Methods: Three hundred and twenty cases of biopsy-proven DPLN with ≥10% crescents (cDPLN) were included in this study. Another

180 DPLN patients without crescents were enrolled as a control group. Their clinicopathological data and long-term outcome were compared. Results: There were 280 females and 40 males with an average age of 31.8 ± 11.3 years followed for a median period of 7 years. Compared with the control signaling pathway group, cDPLN patients had a significant lower rate of clinical remission (CR+PR) (90.3% vs 96.5%, p = 0.036) for longer period (10.1 ± 7.9 vs 8.9 ± 7.6 months, p = 0.154), much higher rate of treatment failure (9.7% vs 3.5%, p = 0.036) and relapse (41.5% vs 37.8%, p = 0.511). The 5-, 10-and 15-year cumulative renal survival rates of cDPLN and the control group were 87% vs 90.8%, 73.3% vs 81.6% and 58.7 vs 81.6%, respectively. At the time of biopsy, higher percentage of crescents (HR 1.030, P = 0. 001), fibro-cellular crescents (HR 1.025, P = 0. 002), glomerular sclerosis (HR 1.033, P = 0. 022), impaired renal function (HR 1.519, P < 0.001), decreased eGFR (HR3.567, P = 0.003), higher levels of NAG enzyme (HR 1.009, P = 0. 014), urinary C3 (HR 1.046, P = 0. 024), serositis history (HR 2.814, P = 0. 013), failure to achieve clinical remission (HR 0.144,

P < 0.001) and relapse (HR 11.634, P = 0. 020), were the independent risk factors for worse renal survival of cDPLN patients. Multivariate learn more Cox analysis showed the percentage of glomerular sclerosis was the most important risk factor of ESRD. Conclusion: cDPLN had worse treatment response and lower probability of renal survival than those without crescents. Ten clinicopathological features including a higher percentage of crescents, fibro-cellular crescents, glomerular sclerosis, impaired renal function, higher NAG enzyme, urinary C3, history of serositis, failure of achieving clinical remission and relapse were independent predictors of an unfavorable renal outcome. IKEUCHI HIDEKAZU, HIROMURA KEIJU, TSHILELA

KADIOMBO A, KAYAKABE KEN, SAKURAI NORIYUKI, SAKAIRI TORU, KANEKO YORIAKI, MAESHIMA AKITO, NOJIMA YOSHIHISA Department of Medicine and Clinical Science, Gunma University 6-phosphogluconolactonase Graduate School of Medicine Introduction: In this study we sought to identify predictive factors for renal insufficiency in patients with lupus nephritis (LN). Methods: We retrospectively analyzed 155 biopsy proven LN patients (21 male, 134 female) at our department between 1976 and 2012. Renal histology was classified by ISN/RPS 2003 classification. A renal endpoint was defined as doubling of serum creatinine (S-Cr) or end-stage renal disease. Results: The mean age at renal biopsy was 36.5 ± 13.2 years.

[8-12] Studies in vivo have also demonstrated a role in colitis and ileitis.[13-17] DR3 regulates immunity to certain bacteria,[18] viruses,[19] tumours[20] and intrinsically maintains Selleck Nutlin3a neurological function.[21] Research in humans has mirrored these findings, primarily showing that DR3 regulates

inflammation and immunity through controlling the development of effector T cells and differentiation of myeloid subsets,[22-30] but it may also have effects on other cell types such as neurons.[31] Local and systemic increases of its ligand are associated with multiple human inflammatory disorders.[32-35] In this respect, the designation ‘Death Receptor 3’ is a misnomer because many of the recognized functions of the gene are associated with cell expansion and differentiation, rather than death. Park et al.[1] clearly describe an increase in cell viability of tumour cell lines following exposure to natural killer (NK) cells when DR3 expression was knocked down; results consistent with DR3 acting to trigger cell death.

To my knowledge, this is the first functional demonstration of a pro-apoptotic role for DR3 in human tumour cell lines, but it is not unique as a general phenomenon. The original DR3 knockout mouse exhibited a defect in negative selection of thymocytes,[36] while DR3-dependent apoptosis HAS1 has been described in renal inflammation in vivo[37] and osteoblast cell lines in vitro.[38] Furthermore, a role in human cancer has been implied from the discovery that Selleckchem ABT263 the DR3 gene is disrupted in ~ 40% of neuroblastomas.[39] It is in this context that clarification is useful on the nature of the DR3 ligand, as its identity is also complicated by a history of diverse nomenclature. Park et al.[1] mention two ligands in their references, Apo3L and TL1A, both of which are distinct tumour necrosis factor superfamily (TNFSF) members. Apo3L was originally named as the ligand for DR3 (i.e. Apo3)[40] and was also called TWEAK (TNFSF12). However,

follow-up studies could not confirm this[41] and indicated that TWEAK signalled in the absence of DR3.[42] A second receptor for TWEAK, Fn14 (TNFRSF12A), was then identified,[43] and TL1A (TNFSF15 and the full-length gene product of the vascular endothelial growth inhibitor, VEGI) was found to bind DR3.[44] All-encompassing work from Bossen et al.[45] involving flow cytometric binding assays between the majority of human and murine TNFSF:Fc proteins and cell lines transfected with TNFRSF members confirmed this, i.e. that TWEAK binds Fn14, whereas TL1A binds DR3 and there is minimal cross-reactivity, findings that have been borne out in later in vivo experiments using gene knockouts.

05, Fig. 1B). We compared the severity of inflammation in the airway between Derf-exposed CD44KO and WT mice. The numbers of total leukocytes, macrophages, and lymphocytes in the BALF of Derf-exposed CD44KO mice were lower than those of Derf-exposed WT mice (p<0.05, Fig. 1B). The number of eosinophils in the BALF of Derf-exposed CD44KO mice was marginally lower than that of Derf-exposed WT mice (p=0.0963, Fig. 1B). Furthermore, accumulation of Th1 and Th2 cells was investigated by counting the number

of CD4+Tim-3+ and CD4+T1/ST2+T cells, respectively, in the BALF. The accumulation of Th2 cells (p=0.0041), but not Th1 cells (p=0.6911), was suppressed in CD44KO mice compared with WT mice (Fig. 1C), after Derf challenge. Therefore, the lack of antigen-induced Selleckchem NSC 683864 AHR in CD44KO mice might be caused Ruxolitinib nmr by the down-regulation of Th2 cell accumulation in the lung. To investigate the possible roles of various cytokines and chemokines in allergic airway inflammation, concentrations in BALF of Th1

(IFN-γ) and Th2 (IL-5, IL-13) cytokines, and chemokines (TARC, IP-10, and eotaxin) were measured by ELISA. The levels of these cytokines and chemokines in the PBS group of both CD44KO and WT mice were under the detection limits (data not shown). Elevated levels of Th1 and Th2 cytokines were observed in both CD44KO and WT mice after Derf challenge. Th2 cytokine (IL-5 and IL-13) concentrations in the BALF of CD44KO mice were lower than those of WT mice (p<0.05, Fig. 2A), while the amount of IFN-γ in the BALF of Derf-exposed CD44KO mice was higher than that of Derf-exposed WT mice (p<0.05, Fig. 2A). Levels of TARC and eotaxin in the BALF of Derf-exposed CD44KO mice were similar to those of Derf-exposed WT mice, while the IP-10 concentration in the BALF of Derf-exposed CD44KO mice was higher than that in Derf-exposed WT mice

(p<0.05, Fig. 2B). These data demonstrate the possibility that CD44 deficiency not only suppresses Th2-mediated airway inflammation, but also facilitates Th1 development in Derf-sensitized and challenged Rho mouse model. To explore the role of CD44 in the development of Th1- or Th2-biased Th differentiation, antigen-specific antibody production, Derf-specific IgE, IgG1, IgG2c, and Th1, Th2 cytokine levels in the serum were determined by ELISA in Derf-immunized CD44KO and WT mice before and after antigen challenge. Serum levels of Derf-specific IgE (p=0.3472), IgG1 (p=0.1172), and IgG2c (p=0.2948) were not significantly different between CD44KO and WT mice before antigen challenge (Fig. 3A), whereas the serum levels of Derf-specific IgG2c (p=0.0109), but not IgE (p=0.5589) and IgG1 (p=0.8494), were significantly higher in CD44KO mice compared with WT mice after Derf-challenge (Fig. 3B). Before antigen challenge, serum levels of IL-5 (p=0.2347) and IL-13 (p=0.

To answer the question of whether affinity or stability is the better correlate of immunogenicity, we extracted 12 affinity-balanced pairs each consisting of an “immunogenic binder” and a “nonimmunogenic binder” according to Sette and colleagues [6]. These peptides were synthesized and affinity and stability of their interactions with HLA-A*02:01 was measured. This representative analysis showed that “immunogenic binders” were significantly more stably bound to HLA-A*02:01 than “nonimmunogenic binders” (p = 0.0007, paired two-tailed

Student’s t-test) (Table 1, Fig. 4B), whereas no significant difference in affinity was observed between the two groups (Table 1, Fig. 4A). Note that one of the reported immunogenic peptides, RTLLGLILFV, in our hands was a low-affinity, low-stability-binding peptide. Upon closer inspection, the N-terminally truncated peptide, TLLGLILFV, appeared to be a likely HLA-A*02:01-binding peptide. This peptide Pexidartinib concentration was synthesized and found to be a high-stability (half-life 33 h) peptide. We would like to suggest that TLLGLILFV is the real HLA-A*02:01-restricted CTL epitope. Depicting this data in a log(stability) versus log(affinity) plot showed that the increased

stability of peptide-HLA-A*02:01 complexes involving “immunogenic binders” (y = 0.65x − 5.1, R2 = 0.65) versus selleckchem “nonimmunogenic binders” (y = 0.75x − 4.5, R2 = 0.53) was seen throughout the binding range KD < 100 nM (Fig. 4C). When we inspected the 2 × 12 affinity-paired peptides (24 Depsipeptide clinical trial in total), we noted that 10 of 12 peptides with optimal amino acids residues in both anchor position 2 (LM) and C-terminal (VLI) had a half-life of more than 5 h, whereas nine of 12 peptides with a suboptimal amino acid residue (typically T or Q in position 2 or C-terminally) had a half-life of less than 5 h. At face value, this highly significant distribution (p = 0.014, Chi-square test with Yates correction) suggests that peptide-HLA-A*02:01 complexes are destabilized by just

one of the anchor positions being occupied with a suboptimal amino acid. For the seven peptides with suboptimal anchor residues, we substituted the suboptimal anchor residue with an optimal residue (leucine or methionine in position 2 and valine in C-terminal), and repeated the stability experiment. In all seven cases, the stability was improved (in six of the seven peptides, stability was increased by seven to tenfold), and four of the seven previously unstable peptides achieved a half-life better than 5 h, see Table 2. Thus, there appear to be a subtle difference in the specificity of high-affinity peptides, which may tolerate a suboptimal amino acid residue in an anchor position, and the specificity of high-stability peptides, which seems to be less inclined to tolerate suboptimal amino acid residue in anchor positions (in particular not in position 2).

19,20 The peak of IFN-I induces an almost global acquisition of a partial activation phenotype in T and B cells which reverts to a resting phenotype within 5 days.19,21 Interestingly, this process Selleck Decitabine is followed by a transient period of partial immune-unresponsiveness (between 5 and 9 days after an acute primary viral episode),22 in which a post-viral expansion of Tregs has been proposed to play a role.23 Although the production of IFN-I after acute infection has a significant role in the acquisition of immune effector functions, whether the transience in IFN-I production may also contribute to the late generation of Tregs is still

unknown. In this study, we found that IFN-α alters the pattern of aTreg (CD4+ FoxP3HI IFN-γNeg) and aTeff (CD4+ FoxP3Low/Neg IFN-γPos) 5-Fluoracil cell generation in anti-CD3 activated peripheral blood mononuclear cells (PBMC), by exerting a negative effect on Treg activation and proliferation while favouring Teff activation. We also demonstrated that IL-2, a critical cytokine involved in Treg survival and proliferation, was

significantly down-regulated by IFN-α, and that the addition of IL-2 was able to reverse IFN-α-induced suppression of Tregs. Finally, we found that the generation of aTregs was suppressed in PBMC from patients with SLE, a condition characterized by chronic IFN-α stimulation and low IL-2 production.24–26 Taken together, these findings provide evidence to suggest that IFN-α has a negative effect on Treg activation and proliferation (probably through inhibition

of IL-2 production by activated Teffs), and that unique patterns of IFN-α production may play a role in defining the balance between Teffs and Tregs in acute and chronic inflammatory conditions. The study was approved by The Johns Hopkins Medicine Institutional Review Board (IRB) and all individuals signed an informed consent Thiamet G form. After IRB approval had been obtained, normal controls were recruited and informed consent obtained. Alternatively, for two of the donors, leucopacks were obtained from the New York Blood Center (New York, NY). Patients with SLE were recruited through the Johns Hopkins SLE cohort, an ongoing, National Institutes of Health (NIH)-funded prospective study. PBMC were purified from healthy controls using Ficoll-Hypaque density-gradient centrifugation. Our system for recapitulating the normal in vivo expansion of Tregs upon immune activation is based on the work of Gavin et al.,4 who described the use of a combination of cell surface and intracellular markers to specifically follow and distinguish CD4+ Tregs from CD4+ Teffs. Purified PBMC were plated at 1 × 106 cells/ml with 5% heat-inactivated human AB serum (Mediatech, Manassas, VA) and stimulated with soluble anti-CD3 (100 ng/ml; OKT3; BD Biosciences, San Jose, CA).

40 Consequently, this study found no evidence for TH2 bias in pregnant sheep, contrary to many previous studies in humans and mice.37 However, as previously mentioned, the TH2 paradigm in pregnancy has been brought into question. A recent study has found no global differences in the production of IFN-γ, IL-4, IL-5, IL-10 or IL-13 production by mitogen-activated PBMC from pre- and post-partum women, concluding that the evidence for a TH2 bias during pregnancy

is certainly debatable and possibly reflective of experimental design.41 This observation is in line with our studies in sheep, and it would therefore appear that other immunological JQ1 in vitro and/or physiological factors (such as placental development) are responsible for the pathogenesis of OEA. For example, we have previously suggested that the placentitis characteristic of OEA originates from the haematomatas in the placentome that create a mechanism for transmission of C. abortus from the maternal blood to the placenta and may not depend on alterations in maternal immune reactivity.21 Consequently, although the TH1/TH2 paradigm provides an attractive explanation

for the pathogenesis of OEA and recrudescence of C. abortus from a peripheral site of latency in mid-gestation, the evidence suggests otherwise and that other factors are involved. Our knowledge of the molecular mechanisms https://www.selleckchem.com/products/r428.html of persistence of C. abortus in ovine cells and of the correlates of immunological protection has greatly advanced our understanding of OEA. Nevertheless, gaps in our knowledge remain that require further investigation if we are developing

more effective control strategies (including vaccination) for this important reproductive disease of sheep and other ruminants. A current area of great interest in vaccine development is the identification of effective delivery strategies that target this website the innate immune system to stimulate an appropriate adaptive host response that confers protection without immunopathology. The particular components of interest in innate immunity are dendritic cells, NK cells, pattern recognition receptors and early cytokine and chemokine production. Several laboratories are actively engaged in the study of these cells and molecules in sheep, with most investing at least some of their effort and resources into the development of immunological tools to define expression and ascribe function. The area of NK cell biology has particular relevance for reproductive biology and definitive moAbs against ovine NK-expressed molecules are expected to become available in the very near future. These probes will help address an unanswered question regarding the relative importance of γδT cells and NK cells in ovine reproduction.

01). Leflunomide suppressed their high expressions in renal tissue of diabetic rats. Conclusions:  Leflunomide can ameliorate the kidney structure

and function injury of diabetic rats through suppressing the expression of NF-κB, TNF-α, MCP-1 this website and macrophage infiltration in renal tissue. “
“Senior-Løken syndrome is a rare syndromic form of nephronophthisis that is associated with retinal dystrophy. Presently, seven genes (NPHP1-6 and NPHP10) have been associated with Senior-Løken syndrome. NPHP5 mutations are known to cause classical Senior-Løken syndrome. Here, we report two sisters (II-4, II-5) from a Chinese Han ethnic family who presented with classical Senior-Løken syndrome. Both affected sisters exhibited Leber’s congenital amaurosis and juvenile nephronophthisis that progressed to end-stage renal disease by the age of 16 years and 9 months in patient II-4 and 12 years and 9 months in patient II-5. Sequence analysis showed a homozygous truncated mutation in NPHP5, c.1090C>T (p.R364X), in the patient II-4. This mutation is predicted to introduce a new open reading frame that results in the truncation of the C-terminal 235 amino acids of nephrocystin-5

and its consequent loss of function. Both parents carried a single heterozygous mutation in the same position, and no homozygous deletion of NPHP1 PS-341 ic50 was found in this pedigree. “
“Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis (PD) that carries a high morbidity and mortality. The ‘two hit theory’ Baf-A1 nmr suggests that long term deterioration of the peritoneum combined with intraperitoneal inflammation is needed in the pathogenesis of EPS. For unclear reasons, post transplantation EPS is being increasingly reported in patients previously on PD. To date, there is no proven effective therapy with an absence of randomised controlled trials. Individual case

reports and small case series have reported on the use of tamoxifen and corticosteroids for medical management of EPS. The use of everolimus has been reported in a single case, and never in the setting of renal transplantation. Here, we present the first case of post-transplant encapsulating peritoneal sclerosis treated successfully with a combination of everolimus, tamoxifen, low dose corticosteroid and surgery. A 37-year-old man of Vietnamese background presented to our hospital in March 2009 for deceased donor renal transplantation. End-stage renal failure was secondary to hepatitis C-related mesangioproliferative glomerulonephritis with cryoglobulinaemia. He had been on automated peritoneal dialysis for over 6 years with a combination of dextrose based peritoneal dialysis solutions. There had been no previous episodes of peritoneal dialysis-related peritonitis. A preceding peritoneal equilibration test showed that he was a high average transporter. In the year prior to transplant he had lost all residual renal function, and had signs of peritoneal membrane failure.

Correspondingly, the Register has very few reports of adverse reactions caused by green pea or soy, a substantial number of reports regarding lupin this website and fenugreek, and many regarding peanut. These data show that there is a need to further investigate cross-allergy in legumes. Most of the work performed on legume allergy has focused on peanut as the major allergenic legume, and information on other

types of legume allergy is limited [4]. As we previously have established mouse models of lupin and fenugreek allergy [25, 26], we used these models to address the clinical cross-allergy between the four most common allergenic legumes: lupin, fenugreek, peanut and soy. We also assessed different serological and cellular responses to explore possible mechanisms related to the cross-allergic reactions. Animals.  Female inbred C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME, USA), 5 weeks old at the start of the experiments, were used. Several experiments have been combined in this study and an account of the animals with immunizations and challenges is therefore given in Table 1. Female Sprague-Dawley rats, 150–200 g (Taconic M&B A/S, Ry, Denmark) were used to perform the passive cutaneous anaphylaxis (PCA) tests. The animals were housed, 3–4

mice or two rats per cage, on NESTPAK bedding (Datesand Ltd, Manchester, UK) in type III macrolon cages in filter cabinets (Scantainers), exposed to a 12-hr/12-hr light/dark cycle Amobarbital (30–60 lux in cages), room temperature of 21 ± 2 °C and 35–75% humidity. Pelleted food (RM1; buy SB203580 SDS, Essex, UK) and tap water ad libitum were given. Before entering the experiments, the animals were allowed to rest for 1 week. The experiments were performed in conformity with the laws and regulations for experiments with live animals in Norway and were approved by the Norwegian Animal Research Authority under the Ministry of Agriculture. Legume extracts.  The National Veterinary Institute of Norway provided all protein extracts. In short, extracts of peanut, lupin and soy were made by extracting

homogenized peanuts, soybeans or lupin (Lupinus angustifolius) in Tris/glycine buffer, pH 8.7, overnight followed by centrifugation. The fenugreek extract was made using an extended protocol utilizing precipitation with (NH4)2SO4, dialysis and freeze-drying [26]. The total protein concentration of the extracts was measured by Lowry’s method. The endotoxin level of the extract was determined with the Limulus Amebocyte Lysate (LAL) Kinetic-QCL Kit (BioWhittaker, Walkersville, MD, USA) and found to be below 0.1 ng/ml for all extracts. Immunizations and challenges.  Immunizations were performed perorally (p.o.) according to the experimental protocols previously established [25, 26]. Briefly, immunizations were performed on days 0, 1, 2, 7, 21 and 28 and challenges on day 35. Lupin immunized mice received 5.

Since

Ig membrane expression on B lymphocytes is required for cell survival 11, 12, targeting IgM exons or the JH locus with ZFN was expected to generate non-homologous end joining mutations resulting in Ig-deficient rats and thus lacking TSA HDAC cell line mature B cells. In this manuscript, we describe the phenotype of rats homozygous for a truncation in Cμ1 and, separately, deletion of the JH locus. Both lines show no detectable Ig production and mature B-cell development. The availability of B-cell-deficient rats will permit to gain new insights of Ig function and development in health and disease. In addition, ZFN technology paves the way for simpler gene replacement and transgenic studies with the immediate aim of expressing human Ab repertoires in the rat. Among several rat lines with IgM CH1 domain mutations 8,

rat line 19 was breed to homozygocity. The mutation in this rat line comprised a 64 bp deletion in both alleles of the IgM CH1 domain gene see more (Fig. 1A, left) and no additional mutations in any of the ten genomic sequences most homologous to the one targeted 8. Analysis of IgM mRNA by RT-PCR of JH1-Cμ transcripts showed a shorter transcript in rats homozygous for IgM mutation (IgM KO rats) compared with WT (Fig. 1B, left). Analysis of IgG transcripts using RT-PCR of JH-Cγ showed the absence of mRNA in IgM KO rats and a strong signal of the expected size in WT rats (Fig. 1B, left). Heterozygous IgM KO rats showed the presence of IgM and IgG transcripts (data not shown). Digestion of the JH-Cμ amplicon with DdeI resulted in the generation of a smaller band due to the 64 bp deletion (Fig. 1B, right). Sequencing of JH-Cμ mRNA isolated from IgM KO rats showed a deletion of 64 bp and the generation of a stop codon (Fig. 1C). Microinjection of rat zygotes with ZFN mRNA specific for the JH locus resulted in the generation of a mutant animal with a 2465 bp DNA deletion, spanning Phloretin the entire locus (Supporting Information Data 1). In homozygous JH locus, mutant rats’ analysis of mRNA using primers spanning several VH or JH sequences to μCH2

(Fig. 1D) or Cγ sequences (data not shown) did not reveal detectable levels of transcripts. These results indicate that IgM KO rats have a deletion in the Cμ1 domain that generated a stop codon, resulting in shorter IgM transcripts and no IgG transcripts. Rats homozygous for J deletion (JH KO rats) showed a large deletion and no detectable IgM or IgG transcripts. ELISA revealed undetectable levels for all Ig isotypes in IgM or JH KO rats analyzed (Fig. 2A). Heterozygous IgM KO animals and WT rats showed normal levels of IgM (1 246±81 μg/mL), IgG (6 060±1 356 μg/mL), IgA (65±5 μg/mL) and IgE (2 845±1 110 ng/mL). In mice, mutations in the IgM Cμ1 exon have resulted in alternative splicing of the mutated region and shorter μ-chains were produced 13.

Exclusion criteria were pregnancy, patients undergoing dialysis or who were severely ill, such as those in the intensive-care unit or who were haemodynamically unstable, patients with infections and patients with drug-induced leucopenia or anaemia. Patient characteristics, including immunosuppressive medications and prednisone dose, are summarized in Table 1. Healthy donors (n = 31) matched by age and sex were included as controls. In both groups, 90% were women and the average ages were 36·1 ± 12·2 and 32·1 ± 9·1 years in the patients with SLE and healthy controls, respectively. In addition, 16 patients with rheumatoid arthritis and five kidney-transplanted patients, undergoing

similar immunosuppressive treatment to the patients with SLE, were included as controls (average ages 59·6 ± 10·41 and 45·4 ± 10·6 years, respectively). Further details regarding patient characteristics and specific medications selleck chemical including prednisone dose are shown

in Tables 2 and 3 for patients with rheumatoid arthritis and transplanted patients, respectively. For additional experiments, including T-cell activation after SEA stimulation, an additional 31 patients with SLE with similar characteristics and treatments were evaluated. Each patient signed an informed consent form before enrolling in the study, in accordance with the regulations of the Ethics Committee from the School of Medicine of the Pontificia Universidad Católica, Ribonucleotide reductase and the study was performed in accordance with the Declaration of Helsinki as emended in Edinburgh (2000). The SLE activity was assessed using Enzalutamide price the Systemic Lupus Erythematosus Activity Index (SLEDAI) 2K. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using the standard Ficoll centrifugation method.

Monocytes were obtained using the adherence method.34 Briefly, PBMCs (3 × 106 cells/ml) were incubated in serum-free X-VIVO-15 medium (Bio-Whittaker, Walkersville, MD) supplemented with 1% autologous serum and 50 μg/ml gentamycin (Calbiochem, San Diego, CA) (DC-medium) for 2 hr at 37°. Adherent cells were washed four times with pre-warmed serum-free X-VIVO-15 medium (Bio-Whittaker) and were then cultured in DC-medium at 37°. Monocytes were differentiated to DCs over 5 days by the addition of 1000 U/ml IL-4 and 1000 U/ml GM-CSF on days 0, 2 and 4. Maturation of the DCs was triggered by the addition of lipopolysaccharide (LPS; 5 μg/ml) for an additional 48 hr. The DC immune-phenotypes were confirmed by flow cytometry using specific monoclonal antibodies against surface markers. Cells were washed with PBS, re-suspended at 2 × 106 cells/ml (50 μl/tube) and incubated with FITC-conjugated, PE-conjugated and APC-conjugated antibodies for 30 min at 4°. The isotype-matched antibodies conjugated with FITC, PE and APC were used as controls.