The authors alone are responsible for the content and writing of the paper and declare no conflicts of interest. “
“Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication

called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical AZD4547 order for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed

mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with Nutlin-3 ic50 mStx2-His produced specific antibodies that neutralized the toxicity of Suplatast tosilate Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens. Enterohemorrhagic Escherichia coli strains cause hemorrhagic colitis and a serious clinical complication called hemolytic uremic syndrome (HUS) that is characterized by hemolytic anemia, thrombocytopenia, and acute

renal failure [1, 2]. Major causative factors of EHEC include two types of Stx, Stx-1 and Stx-2 (also referred to as Vero toxin-1 and Vero toxin-2, respectively), both of which consist of one A subunit (Stx1A and Stx2A) and five B subunits (Stx1B and Stx2B). At the amino acid sequence level, Stx1 is almost identical to Stx produced by Shigella dysenteriae 1, whereas Stx2 shares only 55% and 61% amino acid sequence identity with Stx1 in the A and B subunits, respectively. The B subunits bind to Gb3 on the eukaryotic cell membrane [3-5], whereas the A subunit functions as an RNA N-glycosidase that cleaves off a single adenine in the 28S rRNA component of the 60S ribosomal subunit, leading to cell death by inhibition of protein synthesis [6, 7]. Stx2 toxicity is reportedly greater than that of Stx1, because in mice the LD50 of Stx2 is lower than that of Stx1 [8], and in humans Stx2-producing strains generate more severe symptoms than do other strains [9-11].

For mycobacterial CFP, the membrane was probed with rabbit polyclonal antibodies made against M. tuberculosis CFP (BEI Resources, NR-13809) and then incubated with goat anti-rabbit HRP-conjugated IgG as described above. IT-12 and NR-13809 were obtained from Colorado State University, Colorado, USA, under the TB Vaccine Testing

and Research Material Contract. In exosome-priming experiments, mice were immunized via an i.n. route with a final injection volume of 30 μL (15 μL/nostril) as described previously [21]. Briefly, five mice per group were anaesthetized with isoflurane and administered with PBS alone or with purified exosomes isolated from CFP-treated or untreated macrophages, at a dose of 20 μg/mouse or 40 μg/mouse. The mice were immunized three times at an interval 5-Fluoracil in vivo of 2 weeks. Two weeks after final exosome vaccination, mice were sacrificed and used to measure antigen-specific T-cell activation and 4 weeks after final vaccination, a separate set of mice were infected with M. tuberculosis. As a positive control, M. bovis BCG (1 × 106 CFU/mouse, Pasteur H 89 mw strain) was given i.n. as a single dose 8 weeks prior to M. tuberculosis infection. For BCG priming and exosome boosting experiments, five mice per group were first s.c. immunized with a single dose of M. bovis BCG (1 × 106 CFU/mouse, Pasteur strain) in 50

μL of PBS and subsequently rested for 8 months before boosting. Exosome booster immunization was administrated twice i.n. at 2-week intervals as described above. Another set of BCG-vaccinated mice were also boosted with BCG i.n. at 1 × 106 CFU at the same time as the first exosome boost vaccination. Mice were sacrificed to measure antigen-specific immune

responses or infected with M. tuberculosis H37Rv as described for the exosome-priming experiments. Six weeks following the final vaccination of exosomes, mice were challenged with M. tuberculosis H37Rv using an Inhalation Exposure System (Glas-Col, Terre Haute, IN, USA). Four M. tuberculosis infected mice per group were humanely sacrificed 1 day after infection to determine the bacterial load in the lungs and spleens. The amount of M. tuberculosis used in Ribonucleotide reductase the infection was calculated to give approximately 50 to 150 CFU/lung in mice. For all other infections, mice were euthanized 6 weeks after mycobacterial challenge and the lungs and spleens were removed and homogenized in PBS containing 0.05% v/v Tween-80. The tissue homogenate was appropriately diluted in the same buffer, and then 50 μL of the diluted homogenate was spread on Middlebrook 7H11 agar plates with 10% OADC, 0.5% glycerol and 0.05% Tween-80, and containing a cocktail of fungizone (Hyclone) and PANTA (polymixin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin; BD, Sparks, MD, USA).

[44] On the other hand, very aggressive EAE induction (for example, repeated immunization with high dosages of heat-killed Mtb) completely abrogates IFN-β efficacy GSK-3 beta pathway in wild-type mice (Inoue et al., unpublished data). Hence, EAE induced by moderately aggressive immunization may develop as a mixture of two EAE subtypes; NLRP3 inflammasome-dependent and -independent. When two subtypes of EAE are ongoing, it may be possible that IFN-β efficacy correlates with levels

of NLRP3 inflammasome dependency in EAE development. Although two subtypes of EAE may be occurring simultaneously within some of the disease in WT mice, the findings are summarized as follows: NLRP3 inflammasome-dependent EAE is a disease that responds to IFN-β treatment, whereas NLRP3 inflammasome-independent EAE is a disease that is resistant to IFN-β (Fig. 2). Previous studies have shown that passive EAE induced by Th17 cell transfer is resistant to IFN-β treatment, whereas the disease induced by Th1 cells responds to IFN-β treatment.[81] The result is counterintuitive because IFN-β inhibits Th17 responses;[62, 65] and it will be of great interest to understand why Th17-mediated EAE cannot be treated by IFN-β. Activation status of the NLRP3 inflammasome is not known in the Th17-mediated EAE model, but the result (resistance of Th17-mediated passive EAE to IFN-β) does not conflict with IFN-β resistance in NLRP3 inflammasome-independent

EAE. This is because the Th17 response itself is not the reason

for NLRP3 inflammasome-dependent EAE progression.[44] Further studies will be necessary to determine whether or not these two types Apoptosis inhibitor of IFN-β-resistant EAE (Th17-type EAE and NLRP3 inflammasome-independent Oxymatrine EAE) share the same mechanism. It is currently unknown whether NLRP3 inflammasome-independent MS exists. It is also not known what type of event is an equivalent of ‘aggressive immunization’ in MS. However, if the current findings on the correlation between NLRP3 inflammasome activation and response to IFN-β in EAE can be applied to MS, it might be possible to predict MS patients who do not respond well to IFN-β therapy. For example, the activation status of the NLRP3 inflammasome might be a prediction marker. Or, it might be possible to identify prediction markers by screening molecules that show altered expression in NLRP3 inflammasome-independent EAE. It is also possible to test such molecules for prognosis markers, or even as molecular targets of selected treatment(s). “
“Human Vγ9Vδ2 T cells play a crucial role in early immune response to intracellular pathogens. Their number is drastically increased in the peripheral blood of patients during the acute phase of brucellosis. In vitro, Vγ9Vδ2 T cells exhibit strong cytolytic activity against Brucella-infected cells and impair intracellular growth of Brucella suis in autologous macrophages.

T cell proliferation: Heparin anticoagulated blood (50 ml) was obtained from 10 randomly selected members of each of the three subject groups and centrifuged at 850 g for

20 min. Plasma was removed, and cells were suspended ABT199 in D-Hanks solution. This was layered onto Ficoll separation medium in a tube followed by centrifugation at 850 g for 20 min. Cells in the middle layer were carefully collected, which were peripheral blood mononuclear cells (PBMCs). PBMCs were washed 3 times in RPMI-1640 by centrifugation at 450 g for 10 min and then re-suspended in RPMI-1640 to a density of 1 × 108/ml. A fraction of this cell suspension was loaded onto a prewarmed Nylon Fiber column T (37 °C) with RPMI-1640 medium containing 10% FBS; the volume of the cell suspension was one-third that of the column. After sealing,

MAPK inhibitor the column was kept warm at 37 °C for 1 h, after which prewarmed RPMI-1640 (37 °C) was added at a flow rate of 3–4 ml/min. The opaque medium was collected, which contained T lymphocytes. T lymphocytes were re-suspended in RPMI-1640 containing 10% FBS at a density of 1 × 106/ml. Cell suspensions were added to a 96-well plate (100 μl/well) followed by adding PHA (final concentration: 20 μg/ml; and final volume in each well: 200 μl). As controls, cells without PHA were also included, and three wells were included for each group. Plates were incubated at 37 °C in a 5% CO2 atmosphere for 48 h. At 4 h before the end of incubation, MTT (20 μl; 5 g/l) was added and incubation was continued at 37 °C for the remaining 4 h. The plate was centrifuged, the supernatant was removed, and DMSO (100 μl/well) was added to dissolve the crystals followed by incubation for 15 min. Optical

density (OD) was measured with a DNA Synthesis inhibitor microplate reader (detection wavelength: 570 nm; reference wavelength: 630 nm), and a stimulation index (SI) was calculated: SI = ODexperiment/ODcontrol. Cytokine-induced killer (CIK) cell culture and assessment of tumouricidal activity: PBMCs were suspended in RPMI-1640 at a density of 1 × 106/ml. On day 0, γ-INF (1000 U/ml) was added followed by incubation at 37 °C in a 5% CO2 atmosphere for 24 h. On day 1, IL-1 (100 U/ml), CD3 mAb (50 ng/ml) and IL-2 (500 U/ml) were added followed by further incubation; half of the medium was refreshed every 3 day during which IL-2 was added. On day 6, CD3 mAb (50 ng/ml) was added again. On day 15, cells (CIK cells) were harvested and re-suspended in RPMI-1640 at a density of 1 × 106/ml; these were used as effector cells. K562 cells were used as target cells. Effector cells were mixed with target cells at a ratio of 10:1 and then added to a 96-well plate. As controls, effector cells or target cells alone were also added to three wells for each group. MTT (20 μl; 5 g/l) was added, and plates were incubated at 37 °C in a 5% CO2 atmosphere for 4 h followed by centrifugation.

VIN may be human papillomavirus (HPV)-related classic VIN or -unrelated VIN. The former is by far the most frequent vulvar cancer precursor. It occurs in adult women and tends to be multi-focal. It is caused by high-risk HPV (HR-HPV) types, essentially type 16, and histologically is made of poorly Etoposide research buy to undifferentiated basal cells and/or highly atypical squamous epithelial cells [1]. The involvement

of the entire thickness of the epithelium defines grade 3 of the disease. The disease progresses towards invasion in about 3% of treated patients and 9% of untreated patients, according to a review of more than 3000 cases [2]. Classic VIN can also regress spontaneously [3] in young women presenting with multi-focal pigmented papular lesions. Previously, we studied a patient who presented with multi-focal classic VIN and showed complete clearance of viral lesions 8 months after disease onset and 2 months after electrocoagulation of less than 50% of the classic VIN lesions [4]. Immunohistochemical

study of her initial vulvar biopsy revealed a marked dermal infiltrate containing a majority of CD4+ T lymphocytes and an epidermal infiltrate made up of both CD4+ and CD8+ T cells. She also showed a proliferating response against one peptide from E6 protein and a high-frequency anti-E6 and anti-E7 effector blood T cells by ex vivo enzyme-linked immunospot–interferon-γ (ELISPOT–IFN-γ) assay Dactolisib in vivo just before clinical regression. Such a study of blood cellular immune responses, together with the analysis of vulvar biopsies obtained simultaneously

and correlated with clinical outcome, has not been reported previously. In an anti-HPV vaccine trial conducted by Davidson et al.[5], classic VIN lesions regressed completely in a patient following vaccination. Interestingly, immunostaining of vulvar biopsy prior to the vaccine showed a marked CD4+ and CD8+ T lymphocyte infiltrate of both epithelial and subepithelial sheets. It may be speculated whether the regression of these patient lesions could be related to a spontaneous regression. Therefore, the observation of a CD4+ and CD8+ infiltrate within subepithelial and epithelial sheets in the biopsy and the visualization of very strong blood anti-HPV T cell responses in patients with classic VIN could be predictive of spontaneous clinical outcome. Etomidate It may also be thought that high numbers of blood CD4+ and CD8+ lymphocytes after therapeutic vaccination could allow clearance of HPV-16 lesions in classic VIN, assuming that anti-HPV vaccine-induced T effector cells could home into the HPV cutaneous and mucosal lesions. In the present study, we assessed cellular responses against HPV-16 E6 and E7 peptides in 16 patients presenting with classic VIN with the aim of mapping and characterizing the highest immunogenic regions from these proteins as potential candidates for a peptidic therapeutic vaccination.

The compromised signaling response correlated with the inability of the S291A variant to associate with the chaperone prohibitin. No direct interaction between phosphorylated serine 291 and 14-3-3 proteins was observed in

this study 47 despite the evolutionary conservation selleck inhibitor of the canonical mode 1 motif for 14-3-3 binding in murine and human Syk orthologes. The marked discrepancies to our data cannot be attributed to the use of different experimental systems. It remains however possible that murine and human Syk behave differently. This may also explain why we repeatedly identified prohibitin in our quantitative SILAC-based interactome analysis as unspecific “background” protein (Supporting Information Table 2). Future experiments are needed to directly compare the functions of murine and human Syk. However, the negative-regulatory signal circuit described in this paper for the human Syk ortholog in two different cell lines demonstrates the complexity of the Syk signaling

network. Apoptosis Compound Library screening Moreover, our quantitative proteomic approach to comprehensively identify the Syk phosphoacceptor sites and at least some of the their phosphorylation kinetics as well as the interactome of human Syk in resting and activated B cells provides an indispensable clue to finally decipher Syk-regulated signaling pathways under normal and pathological conditions. B-cell culture conditions, lysis and stimulation procedures have been described 30, 48. Immunoprecipitations of citrine-tagged or endogenous Syk, chicken SLP65 and PLC-γ2 from lysates of 3×107 DT40 cells were performed with antibodies to GFP (Roche), Syk (4D10, Santa Cruz), chicken-SLP65 (kindly provided by T. Kurosaki) or PLC-γ2 (Santa Cruz) find more coupled to protein A/G sepharose

(Santa Cruz). Antibodies for immunoblot analyses were used according to manufacturer’s instructions and recognized Syk (Santa Cruz), 14-3-3γ cell signaling technology (CST), GFP (Roche), 14-3-3-binding motif (CST), GST (Molecular Probes), phosphotyrosine (4G10, Biomol) and PLC-γ (Santa Cruz). For Far Western experiments, immunoprecipitated citrine-Syk was subjected to SDS-PAGE, blotted onto nitrocellulose and incubated with 10 μg GST or GST fusion proteins encompassing 14-3-3γ (plasmids kindly provided by S. Beer-Hammer, Düsseldorf) that were expressed in E. Coli BL21 bacteria upon induction with IPTG for 3 h and purified via glutathione sepharose (GE Healthcare). The cDNA encoding human Syk with an N-terminal OneStrep tag (Iba TAGnologies) was ligated into pAbes-puro vector and transfected via electroporation into Syk-deficient DT40 cells (300 V, 975 μF). For further experiments, three independent clones were selected and pooled. The cDNA of N-terminally citrine-tagged human Syk was ligated into pCRII-Topo.

This NKT cell migration in vivo is arrested in liver sinusoids upon encounter with antigen presented on sinusoidal epithelial cells within minutes after injection of αGalCer.[64, Venetoclax cell line 41, 65-67] In addition to antigen,

the IL-12 and IL-18 pro-inflammatory cytokines also terminate type I NKT cell motility in liver sinusoids of Cxcr6gfp/+ mice in a CD1d-independent manner. The latter arrest in NKT cell movement occurs by 1 hr after exposure to the cytokines and precedes NKT cell activation. Subsequent antigen encounter stabilizes the formation of an immune synapse between NKT cells and interacting APCs. This synapse elicits lymphocyte function-associated-1/intercellular adhesion molecule-1 interactions that enable activated type I NKT cells to be retained in the liver, demonstrating that activated type I NKT cells recirculate less than activated conventional CD4+ T cells.[68] However, after a stroke, type I NKT cells rapidly exit the liver and elicit bacteraemia. Similarly, NKT cells extravasate rapidly from the lung of αGalCer-treated mice and trigger inflammation and adaptive immune responses.[69] Hence, the patterns and kinetics of recirculation of type I mouse NKT cells differ in a tissue- and stimulus-dependent manner. Additional studies are required to unravel the mechanisms involved

and to determine whether this variation in recirculation exists for mouse type II NKT cells and human type I and type II NKT cells. Humans possess both CD4+ and CD4− type I NKT cells.[11] Although both subsets secrete Th1-type cytokines, MLN0128 CD4+ type I NKT cells secrete predominantly Th2-type cytokines. In a population of Th1-like CD4− NKT cells, CD8α+ cells comprise a large subset and CD8αβ+ cells a small subset. CD8α+ typeΙΝΚΤ cells secrete more IFN-γ and possess greater cytotoxic activity than do CD4+ or CD4− NKT cells. In human peripheral blood, type I NKT cells comprise about 0·1–0·2% of T cells, but this proportion is highly variable and can range

from < 0·1% to > 2%.[70-72] Twin studies suggest that the number of human type I NKT cells in PBMCs is genetically regulated.[4] Interestingly, human type I NKT cells are enriched in Farnesyltransferase the omentum (about 10% of T cells) and not in the liver.[73, 74] Reduced numbers of type I NKT cells in PBMCs appear to correlate with several autoimmune or inflammatory conditions and cancers,[75] but this finding remains controversial. Similarly in patients with rheumatoid arthritis, PBMCs[76, 77] and synovia[78] display lower levels of NKT cells as well as a Th1 bias during disease.[77] Interestingly, patients with myasthenia gravis display elevated levels of type I NKT cells in PBMCs, in contrast to those in PBMCs from patients with MS,[75] rheumatoid arthritis[76] and type 1 diabetes[79]. The reason for these differences is currently unknown. Nevertheless, NKT cell levels return to normal levels after treatment.

Nukuzuma, unpublished data). Proliferation characteristics of COS-tat cells may provide important background information for studies using these cell lines. Thus, we first compared the cell proliferation of three COS-tat cell lines

with those of parental Dinaciclib mw COS-7 cells. COS-7 cells (ATCC CRL 1651) and COS-tat cell clones (8) were cultivated in EMEM containing 10% FBS (hereafter called culture medium). Cell cultures were maintained at 37°C in a humidified incubator containing 5% CO2 in air. The relative number of live cells was determined by measuring mitochondrial succinate dehydrogenase activity using MTT assay. COS-7 cells and COS-tat cell clones were each plated in five wells of 96-well culture plates at a concentration of 2 × 103 cells/well in 100 μL culture medium and incubated at 37°C in a CO2 incubator. MTT assay was performed using a Cell Proliferation Kit I (MTT) (Roche, Penzberg, Germany) according to

the manufacturer’s instructions. After an incubation period of 5 days, 10 μL MTT solution was added to each well to a final concentration of 0.5 mg/mL, and the plates incubated for 4 hr. Then, CB-839 price 100 μL solubilization solution was added to each well, and the plates placed in an incubator overnight. The formazan products were solubilized, and spectrophotometric data were measured using an enzyme-linked immunosorbent assay reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 550 nm with a reference wavelength of 650 nm. The significance of inter-group differences was statistically determined by Student’s t-test. As shown in Table 1, the enzyme activity of COS-tat7 and COS-tat15, and COS-tat22 cells was lower than that of parental COS-7 cells and this difference Adenosine triphosphate was statistically significant (P < 0.01). Of note, the enzyme activity of COS-tat22 cells was lower than that of COS-tat7 and COS-tat15 cells (P < 0.01). To measure the doubling time, COS-7 cells and COS-tat cell clones were plated in 6-well culture plates at a concentration of 4 × 104 cells/well in 2 mL culture medium. After an incubation period of 72 hr, cell numbers were counted. The

doubling time of COS-7, COS-tat7, COS-tat15, and COS-tat22 were 21.6, 24.6, 22.8, and 30.8 hr, respectively. The doubling time of COS-7 COS-tat cells were in agreement with the proliferation characteristics of the cells as judged by MTT assay. Taken together, these results indicate that stable expression of Tat leads to down-regulation of cell proliferation. We next compared the production of PML-type JCV in COS-tat cell clones with that in parental COS-7 cells. Since JCV capsids have the property of agglutinating human type O erythrocytes, HA assay has been traditionally employed to determine the virus titer (12). COS-7 and COS-tat cell clones were cultured in 35-mm dishes containing 2 mL culture medium until the cells were 50–80% confluent.

Additionally, more investigation is needed to define how HSV-2 infection might modulate HIV-1 pathology. Support for this work was provided by the National Institute of Allergies and Infectious Diseases (grants NIAID AI060379, AI052731 and AI064520 to DFN and AI64520 to LLL). JDB is supported by

AI-066917 and AI-076014 (NIAID). Additional support was provided by the Brazilian Program for STD and AIDS, Ministry of Health (914/BRA/3014 – UNESCO/Kallas), the São Paulo City Health Department (2004-0·168·922-7/Kallas), Fundação de Amparo a Pesquisa do Estado de São Paulo (04/15 856-9/Kallas), selleck chemicals the John E. Fogarty International Center (D43 TW00003) and the AIDS Research Institute of the AIDS Biology Program at UCSF (grant to DFN). MMS and KIC’s scholarships were supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Ministry of Education. LLL is an American Cancer Society

Research Professor. We thank Skip Virgin for helpful discussions. The authors declare no conflict of interest. “
“Citation Petroff MG, Perchellet A. B7 family molecules as regulators of the maternal immune system in pregnancy. Am J Reprod Immunol 2010 Placental and fetal growth and development are associated with chronic exposure of the maternal immune system to fetally derived, paternally inherited antigens. Because maternal lymphocytes are aware of fetal C59 wnt ic50 antigens, active tolerance mechanisms are required Non-specific serine/threonine protein kinase to ensure unperturbed progression of pregnancy and delivery of

a healthy newborn. These mechanisms of tolerance may include deletion, receptor downregulation, and anergy of fetal antigen-specific cells in lymphoid tissues, as well as regulation at the maternal–fetal interface by a variety of locally expressed immunoregulatory molecules. The B7 family of costimulatory molecules comprises one group of immunoregulatory molecules present in the decidua and placenta. B7 family members mediate both inhibitory and stimulatory effects on T-cell activation and effector functions and may play a critical role in maintaining tolerance to the fetus. Here, we review the known functions of the B7 family proteins in pregnancy. Placental and fetal growth and development are associated with chronic exposure of fetally-derived, paternally inherited antigens to the maternal immune system. Based on studies in mice, this exposure to paternal antigens is thought to occur as early as insemination, wanes until establishment of the fully mature placenta, and again becomes robust when the uterine blood supply to the placenta is established.1–3 Once this occurs, the placenta is inundated with maternal blood, and antigen efflux from the fetus persists for the last 1/2 of pregnancy in mice, and 2/3 of pregnancy in women. In women, the continuous shedding of trophoblast cells and other soluble fetal products is thought to be a major source of antigen to maternal immune cells.

Following stimulation and processing, 5 μl of appropriately Selleckchem Y 27632 diluted IFN-γ Alexa488 (BD), CD3 PerCP·Cy5.5 (BD), CD28 PE-Cy7 (BD), TNF-α V450 (BD), IL-2 Alexa488 (BD), CD45 V500 (BD) and PE-conjugated monoclonal antibodies to CD40L, CD152,

CD137, CD134 or isotype control were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analysed as described above. Aliquots of whole blood were incubated with 10−6 M methylprednisolone for 18 h then stimulated for cytokine production and analysed as reported previously [8]. Statistical analysis was performed using the Kruskal–Wallis test and post-hoc analysis using Mann–Whitney and Spearman’s rho correlation tests using spss software and differences between groups of P < 0·05 were considered significant. Corrections for multiple comparisons were not performed. There was no significant difference

in the absolute lymphocyte counts for controls and transplant patients [1·5 (1·4–1·9), 1·6 (1·3–2·1), 1·6 (1·3–2·2) × 109/l, RO4929097 manufacturer median and range for controls, stable patients and patients with BOS, respectively, P > 0·05]. There was no change in the percentage of CD4 or CD8 T cells between controls or transplant groups (61 ± 11·7, 62 ± 12·8, 60 ± 11·9 CD4 and 39 ± 6·7, 38 ± 6·8, 39 ± 8·1 CD8 T cells for controls, stable transplant and BOS patients, respectively). The percentage of CD28null/CD4+ T cells in stable transplant patients was decreased significantly compared to control subjects (Fig. 1). In BOS, there were significant increases in the percentage Aldol condensation of both CD28null/CD4+

and CD28null/CD8+ T cells compared with both controls and stable transplant patients (Fig. 1). CD28null/CD8+ T cells were increased significantly when compared to CD28null/CD4+ in patients with BOS (Fig. 1). There was a significant increase in the percentage of both CD28null/CD4+ and CD28null/CD8+ T cells expressing perforin in stable transplant patients and in patients with BOS compared with controls (Fig. 2a). A similar increase was noted in the CD28+ subgroup (0·2%, 1·0% and 1·1%; and 0·3%, 2·3% and 2·5% CD28+/perforin+/CD4+ and CD28+/perforin+/CD8+ for controls, stable patients and patients with BOS, respectively) (all P < 0·05). There was an increase in the percentage of both CD28null/CD4+ and CD28null/CD8+ T cells expressing granzyme B (GB) in patients with BOS compared with controls (Fig. 2b). For CD4+ T cells expressing GB, the increase was significantly greater in BOS patients compared with stable transplant patients and controls, and in stable transplant patients compared with controls (Fig. 2b). The percentage of CD28null/GB+/CD8+ T cells was higher in all groups compared to the CD4+ subset (Fig. 2b).