After biotinylation, tetramers are formed by mixing the biotinyla

After biotinylation, tetramers are formed by mixing the biotinylated peptide–HLA complex with fluorophore-labelled avidin [22,46]. The traditional avidin-based tetramers have been superseded by complexes of five HLA/peptides, known as pentamers. HLA class II tetramers have been more

difficult to produce because the peptide complexes are less stable than HLA class I and interactions with the TCR are weaker than HLA class I/ CD8+ T cell interactions [46]. None the less, recombinant class II molecules that incorporate ‘leucine zipper’ motifs can be produced in stably transfected Drosophila cells and purified by affinity chromatography [50]. Because of the very low frequencies of CD4+ T cells specific for self-antigens, this assay often utilizes an in vitro amplification step to increase the threshold of detection [51]. Loading tetramers with modified agonist peptides can increase the tetramer’s binding affinity and allow low-avidity T cell populations to be detected [52,53]. Parallel sorting of tetramer-positive cells, followed by RNA transcription profiling, enables extensive determination of buy BYL719 their functional phenotypes [54]. The recently developed fluorescent quantum dots have been used to label HLA class I tetramers. Quantum dots have narrow emission spectra, making

them ideal for multiplexed tetramer staining [55]. Quantum dots may also prove useful for labelling HLA class II reagents. Advantages. Tetramers and pentamers are unique reagents

because they can identify antigen-specific T cells directly. This property makes them very useful for validating epitopes identified by other means. Ex-vivo tetramer staining (class I and class II) enables direct estimation of the frequency of antigen-specific T cells [56]. Disadvantages.  Class II tetramers are not suitable for use in routine clinical monitoring to detect biomarkers of disease. In vitro expansion of the antigen-specific T cells is required to increase their frequency to detectable levels and may lead to over- or under-estimation of the cell populations depending upon their capacity to proliferate in vitro. Furthermore, large Branched chain aminotransferase volumes (∼50 ml) of blood are required to isolate the required numbers of PBMC. One possible limitation of both class I and class II tetramer assays is that low-affinity TCR-bearing cells may not be detected. Therefore, tetramer staining combined with proliferation and/or cytokine secretion assay may yield more information than either assay alone [57]. HLA-A*0201 pentamers (ProImmune, Oxford, UK) loaded with the autoantigenic epitopes of choice, positive control viral epitope(s) and negative control epitope. Cell sample, e.g. blood sample (RBC-depleted), PBMCs or T cell line. Pro5® recombinant MHC pentamer conjugated to the fluorescent label of choice (note: ensure that the stock pentamer is stored consistently at 4°C in the dark, with the lid tightly closed).

Expression of transcription factors regulating earlier stages (IR

Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells. When B cells are activated by T-cell-dependent antigens, they start proliferating and can form germinal centers (GCs) where affinity maturation and class switch recombination (CSR) of the immunoglobulin (Ig) take place. Secreted and membrane-bound

molecules made by T cells are important for the GC reaction, and CD40L is one of the essential molecules 1. GC B cells can differentiate to Ig-producing plasma cells, and cytokines like IL-4, IL-6, IL-10 and TGF-β direct which Ig isotype Buparlisib chemical structure is produced 2–4. IL-21 has emerged as a strong inducer of B-cell differentiation and Ig production in vitro, and the strength of IL-21 exceeds other positive regulators like IL-2, IL-4 and IL-10 5–8. The combination of CD40L and IL-21 can induce CSR to IgA and IgG 7. The different stages of plasma cell development are regulated by a web of interacting see more transcription

factors. Pax5 and BCL6 are highly expressed in GC B cells, but they are not expressed in plasma cells where B-lymphocyte-induced maturation protein 1 (Blimp-1) and X-box binding protein 1 (XBP-1) are highly expressed 9. BCL6 is required for GC formation 9 and Pax5 upregulates the enzyme activation-induced cytidine deaminase (AID) which is necessary for CSR 10, 11. Another primary function of BCL6 and Pax5 is to repress Blimp-1 and XBP-1 respectively, which are both necessary for plasma cell differentiation 12, 13. To allow terminal B-cell differentiation, Pax5 and BCL6 must be repressed by Blimp-1 14, 15 and the mutual repression of Blimp-1 and BCL6 forms a feedback loop enforcing irreversible plasmacytic differentiation. Blimp-1 induces plasma cell differentiation by repressing genes involved in proliferation and GC functions 15, and indirectly induces XBP-1 expression by downregulating Pax5 16. The role of XBP-1 is to enhance the secretory capacity of plasma cells 17. The

transcription factor interferon regulatory Diflunisal factor 4 (IRF-4), functioning upstream of XBP-1, is also required for plasma cell differentiation and an important role for IRF-4 is to repress BCL6 18, 19. Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily, and mediate their effects by binding to a hetero-oligomeric complex of type I and type II serine-threonine kinase receptors. In humans, three BMP type I receptors and three BMP type II receptors have been identified 20. When BMPs bind to the receptors, the type II receptor phosphorylates the type I receptor, which subsequently phosphorylates the receptor-regulated Smads: Smad1, Smad5 and Smad8. Together with Smad4, Smad1/5/8 form a complex which translocates to the nucleus and induces transcription of BMP target genes including the DNA-binding protein inhibitors (IDs) ID-1, ID-2 and ID-3 20, 21.

Conclusions:  At therapeutically relevant concentrations, rapamyc

Conclusions:  At therapeutically relevant concentrations, rapamycin inhibits VEGF- and PAF-induced microvascular permeability. This inhibition is (i) a direct effect on

the endothelial barrier, and (ii) independent of arteriolar vasodilation. Rapamycin at 10 mg/kg stimulates effectors that increase microvascular permeability. “
“Please cite GPCR Compound Library in vivo this paper as: Michel CC. Electron tomography of vesicles. Microcirculation 19: 473–476, 2012. In this issue of Microcirculation, Wagner, Modla, Hossler and Czmmek [25] describe the use of electron tomography to visualize the three-dimensional arrangement of small endothelial vesicles and caveolae of muscle capillaries. Their images show the well-known clusters of fused vesicles communicating with caveolae at the luminal and abluminal surfaces. The advantages of electron tomography are shown by well resolved images of single cytoplasmic vesicles separate from fused vesicle clusters and also by occasional chains of fused vesicles forming trans-endothelial channels. Twenty five to thirty years ago the existence of both trans-endothelial channels

and single unattached vesicles was disputed. Also, since some single vesicles and all of the trans-endothelial channels are labeled with a lanthanide tracer present in the perfusate Y-27632 in vitro at the time of fixation, this evidence once again raises the question of whether vesicles have a role in vascular permeability to macromolecules. This brief review describes the origin of the vesicle controversy, some of the more recent evidence for and against

the participation of vesicles in macromolecular transport and considers some criticisms of ultra-structural evidence for vesicular transport that still require answers. Two papers in this volume of Microcirculation describe investigations of endothelial cell structure Aspartate using electron tomography. The first [1] highlighted its potential as a tool for examining the structure of the glycocalyx on the luminal surface of endothelia. The second by Wagner et al. [25], which appears in the current issue, uses electron tomography to explore the caveolae (or plasmalemmal vesicles) and shows images that, 25 years ago, would have been highly controversial. Before discussing the vesicle controversy and the relevance of these new observations, it is worth saying a little about electron tomography. Electron tomography is the reconstruction of an object’s three-dimensional structure from a sequence of projections, made as transmission electron micrographs TEMs. The underlying principle is the same as that used in X-ray computerized tomography. Its application in electron microscopy dates from the work of DeRosier and Klug [7] who were aiming to improve electron micrographs of macromolecules. The principle is relatively straightforward.

“Immune-based therapies that prevent type 1 diabetes or pr

“Immune-based therapies that prevent type 1 diabetes or preserve metabolic function remaining at diagnosis have become a major objective for funding agencies and international trial consortia, and receive backing from notable patient advocate groups. The development of immune-based therapeutic strategies in this arena requires a careful balancing of the risks of the therapy

against the potential benefits, because many individuals are diagnosed or identified as being at increased risk of disease in early childhood, a period when manipulation of the developing immune system should be undertaken with caution. In addition, a therapy exists (daily insulin injection) that is life-saving in the acute stages of disease and can be used effectively MK-1775 manufacturer over Gemcitabine a lifetime as maintenance. Conversely, the disease is increasing in incidence; is peaking in ever-younger age groups; carries significant risk of increased

morbidity and early mortality; and remains difficult to manage effectively in many settings. With these issues in mind, in this article we review progress towards immune-based strategies for this chronic autoimmune disease. Other Articles Published in this Series Immunological biomarkers: Catalysts for translational advances in autoimmune diabetes. Clinical and Experimental Immunology 2013, 172: 178–85. With the exception of one or two early attempts at disease modulation, the field of immunotherapy for type 1 diabetes did not develop significant DOK2 momentum until the 1980s, during which a series of studies were initiated that made use of a drug (cyclosporin) which had, by then, revolutionized immune

suppression in the setting of organ transplantation. Some 20 years on from those early successes, in 2007 we reviewed the status of intervention and prevention trials for type 1 diabetes [1]. The timing of our commentary was significant; the first major advance since cyclosporin had recently emerged, notably with the publication of two studies using monoclonal antibodies (mAbs) targeting CD3 and engineered to have limited Fc binding, both of which demonstrated clinically relevant efficacy with manageable toxicity [2, 3]. At that stage we discussed the fact that these drugs (subsequently emerging as teplizumab and otelixizumab) were lead agents at the head of a therapeutic pipeline of immunomodulators. These included several drugs that were emerging from the fields of transplantation immunology and as treatments for other autoimmune and inflammatory diseases, as well as disease-specific, antigen-based therapeutics.

Databases searched: MeSH terms and text words for type 1 and type

Databases searched: MeSH terms and text words for type 1 and type 2 diabetes mellitus were combined with MeSH terms and text words for renal replacement therapy and dialysis. The search was carried out in Medline (1950–March, Week 3, 2008). The Cochrane Renal Group Trials Register was also searched for trials

not indexed in Medline. Date of search/es: 2 April 2008. A prospective study was conducted by Villaret al.5 in order to examine the epidemiology and long-term survival of patients with incident end-stage kidney disease (ESKD) by diabetes status in Australia and New Zealand. The ANZDATA Registry was used to identify patients ≥16 years of age who began dialysis from 1 April 1991 to 31 December 2005. Data collection consisted of information on patient demographics, comorbidites and multiple other parameters CP-673451 nmr (Table 1). This study included 1284 patients with type 1 diabetes (4.5%), 8560 patients with type 2 diabetes (30.0%) and 18 704 non-diabetic patients (65.5%). Rates of coronary artery, peripheral vascular and cerebrovascular disease were higher in diabetic than in non-diabetic patients (Table 1) (P < 0.0001). Multivariate survival analysis showed the risk for death Dinaciclib solubility dmso after the first dialysis treatment was 64.0% (HR 1.64 (1.47–1.84)

greater in type 1 diabetic (P < 0.0001) and 13.0% (HR 1.13 (1.06–1.20) higher in type 2 diabetic (P < 0.0001) patients versus non-diabetic patients. Sex was not associated with survival in type 1 diabetics or Miconazole in non-diabetics; however, older (≥60 years) type 2 diabetic women

had a worse outcome than older type 2 diabetic men, and this difference did not appear to be explained by different comorbid conditions. In type 1 diabetic patients, survival did not alter over time (adjusted HR 0.94 (0.83–1.07) per 5-year period, P = 0.36 but it improved significantly by 9.0% per 5-year period in type 2 diabetics (0.91 (0.87–0.95), P < 0.0001) and by 5% in non-diabetic patients (0.95 (0.92–0.98), P = 0.001). In the DOPPS, a prospective observational study of haemodialysis practices and clinical outcomes among patients treated at randomly selected dialysis facilities in France, Germany, Italy, Japan, Spain, UK and the USA (2004), diabetes was associated with a significantly higher relative risk of mortality (RR = 1.55, P < 0.001).6 Similarly, from the USRDS database, the 5-year survival in diabetic haemodialysis patients is 20% compared with 50% in non-diabetic patients.7 The percentage of all deaths attributed to cardiovascular disease (CVD) in diabetic haemodialysis patients varies from 23% to 54%.

The finding that VCAM-1+ stroma express 4–1BBL, CCL19, CXCL12, an

The finding that VCAM-1+ stroma express 4–1BBL, CCL19, CXCL12, and IL-7 and that adoptively transferred CD8+ memory T cells are often found in

proximity to VCAM-1+CD45− cells in the BM demonstrates the plausibility of the VCAM-1+ stromal cell as A-769662 mw the radioresistant cell that provides 4–1BBL to memory CD8+ T cells in the BM. These data support a model in which a radioresistant VCAM-1+ stromal cell attracts the VLA-4+ CD8+ memory T cells via CCL19, where they can receive 4–1BB-4–1BBL induced survival signals. As the VCAM-1-positive stromal population is very abundant in the BM, there may be heterogeneity in the VCAM-1+ stroma with respect to 4–1BBL, cytokines, and chemokines that contribute to CD8+ T-cell memory maintenance. Further analysis will be required to definitively identify the 4–1BBL-expressing radioresistant cell that contributes to CD8+ T-cell memory. C57BL/6 WT mice were obtained from Charles River Laboratories (St. Constant, QC, Canada).

4–1BB−/− mice [47] extensively backcrossed to the C57BL/6 (n = 10) background were bred in our facility. These mice were previously provided to us by Dr. Byoung S. Kwon (National Cancer Center, Ilsan, Korea). 4–1BBL-deficient (4–1BBL−/−) mice were originally obtained under a materials transfer agreement from Immunex (Amgen, Thousand Oaks, CA, USA) and further backcrossed to the C57BL/6 background in our facility (total n = 9). OT-I

and CD45.1 congenic mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and crossed to Roscovitine generate CD45.1+/+ or CD45.1+/− OT-I mice. TCRα−/– mice were kindly provided by Dr. Cynthia Guidos (Hospital for Sick Children, Toronto). FoxP3gfp knock-in mice on the C57BL/6 background were kindly provided by Dr. Mohamed Oukka (Harvard Medical School) [48]. ACTB-DsRed transgenic mice expressing DsRed protein under control of the β-actin promoter and backcrossed to B6 mice for five generations (B6.Cg-Tg (ACTB-DsRed*MST) 1Nagy/J) were obtained from the Jackson laboratories and crossed with OT-I mice to obtain OT-I ACTB-DsRed mice (OT-I-DsRed). Mice were maintained under specific pathogen-free conditions in sterile microisolators at the University of Toronto. All mouse experiments were approved Orotidine 5′-phosphate decarboxylase by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on animal care (University of Toronto approved protocol #20007828). CD8+ T cells with a central memory phenotype were generated by culture with Ag followed by IL-15 using a variation of a previous protocol [7, 29]. In brief, OT-I splenocytes were stimulated with 0.1 μg/mL SIINFEKL peptide and 1 μg/mL of LPS for 1 day, and then the nonadherent cells were rested for 2 days in fresh media (RPMI-1640 with 10% heat-inactivated FCS, 0.03% L-glutamine, antibiotics, and 2-mercaptoethanol).

e Toxoplasma encephalitis [23, 36] Consistently, blocking NF-κB

e. Toxoplasma encephalitis [23, 36]. Consistently, blocking NF-κB signaling, which is required for astrocyte activation in EAE, by tissue-specific ablation of key signaling molecules including NEMO, IKK2, and

Act1 in the CNS impaired astrocytic production of inflammatory cytokines and chemokines ameliorating EAE as evidenced by decreased leukocyte infiltration and reduced demyelination [5, 37, 38]. Interestingly, in sharp contrast to the proinflammatory function of most astrocyte-derived chemokines, CXCL12, which is upregulated in the CNS of MS patients, particularly produced by astrocytes, suppressed ongoing EAE by redirecting the polarization of effector Th1 cells into IL10-producing Treg cells [39]. Collectively, the present study extends Selleck Enzalutamide the in vivo Selleckchem Sotrastaurin function of astrocytes and illustrates that astrocytes also confer protection against EAE by the

FasL-dependent apoptotic elimination of activated CD25+ Foxp3− and GM-CSF-producing CD4+ T cells and the concomitant inhibition of proinflammatory cytokine production. Thus, augmentation of astrocytic FasL may provide a favorable strategy for treatment of clinically active MS. GFAP-Cre+/− FasLfl/fl mice were generated by crossing C57BL/6 GFAP-Cre transgenic mice [40] with C57BL/6 FasLfl/fl mice [41] and the colony was maintained by breeding of GFAP-Cre+/− FasLfl/fl mice with GFAP-Cre−/− FasLfl/fl mice. Genotyping of offsprings was carried out by PCR of tail DNA with primers targeting GFAP-Cre and FasLfl/fl. Deletion of FasL was analyzed by PCR in various organs and cell types with Del-FasL primers (5′-GTACTTCTTCTGATAAGGACC-3′ Fluorometholone Acetate and 5′-GGAGTTGAACGAGTAGCCTC-3′). C57BL/6 WT mice were obtained from Harlan (Borchen, Germany). Animal care and experimental procedures were performed according to European regulations and approved by state authorities (Landesverwaltungsamt Halle, Germany; IMMB/G/02–994/10). MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from JPT (Berlin, Germany). Active EAE was induced in 8- to 12-week-old

mice by s.c. immunization with 200 μg of MOG35–55 emulsified in complete Freund’s adjuvant (Sigma, Taufkirchen, Germany) supplemented with 800 μg of killed Mycobacterium tuberculosis (Sigma). In addition, mice also received two i.p. injections of 200 ng pertussis toxin (Sigma), dissolved in 200 μL PBS, at the time of immunization as well as 48 h thereafter. Clinical signs of EAE were monitored daily and scored according to a scale of severity from 0 to 5 as described previously [23]. Daily clinical scores were calculated as the average of all individual disease scores within each group. Leukocytes were isolated from the spinal cord and stained for CD4+ T cells, CD8+ T cells, and CD45high inflammatory leukocytes as described before [42].

Another powerful animal model, particularly to study pathogens th

Another powerful animal model, particularly to study pathogens that are only tropic to primates,

are macaques. James Frencher from Zheng Chen’s lab (Chicago, IL, USA) showed evidence for HMB-PP-driven expansion of Vγ9/Vδ2 T cells in macaques infected with Listeria mono-cytogenes, and for priming of anti-microbial Th17 and Th22 responses by HMB-PP-responsive Vγ9/Vδ2 T cells Vorinostat price [15]. Leo Lefrançois (Farmington, CT, USA) presented new data suggesting a memory-like γδ T-cell response to oral Listeria infection in mice. Strikingly, this response is specific to an oligoclonal Vγ6/Vδ1 T-cell population present in mesenteric lymph nodes and lamina propria, which expand more rapidly and robustly to a secondary infection by Listeria but not to an unrelated pathogen, like Salmonella. γδ T cells are highly cytolytic against tumour cells, which has led to clinical trials based on their endogenous activation or adoptive transfer MK-2206 solubility dmso in/ to cancer patients [16]. Telma Lança from Bruno Silva-Santos’s lab (Lisboa, Portugal) stressed the importance of understanding the migratory properties of γδ T cells towards tumours. She showed that both mouse and human γδ T cells migrate in response to CCL2/CCR2 signals, and that these are required for the

in vivo infiltration of murine γδ T cells into tumour lesions. Using the B16 melanoma model, she further showed that mice genetically deficient for either γδ T cells (Trcd−/−) or CCR2 (Ccr2−/−) develop larger tumours (and more rapidly) than controls. Candida Vitale from Massimo Massaia’s lab (Torino, Italy) showed that cells from high-risk chronic SB-3CT lymphocytic leukaemia (CLL) patients with an unmutated tumour immunoglobulin heavy chain variable region

have an accelerated activity of the mevalonate pathway, thereby chronically stimulating peripheral Vγ9/Vδ2 T cells in those patients and driving their differentiation toward terminally differentiated, dysfunctional TEMRA cells, as opposed to patients with low-risk mutated CLL. TEMRA accumulation concurred to non-responsiveness to zoledronate in vitro which was an independent predictor of shorter time to first treatment (TTFT) in the overall patient cohort [17]. John Anderson (London, UK) presented evidence that human Vγ9/Vδ2 T cells effectively kill antibody-opsonised target cells through CD16-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) and that the CD16 interaction is a requirement for the uptake of soluble material by Vγ9/Vδ2 T cells for presentation to antigen-specific CD8+ responder T cells.

32 Only 40% of ESKD deaths from withdrawal of dialysis entered a

32 Only 40% of ESKD deaths from withdrawal of dialysis entered a hospice for care. This study also demonstrated a cost saving associated with dialysis patients dying in a hospice after withdrawal from therapy.

ESKD patients use a hospice at a rate of 25% compared with that seen in cancer patients.55 A pilot study reviewed the charts of 35 dialysis patients that withdrew from therapy and were followed by a palliative care team.23 The mean survival time from dialysis withdrawal to death was 10 days. Symptoms were reduced in the last day with palliative care input. The study suggested improved education of multidisciplinary nephrology staff was required. A small Australian study assessed the abatement of medical treatment in ESKD that encompassed both withdrawal

and non-initiation click here of dialysis treatment.11 This study included four patients that withdrew from dialysis, seven that did not initiate dialysis and five spouses of these patients. The participants undertook semistructured interviews from which the investigators gleaned there would be benefits from a greater discussion of end-of-life issues with acceptance of this as part of standard practice. These findings are supported by a study into the experience of patients after cessation of dialysis that found early palliative care referral could assist the patient and multidisciplinary team to manage areas such as pain and create opportunities to discuss palliative

care options.23 Factors identified as indicators associated with dialysis withdrawal include poor functional status, functional dependency, gender, ethnicity, social Small molecule library purchase isolation and comorbidities.24,34,57 Recently, Kurella Tamura et al. explored dialysis withdrawal preferences and found these varied with race, with blacks less likely to withdraw from dialysis than whites.58 Also they found the elderly did not have an increased preference for dialysis withdrawal whereas younger patients were less likely to record their preferences and be open to end-of-life discussion.58 Symptom control is of paramount importance in ESKD patients on dialysis with pain being the most common.59 The use of the World Health Organization three-step analgesic ladder is effective in pain management in haemodialysis patients.59 A prospective cross-sectional pilot study compared Clostridium perfringens alpha toxin symptom burden and quality of life between patients with advanced ESKD with an eGFR <17 mL/min and a contemporary cohort with terminal malignancy.29 Those patients with ESKD had similar symptom burden and reduced quality of life as the terminal malignancy group. This highlights that the palliative care needs of patients with ESKD are just as important as those with terminal cancer. In a retrospective chart review of conservatively managed stage 4–5 CKD patients Murphy et al. assessed symptom burden using a short patient-completed assessment tool.


CS1 Selleck PLX3397 promotes multiple myeloma cell adhesion, clonogenic growth and tumorigenicity via cmaf-mediated interactions with bone marrow stromal cells [42]. Family-based association studies

in UK and Canadian SLE families identified variants in the promoter and coding region of CS1 contributing to SLE disease susceptibility [43]. Based on the recent finding of a genetic association of SLAM family receptors with SLE, we hypothesized that the alterations in expression of 2B4 and CS1 may mediate the immune dysregulation observed in patients with SLE. In this study, we compared expression levels of 2B4 and CS1 on T, B, NK cells and monocytes in SLE patients versus those of healthy controls. The 2B4-expressing NK cells and 2B4-expressing monocytes were reduced in patients with SLE compared to healthy controls. The proportion of CS1-expressing B cells in patients with SLE was significantly higher than that from healthy controls. Our study also demonstrated differential expression of CS1 and

2B4 splice variants in total peripheral blood mononuclear cells (PBMC) in patients with SLE compared to healthy controls. Blood samples were obtained from 45 patients diagnosed with SLE (two males, 43 females) at John Peter Smith (JPS) Hospital, Fort Worth, TX and from 30 healthy volunteers at University of North Texas Health Science Center (UNTHSC), Fort Worth, TX with prior approval from Internal Review Board of JPS Health Network and UNTHSC. Written informed consents were obtained from all of the study subjects. Patients with SLE were classified according to the 1997 revised criteria from the American College of Rheumatology [44,45]. Clinical and demographic characteristics of SLE patients, including SLE Disease Activity Olopatadine Index (SLEDAI), treatments, major disease manifestations and serological parameters, are

shown in Table 1. Eight patients had active SLE, defined by a SLEDAI score of ≥8 [46]. All 45 patients were positive for anti-nuclear antibody (ANA). PBMCs were isolated from ethylenediamine tetraacetic acid (EDTA)-treated whole-blood samples by Histopaque-1077 (Sigma Chemicals, St Louis, MO, USA) density gradient centrifugation using LeucoSep tubes (Greiner, Monroe, NC, USA). The remaining red blood cells were lysed with ACK lysis buffer. Resulting PBMCs were used for immunostaining or reverse transcription–polymerase chain reaction (RT–PCR). Before starting immunostaining, PBMCs were incubated with human IgG Fc fragments (Rockland, PA, USA) for prevention of possible Fc receptor-mediated fluorescence. The tricolour staining [fluorescein isothiocyanate–phycoerythrin–allophycocyanin (FITC-PE-APC)] method was applied for immunostaining.