[Modulation of RhoA/Rho kinase on migration, invasion and proliferation of fibroblast like synoviocytes from patients with rheumatoid arthritis].

OBJECTIVE: To evaluate the modulation of RhoA/Rho kinase (ROCK), a small Rho GTPase, on migration, invasion and proliferation of fibroblast like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: RA FLS were collected from active RA patients. And 10% fetal bovine serum (FBS) and interleukin-1ß (IL-1ß) were used as stimuli in migration and proliferation experiments respectively. RhoA activity was measured by pull down assay while ROCK activity by Western blot. FLS migration and invasion in vitro were measured by the Transwell chamber method. And thiazolyl blue tetrazolium bromide (MTT) test was used to detect cell proliferation. RESULTS: There were increased activities of RhoA and ROCK in ex vivo FLS from RA versus OA patients and healthy control. The migrated cell number of FBS-induced, C3-treated and Y27632-treated groups was 85 ± 14, 51 ± 15 and 42 ± 11 respectively. The Matrigel invading cell number of 3 groups was 64 ± 13, 39 ± 12 and 26 ± 9 respectively. Statistical differences existed in cell number between FBS-induced, C3-treated or Y27632-treated group (P < 0.05) in above migration and invasion experiments. Inhibition of RhoA and ROCK activity also suppressed the cytoskeletal reorganization and proliferation of RA FLS. CONCLUSION: Increased RhoA/ROCK activity may contribute to abnormal migration, invasion and proliferation of RA FLS. Thus inhibition of ROCK activity may be a new therapeutic target for RA.

[Associated factors for the occurrence of lupus enteritis].

OBJECTIVE: To explore the clinical and laboratory characteristics of patients with lupus enteritis to provide rationales for clinical diagnosis and treatment. METHODS: A retrospective group control study was conducted for systemic lupus erythematosus (SLE) patients with complaints of acute abdominal pain from 2004 to 2011. They were divided into 2 groups: lupus enteritis (n = 66) and non-lupus related abdominal pain (n = 73). The associated factors included demographic, laboratory, clinical and radiographic data. RESULTS: Lupus enteritis (39.3%) was the most common cause of lupus patients with acute abdominal pain. There were no differences in autoantibody profiles, complement, erythrocyte sedimentation rate, C reactive protein and SLE disease activity index (SLEDAI) score between two groups. The level of D-dimer and European consensus lupus activity measurement (ECLAM) score were significantly higher in the group of lupus enteritis than those in non-lupus related gastrointestinal injury. Lupus enteritis had significantly higher percentage of complications with multiple serous cavity effusions and ascites. But after adjusting with logistic regression multivariate analysis, only the level of D-dimer, ECLAM and volume of ascites were associated with occurrence of lupus enteritis. CONCLUSION: Lupus enteritis is the most common cause of acute abdominal pain. D-dimer is an excellent predictor for lupus abdominal pain. As compared with SLEDAI, ECLAM may be more suitable for assessment in SLE patients with alimentary tract injury.

[Modulation of RhoA/ROCK pathway on TLR-2 ligand-induced chemokine secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis].

OBJECTIVE: To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway, a small Rho GTPase that is considered as an important modulator in inflammatory responses, on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: The RhoA activity was measured by a pull-down assay. And the ROCK activity was assessed by Western blot. The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay). MTT test was used to detect the cellular viability. RESULTS: The stimulation of peptidoglycan (PG, 5 mg/L) increased the levels of IL-8 (interleukin-8), RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2) and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS. And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody. Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8, RANTES and MCP-2 in PG-induced RA FLS. CONCLUSION: The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS. It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.

[Clinical analysis of 6 cases of systemic lupus erythematosus complicated by transverse myelitis].

OBJECTIVE: To summarize the clinical features and therapeutic approach of systemic lupus erythematosus (SLE) complicated by transverse myelitis (TM). METHODS: The clinical characteristics, laboratory examinations, treatment and prognosis of 6 SLE cases with TM were retrospectively analyzed with review of the literatures. RESULTS: The 6 patients consisted of 5 females and 1 male aged 14 to 36 years (mean 23 years). The mean duration from symptom onset of SLE to TM was 8 months (1 to 13 months). All the patients had lower limb hypodynamia, and 3 of them developed upper limb hypodynamia. MRI scanning of the spine identified lesions in the cervical spinal cord in 2 cases, thoracic lesions in 3 cases, and multiple involvement of the cervical, thoracic and lumbar cord in 1 case. Examination of the cerebrospinal fluid yielded no specific findings except for leukocytosis in 1 case and hypoglycemia in another. Five cases were treated with high-dose MP+CTX, and the other case was treated with MP (80 mg/day)+CTX. Five patients responded favorably to the treatment, while the other showed no obvious improvement. CONCLUSION: TM is a rare complication of SLE affecting mostly young patients and occurring in the early stage of the disease. Early diagnosis and aggressive treatment might improve the prognosis.

[Cytokine in synovial monocytic cells from rheumatoid arthritis patients].

OBJECTIVE: To evaluate the inhibitory effect of blockade of Rho kinase upon mediating the secretion of proinflammatory cytokine in monocytic cells from rheumatoid arthritis (RA) patients. METHODS: Synovial fluid (SF) monocytic cells and peripheral blood monocytes (PB) from active RA patients were treated with TNFalpha or LPS respectively in the presence or absence of a specific ROK inhibitor, Y27632. ROK activity was assessed by Western blot and cytokine secretion measured by ELISA. RESULTS: Elevated ROK activity was found in synovial fluid monocytic cells from active RA patients. ROK activity was correlated with DAS, an index of disease activity of RA patients. ROK inhibitor Y27632 reduced the secretion of TNFalpha, IL-1beta and IL-6 in RA SF monocytic cells, but had no effect upon the secretion of IL-10, an anti-inflammatory cytokine. CONCLUSION: The present study provides novel evidence that ROK mediates the secretion of proinflammatory cytokines in monocytic cells from RA synovial fluids, suggesting a critical role of ROK in macrophage-mediated synovial inflammation of RA. Thus inhibition of ROK may be a new therapeutic target for RA.

[Abnormal signaling activity of phosphatidylinositol 3-kinase pathway in peripheral blood T cells from patients with systemic lupus erythematosus].

OBJECTIVE: To investigate the activity of phosphatidylinositol 3-kinase (PI3K) signal pathway, a cytoplasmic signaling pathway known to play an important role in T cell activation, in peripheral blood T cells from systemic lupus erythematosus (SLE) patients. METHODS: T cells were isolated from the peripheral blood samples of 28 SLE patients, 5 males and 23 females, with RosettSep T cell purification kit. PI3K activity was determined by immunoprecipitation and ELISA, and Western blotting was used to measure the Akt and phosphorylated Akt protein expression. T cell proliferation and cytokine production was examined by MTT test and ELISA respectively. Fifteen healthy adults and 8 active rheumatoid arthritis patients were used as controls. The T cells from the SLE patients and normal controls were treated with 10% normal control serum of SLE serum for 24 h ("rest") and then to detect the P13K and Akt activity. Some T cells from the SLE patients were stimulated with CD3/CD28 mono-antibodies or CD3/CD28 mono-antibodies + LY294002, a specific P13K inhibitor, and then the proliferation and secretion of IL-6 and IL-10 were analyzed. RESULTS: Compared with the healthy controls and rheumatoid arthritis patients, the activity levels of PI3K and Akt in the T cells of peripheral blood from the SLE patients were significantly increased. T cells allowed to "rest" for 24 hours in culture medium showed a reversal of the changes in activity of PI3K and Akt. The activity of PI3K pathway was increased in the T cells from healthy controls when cultured with SLE serum. The proliferation and IL-6 and IL-10 secretion of the T cells from SLE patients cultured with LY294002 were inhibited. The P13K and Akt activity levels of the T cells from SLE patients were not related to SLE disease activity index (SLEDAI). CONCLUSION: The T cells from SLE patients show an abnormal activation of PI3K pathway which may be due, at least in part, to their exposure to relevant serum factors.

[Comparative study of systemic lupus erythematosus in children and adults].

OBJECTIVE: To discuss the differences in the clinical features, laboratory tests and renal pathology between children and adults with systemic lupus erythematosus (SLE). METHODS: A retrospective study was performed in 198 children and 200 adults with SLE. RESULTS: Fever, rash, arthritis, anemia and renal involvement were the most common symptoms in both groups. The incidence of hepatomegaly, splenomegaly, lymphadenectasis, anemia, renal involvement, nervous system involvement and digestive apparatus involvement were higher in children with SLE. The mean SLE Disease Activity Index score was also higher in the children. Immunological findings showed that a greater proportion of children with SLE were positive for anti-double stranded DNA antibody, anticardiolipin antibody and perinuclear antineutrophil cytoplasmic antibody. Renal pathological examinations showed that children with SLE patients were more likely to have serious renal involvement. The misdiagnosis rate was higher in children with SLE patients. During the hospital stay, 12 (6.1%) children with SLE died, with an average disease course of 6.8 months; 9 (4.5%) adults with SLE died with an average disease course of 4.2 years. CONCLUSION: Children with SLE patients are liable to have systemic involvement and higher misdiagnosis rate, often with poorer prognosis than the adult patients.

[Role of gonadotropin releasing hormone analogues for ovarian protection in systemic lupus erythematosus patients treated with cyclophosphamide].

OBJECTIVE: To evaluate the effectiveness of gonadotropin releasing hormone analogues (GnRH-a) in protection against premature ovarian failure during cyclophosphamide (CTX) therapy for systemic lupus erythematosus (SLE). METHODS: 28 female patients with SLE, aged 35.3 +/- 2.4 (30-39) were treated with prednisone orally 1 mg/kg daily for 8 weeks, and then the dose was decreased by 10% every 10 days. CTX 200 mg with normal saline 200 ml was intravenously injection every other day for 4 months. Peripheral white blood cell (WBC) count was made every week. If the WBC count was less than 3.5 x 10(9)/L, the use of CTX should be stopped temporarily until the WBC count became normal. And then, the CTX administration should be adjusted to 400 mg intravenously every week. All patients were offered treatment with Hypodermic injection of GnRH-a 3.75 mg was given monthly just at the beginning of the standard CTX regimen for 3 months. Follow-up was conducted for 6 months after the last prescription of GnRH-a. RESULTS: All patients developed amenorrhea after treated by GnRH-a. Menstruation recovered 73 days (69-82 days) after the last subcutaneous injection in 25 patients. Among these 25 patients, one developed amenorrhea again after two normal menses periods. The other 3 patients were in persistent amenorrhea during the following 6 months after the GnRH-a treatment. The levels of plasma estradiol (E2) was 998 +/- 308 pmol/L before GnRH-a treatment, and decreased significantly 1, 2, and 3 months after the last injection of GnRH-a (132 +/- 44 pmol/L, 88 +/- 37 pmol/L and 81 +/- 29 pmol/L respectively, all P < 0.05). The level of plasma E2 increased 2 months after the last injection of GnRH-a in the 25 patients with return of menses, and the level of plasma E2 returned to the normal baseline level after 6 months in 24 patients. CONCLUSION: Treatment with GnRH-a during CTX therapy is associated with a significant reduction of premature ovarian failure in most women with SLE.

[Clinical study of etanercept for treating ankylosing spondylitis].

OBJECTIVE: To evaluate the efficacy and safety of etanercept, a tumor necrosis factor (TNF)-alpha inhibitor, in the treatment of ankylosing spondylitis (AS), and investigate its effect on serum levels of matrix metalloproteinase-3 (MMP-3). METHODS: Forty-eight patients with AS received etanercept 25 mg twice a week for a treatment course of 12 weeks. The patients' symptoms, signs, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels and side effects were observed before and after the treatment. The serum levels of MMP-3 was determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: All the patients completed the treatment. The degree of spinal pain and pain at night, the duration of morning stiffness, the finger-to-floor distance, BASDAI and BASFI were significantly improved after the treatment (P<0.05). Etanercept treatment resulted in a significant reduction in serum MMP-3 level in the AS patients to 31.22-/+10.26 ng/ml as compared with the level before treatment (46.17-/+25.74 ng/ml, P<0.05). The reduction of serum MMP-3 was positively correlated to decrement of ESR and CRP (r=0.397 and 0.474, respectively, P<0.05). The most common adverse events of etanercept included injection site reaction and upper respiratory infection. CONCLUSION: Etanercept treatment has obvious therapeutic effects on AS without serious adverse effects. MMP-3 may be a potentially useful indicator to assess the effect of anti-TNF-alpha treatment in AS patients.

[Association between metabolic enzyme genotype of azathioprine and drug tolerance in patients with rheumatic diseases].

OBJECTIVE: To discuss the relationship between the genotype of thiopurine methyltransferase (TPMT) and azathioprine tolerance in the patients with rheumatic diseases. METHODS: Four common mutation alleles of TPMT in 200 patients with rheumatic diseases [TPMT*2 (G238C), TPMT*3A (A719G/G460A), TPMT*3B (G460A), TPMT*3C (A719G)] were detected by allele specific polymerase chain reaction (AS-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP). 194 patients who had used azathioprine finished the 3 months' follow-up. RESULTS: In the 200 patients with rheumatic diseases, 4 cases of heterozygote of TPMT*3C were detected, but no mutation of TPMT*2, TPMT*3B or TPMT*3A was found. The genotypic frequency of wild-type homozygote was 98%, and that of heterozygote (TPMT*1/TPMT*3C) was 2%. Mutation allele frequency in these patients was 1%. Average of TPMT activity of the 4 cases of heterozygote was (2.4 +/- 1.2) U/ml red blood cells, significantly lower than that of the 196 cases of homozygote which was (12.2 +/- 6.8) U/ml RBC. In the 194 patients who had used azathioprine, bone marrow suppression occurred in 18 patients, 2 of which suffered severe crisis of hematopoietic system, and 6 of which were complicated with impaired liver function. In the 4 patients with heterozygote, 3 had used azathioprine, and bone marrow suppression occurred within 1 month of using the drug, including 2 cases of severe crisis of hematopoietic system. CONCLUSION: Patients with mutation alleles of TPMT are intolerant to azathioprine, and likely to have severe crisis of hematopoietic system. To detect the TPMT genotype before using azathioprine is significant to improve the therapeutic safety.

[The profile and clinical significance of azathioprine metabolic enzymes activity in rheumatological patients].

OBJECTIVE: To detect the activity of thiopurine methyltransferase (TPMT) and its relationship with azathioprine (AZA) tolerance. METHODS: 200 patients of rheumatism need AZA were included in the study. RBC TPMT activity was detected with high performance liquid chromatography. Then the patients took AZA doses of 50 mg/d for the first month, 100 mg/d for the second month and 150 mg/d for the third month. RESULTS: TPMT activity of 200 patients ranged from 0.75 - 32.35 U/ml RBC, averaged (12.04 +/- 6.90) U/ml RBC. The activity of TPMT showed a normal skewness distribution and no activity deficiency was founded. 194 patients (97%) completed the 3 month follow-up. 18 showed bone marrow depression including 2 severe hematological crisis and 6 showed hepatic damage during the 3 months. Bone marrow depression was recorded of 7 cases with the TPMT activity of 2.24 - 5.97 U/ml RBC, averaged (3.47 +/- 1.21) U/ml RBC, among the dose of 50 mg/d and 11 cases with the TPMT activity of 4.01 - 11.17 U/ml RBC, averaged (7.08 +/- 2.58) U/ml RBC, among the dose of 100 - 150 mg/d. The other 176 cases did not show bone marrow depression at all, with TPMT activity of 4.47 - 32.35 U/ml RBC, averaged (13.02 +/- 6.07) U/ml RBC. TPMT activities in the 3 groups of patients were significantly different according to statistical analysis (P < 0.01). CONCLUSIONS: Hematological side effects were highly associated with TPMT activity in AZA usage. Patients with low TPMT activity should use low dose of AZA routinely, even though, toxicity may occur. Test of TPMT activity before AZA description was of significance.

[Risk factors of ovarian failure in the patients with systemic lupus erythematosus receiving cyclophosphamide therapy].

OBJECTIVE: To determine the risk factors for ovarian failure after cyclophosphamide therapy in the patients with systemic lupus erythematosus (SLE). METHODS: A case-control study was conducted among 138 female SLE patients, aged 16-45, and treated with cyclophosphamide, 46 of which with premature ovarian failure were included in the case group and 92 of which without menopause after completion of induction therapy with cyclophosphamide were included in the control group. A Logistic regression model was established and step-wise selection was used to analyze different factors, such as age of initiation of therapy, marital history, childbearing history, disease activity index, accumulative dosage of cyclophosphamide, accumulative dosage of azathiopurine, and usage of traditional Chinese medicines etc. RESULTS: Univariate analysis showed that age of initiation of cyclophosphamide treatment (OR = 1.11, 95% CI = 1.06 - 1.17), SLE activity index (OR = 1.11, 95% CI = 1.06 - 1.17), duration of Trypterygium wilfordii treatment (OR = 1.26, 95% CI = 1.05 - 1.51), duration of cyclophosphamide treatment (OR = 0.16, 95% CI = 0.028 - 0.94), and marital situation were associated with ovarian failure; and multivariate analysis showed that age of initiation of cyclophosphamide treatment (OR = 1.24, 95% CI = 1.14 - 1.35), cumulative dosage of cyclophosphamide (OR = 1.13, 95%, CI = 1.05 - 1.23), and duration of treatment of Trypterygium wilfordii agents (OR = 1.36, 95% CI = 1.09 - 1.69)were significantly associated with premature ovarian failure. Only 4 cases in the case group began to receive cyclophosphamide treatment before the age of 20 and their accumulative cyclophosphamide dosage was 28.8 - 32.4 g. The median of cumulative dosage of cyclophosphamide was only 4.8 g for the 11 cases aged 40-45 in the case group. CONCLUSION: The risk factors of premature ovarian failure during the induction therapy in SLE patients include age of initiation of cyclophosphamide treatment, cumulative dosage of cyclophosphamide and duration of treatment with leigongteng.

[Efficacy of oral bromocriptine in protecting the postpartum systemic lupus erythematosus patients from disease relapse].

OBJECTIVE: To explore the efficacy of oral bromocriptine in protecting the postpartum patients with systemic lupus erythematosus (SLE) from disease relapse. METHODS: The research strategy was a randomized controlled trial. 68 consecutive pregnancy patients with SLE from July 1995 to June 2002 followed up in the teaching hospital were included in the study. The patients were randomly divided into the treatment group and the control group according to their expected date of confinement. The patients in the treatment group had bromocriptine (2.5 mg bid) for 14 days started within 12 hours of postpartum and didn't nurse their infants. The patients in the controlled group didn't have any treatment of influence on prolactin or other sexual hormones. 21 patients nursed and 13 didn't nurse their infants in the controlled group. All patients were followed up for 12 months. RESULTS: The serum prolactin and estradiol levels in treatment group were lower than in nursing controlled group and in non nursing controlled groups, at second week and second month after delivery. The relapse rate in the treatment group was lower than in nursing controlled group and in non nursing controlled groups. The result of Log-rank test was chi(2) = 8.90, P = 0.007 5 comparing three group data of following up with SLEDAI increase 3 as endpoint. The number needed treatment was 3.1, 95% CI (1.9-8.5). Accumulative doses of prednisone within the 12 months were (3.90 +/- 1.82) g in the treatment group and (8.92 +/- 3.36) g in the controlled group, P < 0.001, and cyclophosphamide were (1.41 +/- 0.83) g in the treatment group and (4.27 +/- 2.38) g in the controlled group, P < 0.001. CONCLUSIONS: Oral bromocriptine for 2 weeks in postpartum patients with SLE may relieve the disease from hyperprolactinemia and hyperestrogenemia, and may be beneficial in protecting the patients from disease relapse and in reducing the usage of steroid and immunosuppressant.