specificity of the T. rubrum PCR was very high; none of the cases in which a fungal species other than T. rubrum was

grown had a positive T. rubrum PCR. However, neither in scales nor in nails all T. rubrum-infections were detected by the T. rubrum PCR as reflected by probes of scales and nails that yielded a positive T. rubrum culture, BMN 673 purchase but a negative T. rubrum PCR. Furthermore, it remains unknown how

many of the samples with a positive KOH-mount, but negative results of T. rubrum PCR and cultures, might have been caused by an infection by T. rubrum. Depending on the submission of samples, on the workload of the laboratory and on the capacities for analyses, it took about 2–5 days to get a PCR result in our laboratory and 2–3 weeks to obtain a culture result. The samples investigated in this study had been taken under routine conditions and although in most cases the reason for their collection had been to prove a mycotic infection, the exclusion of tinea in case of ambiguous lesions was an indication as well. Therefore, the high percentage of negative results selleck kinase inhibitor with KOH-mounts,

cultures and PCR is not surprising. Our results clearly show that the PCR method used by us allows detecting markedly more infections with T. rubrum than the commonly used combination of KOH-mount plus culture. It is also noteworthy that this PCR assay is feasible in a shorter time than cultural verification even under routine conditions. This improvement of sensitivity and speed applies to infections of the superficial skin and even more to nail infections. It is tempting to calculate exact figures for the sensitivity and specificity of the T. rubrum PCR. However, there is an unquestionable science likelihood that a certain share of the positive KOH-mounts was a result of T. rubrum-infections despite a negative PCR (for reasons of lack of DNA in the probe because of inhomogeneous distribution within the submitted material, degradation of DNA, inhibition of PCR, etc.), and without knowing the rate of missed infections, a calculation of sensitivity and specificity is not sensible. Nevertheless, our data support the conclusion that the T. rubrum PCR improves the detection of T. rubrum. As was mentioned above, this does not mean, however, that all T. rubrum-infections were detected by our T. rubrum PCR. There are at least two reasons that can explain negative culture results despite a positive T. rubrum PCR. First, the fungal elements in the collected samples may not be viable because of previous treatments or incorrect collection.

All authors see more declare no conflicts of interest. “
“Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A488) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin

A488-associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A488 in neutrophils was greater on ice than at 37 °C. Studies using trypan

blue suggested that over 3 h at 37 °C, most of the toxin A488-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A488-associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during NVP-BEZ235 cell line colonic inflammation in C. difficile infection. Clostridium difficile, an anaerobic gram-positive bacterium, is the most common cause of nosocomial diarrhoea and the aetiological agent of antibiotic-associated pseudomembranous colitis, a severe form of the disease that is often characterized histologically by focal inflammation associated

with epithelial ulceration [1–3]. Infection due to C. difficile is a significant clinical problem, especially in hospitalized patients on antibiotics. Studies in hamsters [4, 5] and rabbits [6–8] have shown that the intestinal disease is mediated pheromone by secreted toxins A and B. In vitro studies using human intestinal epithelial cell lines suggest an essential role of toxin A in the disruption of epithelial barrier function [9, 10]. Toxins A and B are among the largest toxins known and consist of three major domains: the N-terminal region with glucosyltransferase activity, a hydrophobic central region (thought to be required for translocation across cell membranes), and a highly repetitive C-terminal region, which is believed to be responsible for binding to cell surface receptors [11, 12] and is required in entirety for binding-induced endocytosis [13]. Monoclonal antibody PCG-4 recognizes epitopes within the C-terminal region and in animal studies has been shown to be capable of neutralizing enterotoxic activity of toxin A [14]. Moreover, this antibody has been shown to block toxin A-induced disruption of epithelial barrier function in vitro [9, 10]. Previous studies have also demonstrated carbohydrate-specific recognition by toxin A [15–17]. During the initial phase of C.

Using TEM, the number of neutrophils and MCs were counted on two intestinal grids for each infected fish. The number of each type of granulocyte was determined in an area measuring 1800 μm2 in close proximity to the point of cestode attachment (i.e. the interface region) and in a second area measuring 1800 μm2 at a distance of approximately 200 μm from the site of cestode attachment. Prior to analysis, the Gaussian distributions (i.e. normality) GS-1101 cost and the homogeneity of variances of the data were assessed; the data were subsequently square

root transformed to meet these assumptions. Using the software package Statistica 7, anovas (Statistica 7, Praha, Cech Republic) were performed to detect significant differences in the number of granulocytes determined from the uninfected and infected tench and in the abundance of neutrophils and MCs at the point of cestode attachment and then at a distance of 200 μm away. Bonferroni post hoc tests and a P < 0·01 level of

significance were used throughout. Fourteen (60·9%) of the 23 tench were parasitized with M. wageneri; identity of the cestodes was confirmed using morphology and standard taxonomic keys. The intensity of infection ranged from 3 to 130 worms per host (39·5 ± 47·7, mean ± SD). The anterior part of the intestine bore the heaviest infections with the vast majority of tapeworms still attached with their scolices embedded within the intestinal wall (Figure 1a). Upon dissection in situ, M. wageneri were noticed in groups of variable numbers and in some portion of the host intestine the presence of more than one foci was frequent (Figure 1a). In tench gut wall, at the site Tyrosine-protein kinase BLK of M. wageneri attachment, ABT-199 cell line a raised plaque-like formation or round nodule encircled the firmly attached scolex (Figure 1b). Histological sections revealed that specimen of M. wageneri had penetrated by means

of bluntly truncated scolex deep into the mucosa and submucosa (Figure 2a, b) and in some instances into the muscularis layer (Figure 2c). This parasite anchoring system provided a secure attachment to the tench intestine (Figures 1a, b and 2b). At the site of attachment, the tapeworms induced necrosis, degeneration and/or loss of the epithelium (Figure 2a). M. wageneri elicited intense immune cells and fibroblasts proliferation within the thickness of the tench gut wall (Figure 2b, c). Diffuse hyperplastic inflammation was noticed in tench with few M. wageneri as well as in those harbouring numerous tapeworms (Figure 2a–c). Within the submucosa layer, beneath the point of M. wageneri scolex insertion, numerous granulocytes (e.g. neutrophils, MCs) (Figure 2d), rodlet cells (Figure 2e) and collagenous fibres were observed. Degranulation of the granulocytes, which was visible by light microscopy (Figure 2d), was common in the submucosa. Parasitized intestines were determined to have a significantly higher number of granulocytes than those that were uninfected (Table 1; anova, P < 0·01).

It is estimated that rates of LTBI in the community

need to be less than 1% to allow TB elimination [94]. At present, there are no accurate tests to predict which of the 2 billion individuals with LTBI will fail to contain the infection and progress to active tuberculosis. Individual testing for genotypes associated INCB024360 solubility dmso with a reduced risk of active tuberculosis, such as autophagy gene variant immunity-related GTPase M (IRGM)-261T [95] and Mal S180L [96], may enable clinicians to target treatment for LTBI to patients at a higher risk of progression. Autophagy plays a key role in immune responses to mycobacteria; it kills intracellular mycobacteria, enhances antigen presentation and modulates the secretion of important cytokines. Moreover, genomewide analysis of host responses to infection with Mtb indicates that survival of the bacilli hinges on its ability to modulate autophagy. Thus, autophagy offers an attractive therapeutic target. Agents that promote autophagy might prove efficacious as an adjunctive treatment for drug-resistant and drug-sensitive tuberculous disease. They might also be used to target latent tuberculosis. In addition, vaccines which specifically stimulate autophagy could prove more effective in protecting against tuberculosis. Effective treatment for tuberculosis could save as many as 1·7 million lives every year:

the stakes are high, and autophagy could be a trump card. CNC is funded by the Health Research Board as part of the National SpR Academic Fellowship Programme; JK, ECL and JH are funded by Science Foundation Ireland as part of the Immunology Research Centre, SFI Strategic Research Cluster. None selleck products of the authors has any conflicts of interest to declare, or any relevant financial interest, in any company

or institution that might benefit from this publication. “
“MHC anchor residue-modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte Carnitine palmitoyltransferase II antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface.

Our current data support previous clinical studies in suggesting a role of E. coli in human PBC. Hopf et al. [63] reported an association between PBC and the presence of rough-form mutants of E. coli in the patients’ fecal RAD001 in vitro samples. In addition, Butler et al. reported reactivity to PDC-E2 in 52% of sera from patients with chronic UTIs [7, 64]. In the first controlled epidemiological analysis for the relationship between

E. coli and PBC, Parikh-Patel et al. showed a positive association between PBC and recurrent UTI [65]. A recent epidemiological study on 1032 PBC patients followed-up in 20 tertiary referral centres in the United States and 1041 demographically matched controls confirms earlier studies indicating a connection SCH727965 molecular weight of UTI with PBC [66]. The discovery of E. coli infection-triggered autoimmunity and liver pathology warrant further consideration in the elucidation of aetiological mechanisms of autoimmune syndromes and may suggest new and simpler ways to diagnose and treat these debilitating diseases. Our data also highlight the importance of microbial

infections in autoimmunity either as primary or co-existing secondary inciting events. This work was supported in part by National Institutes of Health grants DK39588 (M. E. G.) DK067003 (M. E. G.), AI71922 (M. K.) and AI083029 (J. L. V.) The authors have no financial conflicts of interest. “
“Bidirectional signals via Eph receptors/ephrins have been recognized as major forms of contact-dependent cell communications such as cell attraction and repulsion. T cells express EphBs, and their ligands, the ephrin-Bs, have been

known as costimulatory molecules for T-cell proliferation. Recently, another remarkable feature of ephrin-As has emerged in the form of a concentration-dependent transition from promotion to inhibition in axon growth. Here we examined whether this modification plays a role in ephrin-B costimulation in murine primary T cells. Low doses of ephrin-B1 and ephrin-B2 costimulated T-cell proliferation induced by anti-CD3, but selleck inhibitor high concentrations strongly inhibited it. In contrast, ephrin-B3 showed a steadily increasing stimulatory effect. This modulation was virtually preserved in T cells from mice simultaneously lacking four genes, EphB1, EphB2, EphB3, and EphB6. High concentrations of ephrin-B1/B2, but not ephrin-B3, inhibited the anti-CD3-induced phosphorylation of Lck and its downstream signals such as Erk and Akt. Additionally, high doses of any ephrin-Bs could phosphorylate EphB4. However, only ephrin-B1/B2 but not ephrin-B3 recruited SHP1, a phosphatase to suppress the phosphorylation of Lck. These data suggest that EphB4 signaling could engage in negative feedback to TCR signals. T-cell activation may be finely adjusted by the combination and concentration of ephrin-Bs expressed in the immunological microenvironment.

88 Chemotaxis and chemorepulsion GSK1120212 datasheet in the context of T-cell trafficking have been studied in the process of thymic emigration. Egress of mature thymocytes from the medulla to the periphery has been shown to be orchestrated by chemoattraction exerted by S1P and a simultaneous fugetactic function of CXCL12, which induces cells to leave the thymus.81,89 A bimodal effect of chemokines on memory T-cell trafficking has also been demonstrated in cancer. Certain growing tumours initially generate the chemokine CXCL12 at a level that induces T-cell chemoattraction, but ultimately

establish an immune-privileged site through the chemorepellent effect of high levels of CXCL12 on tumour-specific T cells. In this setting, T-cell chemorepulsion impairs cytotoxic T lymphocyte-mediated lysis of tumour cells, which requires that the effector makes direct contact

with the target cell.90 Fugetaxis and chemorepulsion may coexist in situations where the concentration of the chemokine drives cells from chemotaxis to fugetaxis, but dual receptor engagement may take place. In fact, it has been shown that the chemokine CXCL12 mediates a concentration-dependent chemorepulsive effect on diabetogenic T cells by altering firm adhesion. As this effect is G-protein-coupled receptor dependent but is only partially reversed by CXCR4 blockade, it has been suggested that alternative downstream CXCL12 signalling pathways mediated by protein coupled receptor 1 (RDC1)/CXCR7 CRM1 inhibitor may trigger chemorepulsion.91 Memory plasma cells reside on CXCL12-expressing stromal cells of bone marrow and rest there for a long periods.92–94 Until recently, evidence demonstrating the existence of survival niches for memory CD4 T cells has been elusive.95,96 In immune reactions characterized by long-term antigen persistence (virus or adjuvants), memory-phenotype

CD4 T cells are found in the spleen and lymph nodes for long periods.97,98 In contrast, following immunization in the presence of soluble adjuvants (lipopolysaccharide Protein kinase N1 or monophosphoryl lipid A), memory CD4 T cells in the spleen or lymph nodes substantially decrease in number 1 week after immunization.99,100 These T cells have been shown to locate to the bone marrow and rest on IL-7-expressing stromal cells of the bone marrow.99 The relocation of antigen-experienced CD4 T cells to the bone marrow is dependent on integrin α2β1, a collagen receptor. Inhibition of integrin α2β1 on primed CD4 T cells results in defective relocation of antigen-specific CD4 T cells to the bone marrow and reduced B-cell help (e.g. reduced affinity maturation). It is still unknown how the memory T cells migrate to their survival niches in the bone marrow, although they express CCR2 and CXCR6.99 The bone marrow is presumably the most best tissue for long-term localization of CD4 T cells primed by blood-borne antigen.

We evaluated daily doses and trough levels of Tac and serum creatinine levels, and compared pathological findings. Results: Daily doses were higher in the Tac-QD group, but trough levels and serum creatinine levels were comparable. On 3- and 12-month PB, the frequency of subclinical rejection was similar between the groups, while interstitial fibrosis and tubular atrophy (IF/TA) were less common in the Tac-QD group at 12 months (42.2% vs. 20.6%, P = 0.04). Univariate and multivariate logistic selleck chemicals regression analyses revealed allograft rejection (borderline changes or higher) was associated with IF/TA (odds ratio 4.09, 95% confidence interval 1.76–10.10,

P = 0.001). The Tac-QD-based regimen showed a trend toward the absence of IF/TA but it did not reach statistical significance. Tubular vacuolization and arteriolar hyaline changes were also comparable in the two groups. Conclusion: We found a trend toward milder IF/TA, but no significant differences in kidney allograft pathology in patients treated with Tac-QD- versus Tac-BID-based regimens at 12 months. The effects of Tac-QD on chronic allograft injury need to be studied check details by longer observation. FANG DOREEN YP1,2,

LU BO1, HAYWARD SUSAN3, DE KRETSER DAVID3, COWAN PETER1,2, DWYER KAREN1,2 1Immunology Research Centre, St Vincent’s Hospital Melbourne, Victoria, Australia; 2Department of Medicine, The University of Melbourne, Victoria, Australia; 3Monash Institute of Medical Research, Monash University, Victoria, Australia Introduction: Ischemia-reperfusion injury (IRI) accompanies organ transplantation causing inflammation and potentially contributing to poor graft function. Activin is a key driver of inflammation and it is regulated by follistatin. The aim of this study is to investigate the level of activin and the effect of follistatin treatment in renal IRI. Methods: Mice received 5 μg follistatin (n = 4) or

vehicle (n = 4) 30 mins before right nephrectomy and clamping of the left renal pedicle for 20 mins. A sham group (n = 6) ADAMTS5 underwent right nephrectomy without clamping. Mice were sacrificed at 24 hrs. Serum was collected to measure activin A and B by ELISA. Serum creatinine was measured as a marker of renal function. Kidney sections were stained with H&E and scored to evaluate tubular injury on a scale of 0–4. Real-time PCR was performed to analyze the mRNA expression of IL-1β, IL-6, TNFα and kidney injury molecule-1 (KIM-1). Results: Renal IRI increased serum activin A, activin B, creatinine, tubular injury score, and mRNA expression of IL-1β, IL-6, TNFα and KIM-1. Follistatin treatment prior to ischemia reduced activin A, activin B, creatinine, and mRNA expression of IL-6 and KIM-1. There was a trend of improvement in tubular injury score, and mRNA expression of IL-1β and TNFα. [Table 1] Conclusion: Activin is upregulated during renal IRI.

e. expressing at least one of the markers). This clearly confirmed that degranulation became increasingly dominant after transplantation, with a median of 92% of CD8+ pp65-specific T cells and 85% of IE-specific CD8+ T cells expressing this marker (alone or in combination) after transplantation compared with 84% and 71%, respectively, in controls (not shown). However, because of their likely protective role, we were primarily interested in the effect of immunosuppression on the T cells producing IFN-γ, TNF-α and IL-2 simultaneously, or any two

of them.9 For this purpose, the analyses shown in Fig. 1(b) disregard degranulation and focus on IFN-γ, TNF-α and IL-2 alone. They show that

the most dominant CMV-specific CD8+ subset learn more (as defined by these functions) in healthy donors produces just IFN-γ and TNF-α, while the subset producing all three measured cytokines is the only other sizeable subset. Both are strongly reduced in transplant patients. A similar distribution was observed for pp65-specific CD8− T cells. When studying each of 15 non-overlapping functional subsets individually (Boolean gating) it became apparent that T cells exhibiting degranulation as a single function were dramatically increased in transplant patients (Fig. 1c). As all patients received calcineurin inhibitors (but only one-third each received everolimus or mycophenolate mofetil), we attempted to reproduce this effect in vitro by incubating donor-derived cells overnight with the see more calcineurin inhibitors cyclosporin A or tacrolimus before stimulation, because these were the most likely

drugs to cause this change. This resulted in a dose-dependent reduction of polyfunctionality; the subsets producing IFN-γ, TNF-α and IL-2, or IFN-γ and TNF-α decreased (Fig. 2a,b) whereas subsets displaying only single functions emerged and increased (Fig. 2c,d). Dot plots in Fig. 2(e) show a dose-dependent decrease in TNF-α, IFN-γ and IL-2 production, but little effect on degranulation. Our results show that immunosuppression induces marked changes in the CMV-specific T-cell response after heart and lung transplantation. These are reflected in response quality (i.e. the functional response profile) many rather than quantity (i.e. the number of inducible cells). The most obvious effects were reduction of IL-2 and TNF-α production, IFN-γ seemed somewhat less affected and degranulation not at all. This predominantly translated into the generation of T-cell subsets with one single function, most frequently degranulation, at the expense of subsets displaying IFN-γ, TNF-α and IL-2 at the same time. Degranulation was the most inclusive marker of total response size but not the most informative with regard to the effect of immunosuppression.