OBJECTIVE: MicroRNAs (miRNAs) play important roles in many biological processes and recent studies have provided growing evidences that miRNA dysregulation might play important roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate the contribution of miRNAs to altered gene expressions in RA. METHODS: To investigate whether the differential expression of miRNA in RA could account for the altered expression of certain genes, we compared the different expressions of miRNAs and mRNAs in rheumatoid synovial fluid monocytes with that of normal peripheral blood (PB) monocytes by using a gene expression oligonucleotide microarray and a microRNA microarray. RESULTS: Comparative analysis of the mRNA profiles showed significant different expressions of 430 genes in RA synovial monocytes, of which 303 (70%) were upregulated and 127 (30%) were downregulated, as compared with that of normal PB monocytes. Out of differentially expressed 13 miRNAs, 9 miRNAs were upregulated and 4 miRNAs were downregulated in the RA synovial monocytes. A total of 62 genes were predicted as target genes of the 13 differentially expressed miRNAs in the RA synovial monocytes. Among the 62 miRNA-targeted genes, a few genes such as GSTM1, VIPR1, PADI4, CDA, IL21R, CCL5, IL7R, STAT4, HTRA1 and IL18BP have been reported to be associated with RA. CONCLUSION: In the present study, we observed that several miRNAs are differentially expressed in RA synovial monocytes, and we suggest that these different expressions of miRNAs may regulate the expression of several genes associated with the pathogenesis of RA.
Arthritis, Rheumatoid ; Biological Processes ; Gene Expression ; Genes, vif ; MicroRNAs ; Monocytes ; Oligonucleotide Array Sequence Analysis ; Receptors, Interleukin-21 ; RNA, Messenger ; Synovial Fluid

Interleukin-21 (IL-21) is a new member of the interleukin-2 family. It is mainly synthesized and secreted by the activated of CD4(+) T cells and natural killer T cells. IL-21 receptor (IL-21R) is mainly expressed in T cells, B cells, and natural killer (NK) cells. After binding to its receptor, IL-21 can regulate the activation and proliferation of T cells, B cells, and NK cells through activating JAKs-STATs signaling pathways. As a new immunoregulatory factor, IL-21 and its receptor play important roles in the development and progression of various autoimmune diseases. Regulation of the expression levels of IL-21 and IL-21R and blocking of their signal transduction pathways with blockers may be new treatment options for autoimmune diseases.
Animals ; Autoimmune Diseases ; etiology ; immunology ; Dendritic Cells ; immunology ; Humans ; Interleukins ; physiology ; Killer Cells, Natural ; immunology ; Lymphocytes ; immunology ; Receptors, Interleukin-21 ; physiology

OBJECTIVE: To explore the role of IL-21 in the pathogenesis of myasthenia gravis (MG) and its influence on the the class switch of anti-AChR antibodies. METHODS: Blood was taken from 26 patients and 18 healthy controls, and the expression of IL-21R mRNA in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR. The expression of IL-21R on B lymphocytes was measured by flow cytometry, while the concentrations of serum IL-21 and the levels of anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3) were tested by ELISA. RESULTS: The serum concentration of IL-21 in the MG group was higher than that in the control group (31.686±8.499 pg/mL, 15.147±6.366 pg/mL) and the difference was significant (P<0.01). IL-21R mRNA expressed on PBMCs in the MG group was higher than that in the control group (0.139±0.052, 0.101±0.022), and the difference was significant (P<0.05). There was no difference between ocular MG and generalized MG subgroup (P>0.05). Compared with the control group, the expression of IL-21R on B lymphocytes also increased in the MG group (P<0.05). In the anti-AChR-Ab positive MG group, the serum concentration of IL-21 showed positive correlation with anti-AChR-IgG(P<0.05),but no correlation with its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05). Expression of IL-21R mRNA in the PBMCs showed no correlation with the level of serum anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05); however the expression of IL-21R in B lymphocytes showed positive correlation with anti-AChR-IgG and it's isotype IgG(1) and IgG(3) (P<0.05,P<0.01,P<0.05), but no correlation with IgG(2) (P>0.05). CONCLUSION IL-21 might induce the class switch of anti-AChR antibodies to IgG(1) and IgG(3) isotype through IL-21R on B lymphocytes which promotes the pathogenesis of the MG.
Adolescent ; Adult ; Autoantibodies ; classification ; immunology ; Female ; Humans ; Immunoglobulin Class Switching ; immunology ; Immunoglobulin G ; classification ; Interleukins ; blood ; genetics ; Male ; Middle Aged ; Myasthenia Gravis ; blood ; immunology ; RNA, Messenger ; blood ; genetics ; Receptors, Cholinergic ; immunology ; Receptors, Interleukin-21 ; blood ; genetics ; Young Adult

BACKGROUND AND OBJECTIVES: Interleukin-21 receptor (IL-21R) gene polymorphism is related with the development of systemic vasculitis. In this study, we investigated the polymorphisms of IL-21R gene in patients with Kawasaki disease (KD). SUBJECTS AND METHODS: We genotyped the promoter region of IL-21R gene (-2500 bp to +1 bp) in 100 patients with KD and 100 healthy controls. All study subjects were Korean. We designed five pairs of primers and performed polymerase chain reaction (PCR) and direct sequencing. We analyzed whole promoter sequences of 200 individuals with comparison to reference sequences of IL-21R gene (NG_012222.1/NC_000016.9). RESULTS: We found five single nucleotide polymorphisms (SNPs) of which minor allele frequency (MAF) >0.01 in the promoter region of IL-21R gene. Those are -1681 G>T (chromosome site 27411802), -379 G>A (27413104), -332 G>C (27413151, rs2214537), -237 A>T (27413246), and -53 G>A (27413430). There is no significant difference in MAF of each SNP between patients with KD and healthy controls except -237 A>T. Twenty five patients with KD had more than 1 SNP in contrast to only seven healthy controls had. The patients with KD have significantly more IL-21R gene polymorphisms than controls (odds ratio: 3.0, 95% confidence interval: 1.6-5.6, p=0.0005). There was no significant correlation between IL-21R gene polymorphisms and the serum level of IL-21. The serum level of total IgE was not significantly correlated with the presence of IL-21R gene polymorphisms. CONCLUSION: Our data suggest that the genetic susceptibility profile for KD may include IL-21R gene.
Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Immunoglobulin E ; Interleukins ; Mucocutaneous Lymph Node Syndrome ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Receptors, Interleukin-21 ; Systemic Vasculitis

OBJECTIVETo explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.

METHODSMononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-&gamma; (IFN-&gamma;), tumor necrosis factor-&alpha; (TNF-&alpha;), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-&gamma; and TNF-&alpha; in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.

RESULTSAfter IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-&gamma; increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-&alpha; increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-&gamma; in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-&alpha; increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.

CONCLUSIONHumanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-&gamma; and TNF-&alpha;, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.

Cells, Cultured ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; Fas Ligand Protein ; metabolism ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukins ; pharmacology ; K562 Cells ; Perforin ; metabolism ; Receptors, Interleukin-21 ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION The acute myeloid leukemia NOD-SCID-IL2rg
Animals ; Disease Models, Animal ; Interleukin Receptor Common gamma Subunit ; Leukemia, Myeloid, Acute ; Luciferases/genetics* ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID

Animals ; Cell Line ; Cytological Techniques ; methods ; Embryonic Stem Cells ; cytology ; Interleukin Receptor Common gamma Subunit ; genetics ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mutation

BACKGROUNDThe common &gamma; chain (&gamma;c) plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the &gamma;c signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism.

METHODSSplenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE). After 24 hours, recipients received i.p. injection of mixture of anti-&gamma;c mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry.

RESULTST-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point.

CONCLUSIONSThe results suggested that blockade of &gamma;c signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Cells, Cultured ; Flow Cytometry ; Fluoresceins ; Immune Tolerance ; drug effects ; Interleukin Receptor Common gamma Subunit ; antagonists & inhibitors ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Signal Transduction ; drug effects ; Spleen ; cytology ; Succinimides ; T-Lymphocytes ; cytology ; drug effects

This research investigated the clinical features of immunodeficiency disease and the features of the mutation of its pathogenic genes. All 7 patients were boys aged 5 months to 4 years and 6 months and had a history of recurrent respiratory infection and pneumonia, low levels of IgM and IgG, and abnormal absolute values or percentages of lymphocyte subsets. High-throughput sequencing showed c.1684C>T mutations in the BTK gene in patient 1 and IVS8+2T>C splice site mutations in the BTK gene in patient 2. Both of these mutations came from their mothers. Patients 3, 4, and 5 had mutations in the IL2RG gene, i.e., c.298C>T, IVS3-2A>G, and c.164T>A, among which c.164T>A mutations had not been reported. Patient 6 had c.204C>G mutations in the RAG2 gene. Patient 7 had complex heterozygous mutations of c.913C>T and c.824G>A in the RAG2 gene, which came from his father and mother, respectively. Patients with immunodeficiency disease have abnormal immunological indices, and high-throughput sequencing helps to make a definite diagnosis.
Agammaglobulinaemia Tyrosine Kinase ; Agammaglobulinemia ; genetics ; Child, Preschool ; Computational Biology ; DNA-Binding Proteins ; genetics ; Genetic Diseases, X-Linked ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Immunologic Deficiency Syndromes ; genetics ; therapy ; Infant ; Interleukin Receptor Common gamma Subunit ; genetics ; Male ; Mutation ; Nuclear Proteins ; genetics ; Protein-Tyrosine Kinases ; genetics

OBJECTIVETo analyze the mutation of IL2RG gene in a Chinese family with a birth history of a dead child suspected of X-linked severe combined immunodeficiency (X-SCID), and to perform prenatal diagnosis with DNA sequencing.

METHODBlood samples of the parents of the dead child and chorionic villi at gestational age 11 weeks were collected. Eight exons comprising the open reading frame as well as their exon/intron boundaries of IL2RG gene were analyzed by PCR and bi-directional sequencing.

RESULTA heterozygous nucleotide substitution c.690C > T (R226C) in exon 5 was detected in the mother, but not in the father. In the second pregnancy of the mother, the mutation of R226C was not detected in the male fetus by prenatal diagnosis, and the heterozygous mutation was detected in the female fetus of the third pregnancy. The reliability of the prenatal genetic diagnosis was confirmed by the one-year follow-up after the neonates were born.

CONCLUSIONThe mutation of c.690C>T in IL2RG gene may be the pathologic cause of the proband with X-SCID. DNA sequencing combining sex determination is a valid strategy for prenatal diagnosis of X-SCID.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Exons ; genetics ; Female ; Heterozygote ; Humans ; Infant ; Interleukin Receptor Common gamma Subunit ; genetics ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis ; methods ; X-Linked Combined Immunodeficiency Diseases ; diagnosis ; genetics