Nakashima et al.[48] showed the accumulation of IL-17+ T cells in the deciduas in women GSK3235025 with inevitable abortion. Decidual IL-17+ T cells were mostly CD4+ T cells and a few CD8+ cells also expressed IL-17 in this study. In addition, the number of decidual IL-17+ cells was positively correlated with the number of decidual neutrophils. However, they could not find any difference in the number of decidual IL-17+ T cells between women with missed abortion and normal pregnancy. From these results, the authors concluded that decidual IL-17+ cells might be involved in the inflammation of the late stage of abortive process, not the causative factor of abortion.[48] Because their data of IL-17+

cells were limited to inevitable abortion, not to RPL, it may be difficult to generalize the results as the immunologic mechanism of RPL. A series of studies concerning Th17 cells have been reported regarding RPL in the past 2 years. Wang et al.[70] found an increase in Th17 cells in the peripheral blood and decidua of women with unexplained RPL as compared to normal pregnant women. Serum IL-17 and IL-23 levels were significantly higher in women with RPL. Furthermore, Th17-related selleck products molecules such as IL-17, IL-23, and retinoid orphan receptor C (RORC) were significantly expressed in the deciduas of women with RPL. The number of Th17 cells inversely correlated

with that of regulatory T cells in the peripheral blood and deciduas. The same group has reported another Th17 cell study in women with RPL.[73] They found that the proportions of peripheral blood CCR6+ CD4+ T and CCR6+ IL17+ T cells were significantly

elevated in women with RPL as compared to healthy pregnant women undergoing elective abortion. In ex vivo culture study, IL-17 production from CD4+ T cells was significantly higher Dapagliflozin in women with RPL and regulatory T cells from women with RPL were less suppressive to the expression of IL-17 as compared to control women. Similarly, a decrease in CD4+ CD25bright Foxp3+ regulatory T cells and increase in Th17 cells have been reported in the peripheral blood of women with RPL in comparison with normal healthy pregnant women.[64] The ratio of Th17/regulatory T cells was significantly increased in women with RPL as compared to normal pregnant and non-pregnant women. The proportion of regulatory T cells negatively correlated with the proportion of Th17 cells (Table 1). Serum IL-17 levels correlated positively with Th17 cells and the ratio of Th17/regulatory T cells.[64] These results suggest that regulatory T cells inhibit IL-17 expression and suppressive function of regulatory T cells on Th17 cells may decrease in women with RPL. Our group recently published a study that investigated pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17), anti-inflammatory cytokine IL-10, and Foxp3 in the PBMCs of idiopathic women with RPL.

In contrast, in autoimmune diseases, the pathogenic epitope(s) might bind to one or a restricted set of HLA class II molecules (such as DR*0101, *0401, *0404 in RA), with different binding rules compared to most of the peptides and, perhaps, with low affinity. Thus, in the present study, we used the TEPITOPE program in combination with binding assays to increase the probability to obtain an exhaustive list of epitopes binding to RA-associated HLA class II molecules. Although the dominant hnRNP-A2 core sequence 123–131 found here to be recognized by RA patients was also identified

by TEPITOPE and appears to be promiscuous, this may not be a general rule for various autoantigens and autoimmune diseases. In addition, we found that the flanking amino acid residues were essential since the two overlapping dominant T-cell epitopes 117–133 and 120–133 were differently recognized EPZ-6438 purchase by the patients’ PBMC. This subtle difference highlights the necessity of performing a very detailed peptide

analysis, in addition to the use of computer programs, when searching for disease-relevant T-cell epitopes. Recognition of MHC-self-peptide complexes by T lymphocytes is a central event in autoimmunity. Although many potential epitopes on an antigen selleck products may be able to bind class II molecules, one determinant is usually preferentially processed, the so-called immunodominant epitope 23. The mechanisms of determinant selection likely

involve the availability of the determinant, its class II affinity, including the competition for binding to different MHC molecules, and the proteolytic system of the various APC types 23, 24. For organ-specific autoimmune diseases, APC involved in presenting the appropriate self-epitope are likely located in the draining lymph nodes 25, 26 or in the organ itself 24. Although pathogenic T cells may recirculate throughout the body, they may not be detectable using PBMC since appropriate APC able to process the antigen in its pathogenic determinant may be absent 24. Therefore, Phospholipase D1 our method consisted of using short peptides able to bind directly on the cell surface to MHC class II molecules and of selecting RA-associated HLA epitopes. In addition, we used a high number of PBMC to detect a low frequency of antigen-specific T cells. This approach led to the selection of about 12 determinants within the 341-amino-acid-long hnRNP-A2 protein. These few epitopes appear to be of physiological relevance because the determinant hnRNP-A2 293-310 was recently found naturally processed and presented by I-E(p) molecules 27, the mouse homologue of DR molecules. This determinant has the core sequence 294–302 (Table 1) contained in the peptide 289–306 selected in our study and recognized by cells from patient 12 (Table 2) and from primed DR4-Tg mice (Table 1, Fig. 2).

27 The reduced plasma volume may be explained by the capillary leak syndrome and volume redistribution into the extracellular space, as there is no reduction in extracellular fluid volume.28 Therefore, the elevation in blood pressure may be more closely related to endothelial dysfunction and later, vasoconstriction rather than any direct effect of the RAAS.11 Alternative locally vasoactive compounds such as endothelin, nitric oxide inhibition, oxidative stress or cytokines have been implicated as vasoconstrictors in preeclampsia but selleck compound are not proven.29 The use

of antioxidants in humans has not been shown to treat or prevent preeclampsia.30 Interest in the endothelial cell integrity provided by angiogenic factor vascular VEGF (vascular endothelial growth factor) in pregnancy and its potential role in preeclampsia are not new.31,32 There was a resurgence in interest in angiogenic molecules after the elegant demonstration of a mechanistic role for the soluble VEGF receptor, soluble fms-like tyrosine kinase-1 (sFLT-1)33 in preeclampsia. The infusion of sFLT-1 in pregnant rodents induced

RXDX-106 price hypertension and proteinuria in pregnancy. The pathological feature of renal biopsies in this model is endothelial disruption similar to that seen in human preeclampsia. The same renal lesion, however, was seen in non-pregnant animals, thus providing evidence of direct renal toxic effect of sFLT-1. The specificity of this pathological mechanism in pregnancy rests with the placenta as the likely site of production. Zhao et al. have demonstrated a net increase in sFLT-1 binding in human renal tissue in preeclampsia.34 We and others have shown that the likely source of the sFLT-1 is acute placental ischaemia35,36 and that the effect of ischaemia and sFLT-1 on the renal capillary loops mimic those seen in human de novo disease (Fig. 1). The clinical importance of the increased sFLT-1 in humans

was demonstrated subsequently Thiamet G in a longitudinal retrospective study. It was found that the maternal circulating sFLT-1 was significantly increased in women who were to develop preeclampsia later in the pregnancy. The elevation in sFLT-1 was noted about 5–6 weeks prior to the onset of clinically apparent disease.37 The correlation of high sFLT and low binding proteins VEGF and its co-agonist placental growth factor (PlGF) confirm the binding activity of the sFLT-1. The relative reduction in free VEGF (resultant from the increased sFLT-1) has a potentially important role in mediating the renal involvement in preeclampsia as outlined above. Other recently identified toxins in preeclampsia such as soluble endoglin38 do not appear to be a direct glomerular cell toxins,39 at least in animal studies where its effect is most potent in the presence of sFLT-1.

13 months vs 19.63 months, P = 0.019). The rate of 24-h urinary protein decrease after valsartan treatment in the present study is consistent PF-562271 with previous studies. For example, the HKVIN study of patients on ARBs showed that the 24-h urinary protein was reduced from 1.80 ± 1.24 g at baseline to 1.26 ± 1.21 g at week 52, and to 1.23 ± 1.25 g at week 104 (P < 0.001).[15] Previous study in the rat indicated that the antioxidant probucol, when added to an Ang II receptor blockade, fully arrests proteinuria and disease progression

in GN.[16]Another study also demonstrated that treatment with an anti-oxidant, alpha-Tocopherol, alone reduced urinary protein in IgA nephropathy in the rat.[17]Consistent with these animals’ results, our data also indicated that during the first 2 years of treatment, probucol Fluorouracil cell line (an antioxidant and anti-hyperlipidemic agent) in combination with valsartan rapidly reduced urinary protein, a response known to decrease the risk for ESRD in high risk IgA nephropathy patients.[12] This more rapid reduction in urinary protein in the combined therapy group compared to valsartan treatment alone might be due to the potent anti-oxidative effects of probucol.[16] In addition, patients receiving probucol had a decline in plasma cholesterol in the early phases of treatment, but an increase in the later

phases of treatment. These changes in plasma cholesterol paralleled the changes in urinary protein excretion. Previous clinical trials also demonstrated that lowering cholesterol with statin regimens were able

to decrease proteinuria and to improve renal function.[18, 19] Therefore, we cannot rule out the probability of urinary protein reduction is due to the effect of probucol in lowering cholesterol. During the first 2 years of follow-up in our patients, urinary protein tended to decrease. Previous studies reported that urinary protein decreased at 3 months to 2 years after initiation of therapy,[20-22] which was consistent with our findings. However, we noted that urinary protein had increased Acesulfame Potassium at the 2- and 3-year follow-ups, especially in the valsartan (control) group at 2 years. At the end of the study, the 24-h urinary protein levels were comparable to the baseline levels in both groups. This suggests that 750 mg probucol combined with 160 mg valsartan may decrease proteinuria, but the long-term effect remained less convincing. This might be a result of an increase in oxidative stress due to the development of compromised endogenous anti-oxidative responses over the course of 3 years. Disruption of the immune response has a role in the pathogenesis of IgA nephropathy.[23, 24] oxidative stress was only as a minor reason. So the absence of steroids and/or immunosuppressants fails to forestall disease progression.

In HD, halting immunosuppression did not Pexidartinib manufacturer correlate with graft rejection [160] at early or later time points, except in one case [51]. As we mentioned in previous sections, the post-mortem analyses of HD transplanted cases a decade following grafting revealed a strong immune response cuffing the grafts [43], in conditions where the immunosuppression began 2 weeks before the surgery and continued for 6 months [17]. It is possible that solid tissue grafts, following the withdrawal of the immunosuppressive therapy, may present enhanced antigenic stimulation triggering a more robust inflammatory reaction, as compared with cell suspension grafts [155,156,161–163].

Solid grafts may trigger a stronger immune response also because they still contain the donor vasculature which is highly immunogenic [139]. Finally, the MI-503 in vitro use of multiple donors may represent another important

variable in introducing a number of mismatched HLA tissues [160]. Some of the critical steps to insure the success of the transplant surgery involve the choice of suitable candidates, and the identification of the exact patient characteristics that can predict treatment outcome. Drawing conclusions from the very limited number of studies currently available is obviously a difficult task. Patients recruited so far show important variability regarding their age at the time of transplantation, their symptom duration, their number of CAG repeats, the time of transplantation from diagnosis and their UHDRS motor score. Nevertheless, we have attempted to analyse these parameters to determine whether any factor might account for

the various behaviours observed for transplants between studies. For this purpose, we have excluded the cases analysed at early time points after transplantation [43–46]. We thus arbitrarily assessed graft survival giving a score from 0 (no graft survival) to 5 (all grafts having survived) and performed Spearman correlation analysis with the selected parameters. These analyses, although performed on PD184352 (CI-1040) a very limited number of cases (n = 7), suggested that grafts survived better when implanted in younger patients (Figure 2A; Spearman r = −0.97101, P = 0.0012) who manifested symptoms for a shorter period of time (Figure 2B; Spearman r = −0.9255, P = 0.008). Surprisingly, patients with the higher number of CAG repeats showed better graft survival (Figure 2C; Spearman r = 0.93796, P = 0.0057). Although it is difficult to explain how higher CAG repeats may not be detrimental to graft survival, our analysis suggests that younger patients at earlier phases of disease progression may be better candidates for transplantation, as severe brain atrophy may represent a less than favourable niche for graft survival and integration. Cell therapy offers the possibility to replace degenerated neurones and thereby to improve symptoms and signs in neurodegenerative diseases such as HD.

8 The continuous

wakefulness condition was performed in order to distinguish sleep-dependent and diurnal variations in T-cell responses. Inclusion criteria for volunteers were as follows: mental and physical health (determined from medical history, physical examination and routine laboratory testing); a body mass index between 18 and 26 kg/m2; no sleep disturbances; non-smoker; and not taking medication. Each subject participated in two experimental sessions, each covering 24 hr R788 datasheet and starting at 20:00 hr. Each subject spent an adaptation night in the sleep laboratory, where sleep was determined offline from polysomnographic recordings according to standard criteria.32 All subjects received standardized meals and blood samples were processed immediately. An intravenous forearm catheter (Braun, Melsungen, Germany) was connected to a long thin tube, allowing blood collection from an adjacent room without disturbing the subject’s sleep. Blood samples, taken at five time-points (20:00, 02:00, 07:00, 15:00 and 20:00 hr) into heparin anticoagulant, were used for isolation and functional analyses of CD4+ CD25high nTreg and CD4+ CD25− Tres. Hormone levels were measured periodically every 3 hr. The protocol

was approved by the local ethics committee and all subjects signed informed consent forms. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood applying into CPT® Vacutainer (BD Biosciences, Heidelberg, Germany), according to the Metformin in vitro manufacturer’s instructions. Plasma was collected, inactivated by heating at 56° for 30 min

and then centrifuged at 4500 g. The supernatant was designated Florfenicol as autologous inactivated plasma. T cells were isolated from PBMC and separated into nTreg and Tres populations using the CD4+ CD25+ Regulatory T Cell Isolation Kit® (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions, in combination with an autoMacs® Separator (Miltenyi Biotec). We subsequently refer to this isolation protocol as MACS®. For logistical reasons we performed this protocol for the diurnal analysis. Cell purities were examined using flow cytometry. As a control for the results obtained with MACS-isolated Tres and nTreg we also performed an isolation protocol where negatively MACS isolated CD4+ T cells were sorted in CD25− and CD25high T cells by fluorescence-activated cell sorting (FACS), using MoFlo® (DakoCytomation, Hamburg, Germany). We will refer to this isolation protocol as MACS + Sort. The CD4− cells were enriched for monocytes by plastic adherence for 2·5 hr and, after harvesting, were irradiated with 60 Gy using a cobalt source. For proliferation assays, half of the Tres obtained were stained with carboxyfluorescein diacetate (CFSE) and the other half were left unstained for control purposes. For analysis of the suppressive activity of nTreg on Tres, we employed a procedure described previously33 with minor modifications.

Results: In LN tissues, CD147 induction was striking in injured glomeruli and infiltrating inflammatory cells, but not in damaged, atrophic tubules. Plasma CD147 levels accurately reflected the histological disease activity in both acute and chronic phase of LN. Since prediction of disease activity with a single biomarker might be difficult because of complex pathogenesis SAHA HDAC molecular weight of LN, we further evaluated encouraging combinations of multiplex markers. Interestingly, higher the area under the curve (AUC)

scores were shown in the combination of marker such as plasma CD147+ component C3 (AUC. 0.92). In addition, inactive LN patients treated with immunosuppressive therapy exhibited the reduction of plasma CD147 values compared to active LN patients before treatment. LN patients tended to show the higher levels of plasma CD147 than SLE patients without renal involvement. Conclusion: Plasma CD147 levels might offer useful insights into disease selleck chemicals llc activity as a crucial biomarker in patients with LN. TAKAHASHI KAZUO1, KONDO AYAKO1, HIRANO DAISUKE2, AKIYAMA SHINICHI1, HAYASHI HIROKI1, KOIDE SHIGEHISA1, HASEGAWA MIDORI1, YOSHIDA SHUNJI2, HIKI YOSHIYUKI3, MIURA KEIJI4, YUZAWA YUKIO1 1Department of Nephrology, Fujita Health University School of Medicine; 2Rheumatology, Fujita

Health University School of Medicine; 3Fujita Health University School of Health Sciences; 4Fujita Health University, Institute of Comprehensive Medical Science Introduction: Although anti-endothelial cell antibodies (AECA) against

human umbilical vein endothelial cells (HUVEC) have been detected in systemic lupus erythematosus (SLE), their pathological role remains unclear. Because antigens expressed on the endothelial cell (EC) surface are pivotal for autoimmune reactions, methods that detect antibodies only to EC surface molecules are required. Therefore, we developed a solubilized cell surface protein capture enzyme-linked immunosorbent assay (CSP-ELISA) that is able to detect antibodies against membrane proteins. We also aimed to elucidate the clinical importance of AECA for tissue-specific EC. Methods: Sera from 52 patients with biopsy-proven lupus nephritis (LN), 25 with SLE without renal involvement (non-LN Bumetanide SLE), 10 disease controls (DC) and 81 healthy controls (HC) were tested for IgG- and IgA-AECA to human glomerular EC (HGEC) by CSP-ELISA. Results: Titers of IgG- and IgA- AECA to HGEC were significantly higher in LN and non-LN SLE patients than in the combined DC and HC (P < 0.001) groups. The level of IgG-AECA did not correlate with active lesions defined by ISN/RPS classification, but the level of IgA-AECA to HGEC did correlate with histological evidence of active lesions in LN patients (P < 0.001). Immunocytochemical analysis demonstrated that AECA recognized membrane proteins on HGEC. The significant correlation of titer of AECA to both HGEC and HUVEC (R2 = 0.90 for IgG-, 0.

All experimental protocols were approved by the Animal Experimentation Ethics Committee, Faculty of Chemical Sciences, www.selleckchem.com/products/poziotinib-hm781-36b.html National University

of Cordoba (resolution number 1135/09). Serotype A C. neoformans strain 102/85 (National University of Cordoba stock culture collection) was used. This strain of Cryptococcus is a clinical isolate with a large capsule, typified by a polymerase chain reaction (PCR) multiplex and PCR fingerprinting (Centro de Biotecnologia da Universidade Federal do Rio Grande do Sul, Brasil) as C. neoformans var. grubii, which has been used in previous studies.6,20–23 To perform the experiments, living yeasts of C. neoformans were expanded in liquid Sabouraud media for 24 hr in a gyratory shaker at 30°. Then, the yeasts were washed three times with phosphate-buffered saline (PBS), resuspended at 107 cells/ml and opsonized with 5 μg/ml of mAb 3C2 for 30 min at 37°. After this, the yeasts were washed with PBS and finally resuspended in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine and 50 μg/ml gentamycin for subsequent cultures with eosinophils. Eosinophils were purified from the peritoneal cavity

of normal rats by washing it with cold PBS, pH 7·3, containing 0·1% FBS. The cells thus obtained were centrifuged at 400 g for 10 min and resuspended in Carfilzomib cost 2 ml of 1× Hanks’ balanced salt solution (HBSS). Then, the cells were separated on a discontinuous Percoll gradient (2 ml of a solution of Percoll with a density of 1·090 g/ml Demeclocycline and 2 ml with density of 1·080 g/ml, carefully

overlaid). The tubes were centrifuged at 400 g for 25 min, and the eosinophils were collected from the middle interface between the Percoll layers.24 The percentage of eosinophils was > 90%, as determined by May–Grünwald–Giemsa staining. This population was further purified by negative selection, by incubation for 30 min with anti-CD11b/c- and anti-OX-62-labelled fluorescein isothiocyanate (FITC), and then for a further 15 min with anti-FITC MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The eosinophil population contained < 1% OX-62+ cells and < 2% CD11b/c+ cells, which was not significantly different from the isotype control (Fig. S1). Finally, the percentage of eosinophil viability was > 95%, as determined by the Trypan Blue dye-exclusion test. Purified eosinophils were incubated in supplemented RPMI-1640 alone, or with opsonized or non-opsonized live yeasts of C. neoformans at 37° and a 5% CO2 humidified atmosphere, in the presence or absence of GM-CSF (5 ng/ml). For some comparative experiments, rat peritoneal Mφ were used. These cells were purified from the upper interface of the Percoll layers. Phagocytosis assays were performed as described in previous studies with some modifications.

Several lines of evidence indicate an important role of miRNAs during innate and adaptive immune responses 14. MiR-146 has been shown to regulate Selleck p38 MAPK inhibitor TLR-mediated inflammatory responses, whereas miR-155 and miR-181a have been implicated in B- and T-cell-mediated immune responses 14–16. To date, most of the information regarding the role of the miRNAs during an immune response has been obtained through either genetic ablation or overexpression. However, how individual miRNAs are altered during breakdown of tolerance and initiation of an autoimmune response at the cellular level remains unclear. In this study, using PD-1−/− mice, we demonstrate that Ag-primed PD-1−/− T cells

upregulate microRNA 21 (miR-21) expression upon TCR ligation. In vitro inhibition of miR-21 activity results in reduced proliferation and cytokine secretion

by Ag-specific T cells. Importantly, PD-1 inhibition results in phosphorylation of STAT5, which binds in the promoter area of miR-21 and upregulates miR-21 expression. Collectively, our findings suggest that PD-1 through a microRNA signaling cascade regulates the balance Seliciclib between tolerance and immune activation. To assess the breakdown of tolerance and development of AIA in the absence of PD-1 pathway, PD-1−/− and WT control mice were subcutaneously (s.c.) immunized with ovalbumin (OVA)/CFA followed Tangeritin by an intra-articular injection of OVA/PBS. One day after the intra-articular challenge, both group of mice developed an acute inflammatory reaction and severe arthritis (Fig. 1A). The extend of joint swelling decreased in WT mice starting day 2 after AIA induction in contrast to PD-1−/− mice where disease severity remained high during the entire period of monitoring. As shown in Fig. 1B, on day 8 after challenge the affected paw of PD-1 KO mice was observed to have severe swelling and loss in the range of motion, in contrast to WT mice in which the inflammatory reaction had diminished. To characterize the T-cell immune responses

in OVA-immunized PD-1−/− mice, draining lymph nodes (dLNs) were isolated 9–10 days after antigenic challenge and cells were cultured in vitro in the presence of varying concentrations of OVA. T cells from PD-1−/− mice exhibited higher proliferation than T cells from WT mice in response to Ag (stimulation index=18 for PD-1−/− mice versus 5.5 for WT at 50 μg/mL OVA) (Fig. 1C). The T-cell proliferation profile correlated with the capacity of OVA to elicit IFN-γ and IL-17 production by OVA-primed T cells. Thus, the analysis of culture supernatants revealed that OVA-primed LN cells from PD-1−/− mice secrete increased amounts of IFN-γ upon stimulation as compared with WT T cells (2400±76 pg/mL for PD-1−/− versus 1790±5 pg/mL for WT) (Fig. 1D).

The serine protease CatG uniquely was able to cleave MHC II molecules in vitro. CatG is abundant in storage granules of neutrophils; it is released in inflammatory sites and contributes to innate

protection from bacterial infection. Non-immune roles for CatG are suggested by subtle developmental defects in CatG-deficient mice.18 Notably, CatG is expressed in primary human APCs, such as B cells, monocytes, and myeloid and plasmacytoid DCs,19,20 where it has been shown to contribute to proteolytic antigen processing.21 Here, we characterized the specificity of CatG cleavage of MHC II molecules in vitro, and examined whether CatG contributes to MHC II turnover in vivo. The HLA-DM-deficient human B-LCLs 9.5.3 and 5.2.4, their parent line 8.1.6 and the 5.2.4-DR3 transfectant have been described previously.22–24 Transduced find protocol B-LCL 5.2.4 expressing the mutant HLA-DR3 molecules

DRB R74Q, DRB D152N, DRB S197N and DRB E187K have been described.24,25 Schneider-2 Drosophila melanogaster (S2) cells expressing recombinant soluble HLA-DR molecules have been described previously.26,27 Mammalian cells were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) and 2 mm l-glutamine (Life Technologies, Carlsbad, CA). S2 cells were cultured as described previously.28 Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donor blood. B cells and myeloid type 1 dendritic cells (mDC1s) were positively selected using immunomagnetic selleck chemicals beads specific for CD19 and CD1c, respectively [magnetic-activated cell sorting (MACS); Miltenyi Biotec, Auburn, CA] according to the manufacturer’s protocols. The purity of primary cell preparations routinely exceeded 90%. Cells were cultured in the presence or absence

of the CatG-specific inhibitor I (10 μm; Calbiochem, San Diego, CA; Compound 7 in29) or E64d (10 μm; Calbiochem) for 4·5, 24 or 72 hr at 37°, and either analysed by flow cytometry or prepared for western blotting by lysis in 10 mm Tris (pH 7·5), 150 mm NaCl, 0·5% NP-40, and CatG-specific inhibitor (1 μm), Janus kinase (JAK) followed by adjustment for equal total protein content (quantified by the Bradford assay). Purification of full-length native HLA-DR molecules was performed essentially as described previously.26,27 Briefly, B-LCLs were lysed in 10 mm Tris (pH 7·8), 140 mm NaCl, and 0·5% NP-40. The lysate was pre-cleared by centrifugation and filtration and passed over an anti-DR (L243)-sepharose immunoaffinity column (L243: IgG2a anti-DR). The column was washed extensively (50 mm Na-phosphate, 150 mm NaCl and 1% octylglucoside, pH 8) and eluted at high pH (100 mm glycine-NaOH and 1% octylglucoside, pH 11). Soluble HLA-DR was purified from insect cell supernatants by a similar method, except that detergents were omitted.