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Background and aims Neutrophils are important source of high interferon in SLE, we aimed to identify Long noncoding RNAs (LncRNAs) that can be strongly induced by interferon and simultaneously show different expression in neutrophils of SLE and healthy controls. We also investigated how this LncRNA modulate neutrophils interferon production. Methods RNA-seq was performed in two series of samples, interferon stimulated neutrophils samples and SLE versus healthy controls neutrophils samples. LncRNA-CMPK2 was screened out by cross-reference the two RNA-seq results. Neutrophils interferon production was measured by qPCR and ISRE report gene assay after LncRNA-CMPK2 was knocked down using antisense oligos electrotransfection. Results SLE neutrophils produced more interferon when stimulated by TLR7/8 agonist R848 as compared to healthy controls. Neutrophils enhanced interferon production capacity after interferon prime. LncRNA-CMPK2 was an interferon stimulated LncRNA in neutrophils and had an expression level correlated with SLE disease activity. Knock down LncRNA-CMPK2 attenuated neutrophils interferon production. Conclusions Interferon can augment neutrophils interferon production capacity in regenerative feedback. LncRNA-CMPK2 was an important interferon stimulated LncRNA and can facilitate neutrophils interferon production in SLE. Accommodate the expression of LncRNA-CMPK2 could probably supply a new thread of thought to SLE treatment.

©2014 Turkish League Against Rheumatism. All rights reserved. Neuropsychiatric systemic lupus erythematosus (NPSLE) is a specific disease, and its clinical manifestations involves the central and the peripheral nervous system range from life-threatening manifestations to subtle abnormalities.1 Neuropsychiatric systemic lupus erythematosus may be the first indication of systemic lupus erythematosus (SLE) in patients without disease activity in other organ systems.2 It represents a major cause of morbidity and mortality in SLE patients.3-8 Therefore, recognition, early diagnosis and treatment of NPSLE are of great significance.

Objective MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)–related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells. Methods Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects. Results The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels. Conclusion MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.

The recent discovery of microRNAs (miRNAs) has revealed a new layer of gene expression regulation, affecting the immune system. Here, we identify their roles in regulating human plasmacytoid dendritic cell (PDC) activation. miRNA profiling showed the significantly differential expression of 19 miRNAs in PDCs after Toll-like receptor 7 (TLR7) stimulation, among which miR-155* and miR-155 were the most highly induced. Although they were processed from a single precursor and were both induced by TLR7 through the c-Jun N-terminal kinase pathway, miR-155* and miR-155 had opposite effects on the regulation of type I interferon production by PDC. Further study indicated that miR-155* augmented interferon-α/β expression by suppressing IRAKM, whereas miR-155 inhibited their expression by targeting TAB2. Kinetic analysis of miR-155* and miR-155 induction revealed that miR-155* was mainly induced in the early stage of stimulation, and that miR-155 was mainly induced in the later stage, suggesting their cooperative involvement in PDC activation. Finally, we demonstrated that miR-155* and miR-155 were inversely regulated by autocrine/paracrine type I interferon and TLR7-activated KHSRP at the posttranscriptional level, which led to their different dynamic induction by TLR7. Thus, our study identified and validated novel miRNA-protein networks involved in regulating PDC activation.

Objective MicroRNA have recently been identified as regulators that modulate target gene expression and are involved in shaping the immune response. This study was undertaken to investigate the contribution of microRNA-146a (miR-146a), which was identified in the pilot expression profiling step, to the pathogenesis of systemic lupus erythematosus (SLE). Methods TaqMan microRNA assays of peripheral blood leukocytes were used for comparison of expression levels of microRNA between SLE patients and controls. Transfection and stimulation of cultured cells were conducted to determine the biologic function of miR-146a. Bioinformatics prediction and validation by reporter gene assay and Western blotting were performed to identify miR-146a targets. Results Profiling of 156 miRNA in SLE patients revealed the differential expression of multiple microRNA, including miR-146a, a negative regulator of innate immunity. Further analysis showed that underexpression of miR-146a negatively correlated with clinical disease activity and with interferon (IFN) scores in patients with SLE. Of note, overexpression of miR-146a reduced, while inhibition of endogenous miR-146a increased, the induction of type I IFNs in peripheral blood mononuclear cells (PBMCs). Furthermore, miR-146a directly repressed the transactivation downstream of type I IFN. At the molecular level, miR-146a could target IFN regulatory factor 5 and STAT-1. More importantly, introduction of miR-146a into the patients' PBMCs alleviated the coordinate activation of the type I IFN pathway. Conclusion The microRNA miR-146a is a negative regulator of the IFN pathway. Underexpression of miR-146a contributes to alterations in the type I IFN pathway in lupus patients by targeting the key signaling proteins. The findings provide potential novel strategies for therapeutic intervention.

Objective To explore whether there are extrinsic factors that impair the suppressive function of CD4+,CD25+ regulatory T cells in patients with untreated active systemic lupus erythematosus (SLE). Methods We studied 15 patients with untreated active SLE, 10 patients with SLE in remission, and 15 healthy control subjects. Percentages of CD4+,CD25+,FoxP3+ Treg cells and levels of forkhead box P3 (FoxP3) protein were analyzed by flow cytometry. Expression of messenger RNA (mRNA) for FoxP3 in purified Treg cell populations was assessed by real-time polymerase chain reaction analysis. Experiments examining Treg cell function in SLE were designed to distinguish primary from secondary T cell dysfunction. Levels of interferon-α (IFNα) in supernatants from the function assays were determined with an IFN-stimulated response element–luciferase reporter assay. Results The percentage of CD4+,CD25+, FoxP3+ cells in peripheral blood was significantly increased in SLE patients as compared with controls (mean ± SEM 9.11 ± 0.73% versus 4.78 ± 0.43%; P < 0.0001). We found no difference in FoxP3 expression at either the mRNA or protein level in any CD4+,CD25+ T cell subset from SLE patients as compared with controls. Antigen-presenting cells (APCs) from SLE patients were responsible for decreased Treg cell activity and could also render dysfunctional Treg cells from healthy control subjects. CD4+,CD25+ Treg cells from SLE patients exhibited normal suppressive activity when cultured with APCs from healthy controls. A partial Treg cell blockade effect was induced by the high levels of IFNα derived from SLE patient APCs. Conclusion We suggest that blockade of Treg cell–mediated suppression by IFNα-producing APCs in SLE patients may contribute to a pathogenic loss of peripheral tolerance in this disease.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)2D3 levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)2D3 on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)2D3, although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1α-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)2D3 and/or activation. Importantly, 1,25(OH)2D3 up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)2D3 may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.

The combined presence of anti-phospholipid Ab (aPL), thrombosis, and/or fetal loss is recognized as the antiphospholipid syndrome (APS). aPL include anti-cardiolipin Ab (aCL) and/or lupus anticoagulants (LAC, detected as Ig that prolong certain in vitro phospholipid (PL)-restricted blood clotting tests); both aCL and LAC are the diagnostic Ab for APS. Studies show that aPL represent a heterogeneous group of Ab, which recognize various PL, PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found that five of seven patient-derived IgG monoclonal aCL react with thrombin, activated protein C, and plasmin. All three proteins are trypsin-like serine proteases (SP), and are highly homologous in their catalytic domains. Importantly, among these SP autoantigens, the reactive aCL bind to plasmin with the highest affinity, suggesting that plasmin may serve as a major driving autoantigen for some aCL in ∼30% of APS patients who are positive for IgG anti-plasmin Ab. To test this hypothesis, we immunized BALB/c mice with human plasmin and analyzed immune sera for aCL activity and reactivity with relevant SP. We found that some immune sera displayed aCL activity and/or bound to test SP. Subsequently, eight mAb were obtained and studied. The results revealed that one mAb displayed the aCL and the LAC activities and induced fetal loss when injected into pregnant mice. Immunohistological analyses of placentas revealed extensive deposits of activated C3 components. Combined, these data demonstrate that plasmin may serve as a driving Ag for some pathogenic aPL.

Despite growing evidence that large intergenic noncoding RNAs (lincRNAs) can regulate gene expression and widely take part in normal physiological and disease conditions, our knowledge of systemic lupus erythematosus (SLE)-related lincRNAs remains limited. The aim of this study was to detect the levels of four lincRNAs (ENST00000500949: linc0949, ENST00000500597: linc0597, ENST00000501992: linc1992, and ENST00000523995: linc3995) involved in innate immunity in the peripheral blood mononuclear cells (PBMCs) of patients with SLE and correlate these lincRNA levels with disease activity, organ damage, clinical features and medical therapies. PBMCs were obtained from 102 patients with SLE, 54 patients with rheumatoid arthritis (RA) and 76 healthy donors. lincRNA expression levels were measured by real-time quantitative polymerase chain reaction. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) scores, and organ damage was evaluated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. linc0949 and linc0597 were significantly decreased in patients with SLE compared with patients with RA and healthy control subjects. linc0949 was correlated with SLEDAI-2K score (r = −0.329, P = 0.0007), as well as with complement component C3 level (r = 0.348, P = 0.0003). The level of linc0949 was also reduced in patients with SLE who had the presence of cumulative organ damage. In addition, decreasing expression of linc0949 was associated with lupus nephritis. linc0949 expression significantly increased after treatment, whereas neither disease activity nor organ damage correlated with linc0597 expression. Our results provide novel empirical evidence that linc0949 could be a potential biomarker for diagnosis, disease activity and therapeutic response in SLE.

Aim: To investigate the long-term prognosis of systemic lupus erythematosus (SLE) and its risk factors. Methods: An 18-year (1980–1998) clustering follow-up study of 50 patients with SLE was performed. Results: The overall survival rate at 1, 5, 10, 15 and 18 years after the onset of illness was 98%, 98%, 84%, 76%, and 70%, respectively. The two major causes of death were infection and renal failure. Cox proportional hazard regression analysis showed that the presence of ≥ 7 of the American College of Rheumatology criteria for SLE at diagnosis and vasculitis were associated with worse survival. Thirteen (26%) patients were in remission with complete discontinuation of drugs, with the mean remission duration being 12 years (range: 2–17 years). Conclusion: It is possible for patients with SLE to get long-term remission through timely and effective treatment. Prevention of infection and renal failure and reducing dosage of corticosteroids by combined treatment with immunosuppressive drugs may enhance prognosis and survival in SLE.

Objective: To evaluate the differences in the renal survival of lupus nephritis (LN) diagnosed either during 1985–1994 or 1995–2004 and to analyse the possible causes. Methods: Fifty-two patients with biopsy-confirmed LN were followed up between 1985 and 1994 and 130 patients were followed up between 1995 and 2004. Renal survival was studied with Kaplan-Meier analysis and the log rank test. Status at diagnosis and treatment schedules were also analysed. Results: Renal survival was significantly better in the patients who were diagnosed between 1995 and 2004 (P = 0.0233). The mean time from initiation until first diagnosis of SLE, from the initiation of SLE until referral to our centre, and from first detection of proteinuria until kidney biopsy was significantly shorter in the later decade (P

The promyelocytic leukemia zinc finger gene ( PLZF ) is involved in chromosomal translocation t(11;17) associated with acute promyelocytic leukemia. In this work, a 201-kilobase genomic DNA region containing the entire PLZF gene was sequenced. Repeated elements account for 19.83%, and no obvious coding information other than PLZF is present over this region. PLZF contains six exons and five introns, and the exon organization corresponds well with protein domains. There are at least four alternative splicings (AS-I, -II, -III, and -IV) within exon 1. AS-I could be detected in most tissues tested whereas AS-II, -III, and -IV were present in the stomach, testis, and heart, respectively. Although splicing donor and acceptor signals at exon–intron boundaries for AS-I and exons 1–6 were classical (gt–ag), AS-II, -III, and -IV had atypical splicing sites. These alternative splicings, nevertheless, maintained the ORF and may encode isoforms with absence of important functional domains. In mRNA species without AS-I, there is a relatively long 5′ UTR of 6.0 kilobases. A TATA box and several transcription factor binding sites were found in the putative promoter region upstream of the transcription start site. PLZF is a well conserved gene from Caenorhabditis elegans to human. PLZF paralogous sequences are found in human genome. The presence of two MLL/PLZF- like alignments on human chromosome 11q23 and 19 suggests a syntenic replication during evolution. The chromosomal breakpoints and joining sites in the index acute promyelocytic leukemia case with t(11;17) also were characterized, which suggests the involvement of DNA damage-repair mechanism.

Introduction Glucocorticoid (GC) therapy remains important in improving the prognosis of patients with systemic lupus erythematosus (SLE). However, some patients do not achieve an effective response with GC treatment, creating an obstacle to the remission of SLE. Identification of the underlying mechanisms responsible for steroid resistance can be significant. Macrophage migration inhibitory factor (MIF) arouses our interest because of its reciprocal relationship with GCs. In the present study, we investigated for the first time whether MIF correlated with steroid resistance in SLE and explored potential mechanisms of action. Methods Sixty-two patients with SLE (40 steroid sensitive and 22 steroid resistant) and 21 normal controls were recruited. Serum levels of MIF were measured by ELISA. Cytosolic MIF and IκB expression in peripheral blood mononuclear cells (PBMCs) were determined by western blotting. The electrophoretic mobility shift assay was assessed by NF-κB in nuclear aliquots. Gene silencing was applied to reduce expression of MIF in PBMCs in steroid-resistant patients. PBMCs obtained from steroid-sensitive patients were treated with recombinant human MIF of different concentrations. Results MIF levels in serum and PBMCs were higher in steroid-resistant patients compared with steroid-sensitive patients and controls. In contrast to the steroid-sensitive group, NF-κB levels were significantly higher and IκB levels lower in steroid-resistant patients. After MIF gene silencing, IκB levels in cells from steroid-resistant patients were increased. In steroid-sensitive patients, a decrease in IκB levels and an increase in NF-κB expression from baseline were detected in PBMCs treated with a higher concentration of recombinant human MIF. Treatment with recombinant human MIF did not regulate expression of IκB and NF-κB in PBMCs from patients treated with an anti-MIF monoclonal antibody. Conclusions Our results indicated that MIF may play a role in the formation of steroid resistance in SLE by affecting the NF-κB/IκB signaling cascade. As a regulator of glucocorticoid sensitivity, MIF may be a potential target for steroid sparing.

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The prognosis of APL is changing, from the worst among AML as it used to be, to currently the best. The application of all-trans-retinoic acid (ATRA) to the induction therapy of APL decreases the mortality of newly diagnosed patients, thereby significantly improving the response rate. Therefore, ATRA combined with anthracycline-based chemotherapy has been widely accepted and used as a classic treatment. It has been demonstrated that high doses of cytarabine have a good effect on the prevention of relapse for high-risk patients. However, as the indications of arsenic trioxide (ATO) for APL are being extended from the original relapse treatment to the first-line treatment of de novo APL, we find that the regimen of ATRA, combined with ATO, seems to be a new treatment option because of their targeting mechanisms, milder toxicities and improvements of long-term outcomes; this combination may become a potentially curable treatment modality for APL. We discuss the therapeutic strategies for APL, particularly the novel approaches to newly diagnosed patients and the handling of side effects of treatment and relapse treatment, so as to ensure each newly diagnosed patient of APL the most timely and best treatment.

The molecular characterization of cytogenetic abnormalities has not only provided insights into the mechanisms of leukemogenesis but also led to the establishment of new treatment strategies targeting these abnormalities and thereby further improve the prognosis of patients. We analyzed the prognosis of 1091 Chinese patients with newly diagnosed acute lymphoblastic leukemia (ALL) and explored the prognostic impacts of a large number of cytogenetic/molecular abnormalities. It was demonstrated that, in both B- and T-ALL settings, the prognosis was negatively correlated to the age as reported to date. For childhood T-ALL patients, it was also documented that the HOX11 expression represented a favorable prognostic factor as it was in adult ones. We identified CRLF2 overexpression as an intermediate-risk marker and Ik6 variant of IKZF1 gene as a high-risk one when stratifying pediatric B-ALL cases according to cytogenetic/molecular risks. We also found that Ik6 variant and CRLF2 overexpression had an important role in dictating the prognosis of Ph-negative patients, which may be useful markers in guiding the treatment of ALL in the future, with tyrosine kinase inhibitors on the other hand reversing the fate of Ph-positive ALL patients.

Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3′UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (P combined = 6.5 × 10 -10), with a stronger effect in male than female subjects [odds ratio, male vs. female = 2.33 (95% CI = 1.64-3.30) vs. 1.24 (95% CI = 1.14-1.34); P = 4.1 × 10 -4]. G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects., link_to_OA_fulltext

Objective MicroRNA (miRNA) have received increasing attention as posttranscriptional regulators that fine-tune the homeostasis of the inflammatory response. This study aimed to clarify whether miR-125a, which was identified in a pilot expression profiling step, is involved in the inflammatory chemokine pathway in systemic lupus erythematosus (SLE). Methods Independent verification of miR-125a expression in amplified samples from SLE patients and normal controls was performed by TaqMan quantitative polymerase chain reaction (PCR) analysis. A combination of 3 bioinformatic prediction techniques and reporter gene assays was used to identify miR-125a targets. In vitro systems of overexpression by transfection and inducible expression by stimulation were performed to investigate the function of miR-125a, which was followed by real-time quantitative PCR and enzyme-linked immunosorbent assay. Results In SLE patients, the expression of miR-125a was reduced and the expression of its predicted target gene, KLF13, was increased. Bioinformatics predicted that miR-125a base-paired with sequences in the 3′-untranslated region of KLF13. Overexpression of miR-125a led to a significant reduction in the expression of RANTES and KLF13. MicroRNA-125a inhibited endogenous KLF13 expression in a dose-dependent manner, as determined using gain- and loss-of-function methods. A luciferase reporter system confirmed the miR-125a binding sites. Notably, miR-125a expression was induced in T cells in a dose- and time-dependent manner. Finally, the introduction of miR-125a into T cells from SLE patients alleviated the elevated RANTES expression. Conclusion MicroRNA-125a negatively regulates RANTES expression by targeting KLF13 in activated T cells. The underexpression of miR-125a contributes to the elevated expression of RANTES in SLE. Our findings extend the role of miRNA in the pathogenesis of lupus and provide potential strategies for therapeutic intervention.

To evaluate the efficacy and safety of leflunomide in comparison with methotrexate (MTX) on patients with rheumatoid arthritis (RA) in China.Five hundred and sixty-six patients with active rheumatoid arthritis were randomly assigned to receive leflunomide at 20 mg once daily or MTX at 15 mg once weekly in a controlled trial. Five hundred and four patients completed the 12-week treatment and some patients continued the treatment for 24 weeks.Both leflunomide and MTX could improve the symptoms, signs, and joint function, but there were no changes in X-ray observations of patients with rheumatoid arthritis. In the leflunomide group, the overall rates of effectiveness at 12 weeks and 24 weeks were 86.94% and 92.31% respectively; the rates of remarkable improvement were 64.95% and 79.81% respectively. In the MTX group, the overall rates of effectiveness at 12 weeks and 24 weeks were 84.04% and 83.15% respectively; the rates of remarkable improvement were 56.81% and 75.28% respectively. According to intent-to-treat analysis, the ACR 20% response rates at 12 weeks and 24 weeks in the leflunomide group were 62.54% and 67.18% respectively, compared with 60.08% and 61.32% respectively in MTX group. No statistical differences were shown in the efficacy between the two groups (P0.05). The adverse events in the leflunomide group were gastrointestinal symptoms, skin rash, alopecia, nervous system symptoms, decreased leukocyte count, and elevation of alanine aminotransferase (ALT). Most of these side effects were mild and transient. The incidence of adverse events in the leflunomide group was 16.84%, significantly lower than that in MTX group (28.17%, P = 0.002).Leflunomide is effective in the treatment of RA with less adverse events than MTX. Its efficacy is similar to MTX, but the incidence of adverse events and the rate of withdrawal due to adverse events were lower in the leflunomide group than in MTX group.

To develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.Stable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight over ten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60.As a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.

To study the association between bcl-2 gene polymorphism and systemic lupus erythematosus (SLE) in Chinese and detect the synergism between IL-10 and bcl-2 genotypes in determining susceptibility to SLE.The peripheral white blood cells from 232 nuclear families of SLE and 106 ethnically matched normal controls were collected and the DNA extracted. The allelic distribution of one microsatellite located in Bcl-2 gene was determined by using fluorescent-labeled primer and genotyping method.(1) The distribution frequency of Bcl-2 alleles and its major genotypes were significantly different between SLE group and normal controls (P = 0.001 and 0.0029 respectively). (2) Transmission disequilibrium existed in Bcl-2 alleles. Bcl-2-195 bp allele was preferentially transmitted to affected offspring (t: non-t = 122:82, P = 0.0051) by TDT approach. Individuals carrying specific genotypes with both Bcl-2-195 bp allele and IL-10G138 bp allele were at higher risk of developing SLE than those carrying only Bcl-2-195 bp allele or IL-10G138 bp allele (OR = 3.00, 1.77, 1.07 respectively).Bcl-2 gene polymorphism is associated with SLE and may be directly involved in the process of SLE or in linkage disequilibrium with some susceptibility loci nearby. Synergistic effect may exist between IL-10 and bcl-2 genotypes in determining susceptibility to SLE.

To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to beta(2)-glycoprotein.Full-length beta(2)GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT-PCR and cloned into the mammalian expression vector pEGFP-C1. The recombinant plasmid pEGFP-beta(2)GP was transfected into HEp-2 cells. RT-PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP). Serum specimens from 19 patients suspected as with secondary antiphospholipid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF-IgG-beta(2)GP1, ELISA-IgG-ACL, and ELISA-IgG-beta(2)GP I simultaneously.(1) The HEp-beta(2)GP I cells thus obtained retained their ability of expression of beta(2)GP-I-GFP for more than ten generations. This beta(2)GP-I-GFP showed the antigenicity of beta(2)GP-I with a characteristic feature. (2) Seven of the 20 serum specimens from APS patients showed characteristic immunofluorescent pattern. No serum specimen from normal persons showed immunofluorescent staining. The comparison of results of the three methods showed that the concordance between IIF-IgG-beta(2)GP I and ELISA-IgG-beta(2)GP I was the most perfect (Kappa = 0.886). (3) HEp-beta(2)GP I retained the immunofluorescent property of HEp-2 cell.As a new kind of substrate of IIF, beta(2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies. Transfeted HEp-2 cells keep the immunofluorescent property of HEp-2 cells in IFANA test and can be used as substrate for routine IFANA detection.

Introduction Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown. Methods In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting. Results We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls. Conclusion Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.