Influences of IL-6R antibody on PMMA bone cement-mediated expression of OPG and RANKL in synovial fibroblasts

Effect of interleukin-6 receptor （IL-6R） antibody on polymethyl methacrylate （PMMA） bone cement-mediated expression of osteoprotegerin （OPG） and ：receptor activator of nuclear fac- tor-kappaB ligand （RANKL） in synovial fibroblasts was investigated. Synovial tissue obtained from to- tal knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry （SABC method） staining was used to identify synovial fibro- blasts. This experiment was divided into three groups according to different culture media： PMMA group （75μg/mL PMMA bone cement particles）, IL-6R antibody group （10 ng/mL IL-6R antibody＋75 μg/mL PMMA bone cement particles）, and control group （no IL-6R antibody or PMMA bone cement particles）. Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was meas- ured by cell counting kit-8 （CCK-8）. ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR （FQ-PCR） was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance （A） value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group （P〈0.01）, but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group （P〉0.05）. Re- suits of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group （P〈0.01）. The expression of RANKL was inhibited （P〈0.05）, and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group （P〉0.05）, but the expression of RANKL was higher in PMMA group than in control group （P〈0.05）, and there was a significant difference in the ratio of OPG/RANKL be- tween them （P〈0.05）. Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited （P〈0.01） and the expression of OPG mRNA was significantly increased （P〈0.01） in IL-6R an- tibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group （P〈0.05）, but the expression of OPG mRNA had no sig- nificant difference between them （P〉0.05）. IL-6R antibody could significantly increase the expression of OPC~ but inhibit the expression of RANKL, which might provide a theoretical basis of molecular bi- ology for the prevention and treatment of aseptic loosening of prosthesis.     

COA theme issue of the Chinese Medical Journal: A call for papers

Background Rheumatoid arthritis （RA） is characterized by inflammation of the synovial membrane, leading to invasion of synovial tissue into the adjacent cartilage matrix with degradation of articular cartilage and bone as a consequence. Dickkopf-1 （DKK-1） and osteoprotegerin （OPG） have been demonstrated to be key molecules involved in bone erosion and bone remodeling. The aim of this study was to explore the potential role of DKK-1 and OPG in different stage of RA. Methods The protein levels of DKK-1 and OPG were detected by ELISA. The serum samples were collected from 300 patients with RA and 60 healthy controls. Of which, 150 RA patients were defined as early RA （disease duration 〈1 year）, and other 150 RA patients were defined as Ionglasting RA （disease duration 〉5 years）. At the time of serum sampling, various clinical and laboratory parameters were assessed. The correlations of DKK-1 or OPG and clinical/laboratory parameters were analyzed. Results The serum level of DKK-1 was elevated in patients with longstanding RA compared with healthy controls, while no significant difference was observed between the two groups in the level of OPG. In contrast, in early RA patients, the circulating OPG was elevated, while there was no significant difference between the two groups in expression of DKK-I. The serum DKK-1 was correlated with Sharp score and DAS28 in longstanding RA patients. In early RA, age was the only parameter that was significantly related to serum OPG. Conclusions There was a cross-talk between DKK-1 and OPG, which involved in bone destruction in RA. In different stage of RA, DKK-1 and OPG may play different roles in the pathogenesis of RA.     

Bone Marrow Urokinase Plasminogen Activator Receptor Levels are Associated with the Progress of Multiple Myeloma△

Objective To determine the mRNA and protein levels of urokinase plasminogen activator receptors (uPAR) in bone marrow fluid and bone marrow tissue from multiple myeloma (MM) patients and assess association of uPAR level with prognosis of MM. Methods uPAR levels in bone marrow fluid of 22 MM patients at the stable and progressive stages and 18iron deficiency anemia patients with normal bone marrow (control) were examined by ELISA. Furthermore, uPAR expression in bone marrow tissue was investigated by RT-PCR and Western blot, respectively. The distribution of uPAR in MM cells was examined using immunofluorescence staining. The pathological changes in different stages of MM patients were studied by HE staining. Results uPAR level in bone marrow fluid of MM patients (1.52±0.32μg/ml) was found to be higher than that in the control group (0.98±0.15μg/ml). Interestingly, uPAR protein (0.686±0.075vs. 0.372±0.043, P<0.05) and mRNA (2.51±0.46vs. 4.46±1.15,P<0.05) expression levels of MM patients at the progressive stage were significantly higher than those at the stable stage. The expression of uPAR in MM bone marrow was confirmed by immunofluorescence staining.Moreover, HE staining revealed a great increased number of nucleated cells and severe impairment of hematopoietic function in the bone marrow of patients with progressive-stage myeloma. Conclusion Our study reveals that uPAR expression is positively correlated with the development and progress of MM.     

ROLE OF B LYMPHOCYTE AND ITS SUBPOPULATIONS IN PATHOGENESIS OF IMMUNORELATED PANCYTOPENIA

Objective To measure the quantities and apoptosis-related protein levels of B lymphocyte in the patients with immunorelated pancytopenia(IRP)and explore the action of B lymphocyte in the pathogenic mechanism of IRP. Methods Quantities of whole B lymphocytes and CD5 B lymphocytes as well as the expressions of Fas and Bcl-2 in B lymphocytes in 35 patients with untreated IRP, 15 IRP patients in complete remission (CR), and 10 normal controls were assayed by flow cytometry. Results The percentages of B lymphocyte and CD5 B lymphocyte were significantly higher in untreated IRP patients than in CR IRP patients and normal controls (P<0.05), and there was no significant difference between the latter two groups (P>0.05). There was no significant difference of Fas expression in B lymphocyte among three groups (P>0.05). The expression of Bcl-2 in B lymphocyte was significantly higher in untreated patients than in CR patients or normal controls (P<0.01), and significantly higher in CR patients than in normal controls (P<0.01). The apoptosis-related index was significantly lower in untreated patients than in CR patients or normal controls (P<0.05), and significantly lower in CR patients than in normal controls (P<0.05). The percentage of B lymphocyte was positively correlated with post-treated response time(r=0.53, P<0.01). Conclusion The production of auto-antibodies in IRP patients probably has some relationship with the abnormal quantities of B lymphocyte and its subpopulations as well as with the inhibition of B lymphocyte apoptosis.     

Correlation of Serum Leptin Level with Bone Mineral Density and Bone Turnover Markers in Chinese Adolescent Dancers

Objective To investigate plasma leptin concentrations in adolescent female dancers and to determine whether leptin has some effects on their bone mineral density （BMD） and bone turnover markers. Methods Sixty dancers aged 15-17 years and 77 healthy controls were enrolled in the study. Bone mineral density （BMD） and body composition were detected by dual energy X-ray absorptiometry. Serum leptin concentrations were measured by radioimmunoassay （RIA）. Two bone turnover markers, bone-specific alkaline phosphatase （BAP） and tartrate-resistant acid phosphatase（TRACP）, were determined by ELISA. Results The dancers had a lower fat mass and a lower leptin level than the controls, while they had a relatively higher BMD of the total body and legs after adjustment for BMI and age. The levels of bone resorption and formation of markers were higher in the dancers than in the controls. Leptin was positively correlated with BMI, body weight, fat mass, and percentage of body fat. In dancers, Leptin was positively correlated with the BMD of the total body and the left leg. However, after adjustment for BMI, no correlation of serum leptin concentrations with BMD values was found in either dancers or controls. Nor correlation was found between leptin and bone turnover markers after adjustment for BMI. Conelusion The leptin profile is different between the controls and the dancers with a lower BMI and a lower fat mass. Circulating plasma leptin level depends on BMI and is not a direct determinant of BMD in Chinese adolescent dancers.     

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis: possible association with inflammatory cells

Background  Receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in chronic apical periodontitis.

Methods  The samples of chronic periapical lesions (n=40) and healthy periapical tissues (n=10) were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.

Results  The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues (P <0.001). The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration (P <0.05). Significantly increased RANKL expression was found with T lymphocytes (CD3⁺), macrophages (CD68⁺) and B lymphocytes (CD20⁺) infiltration (P <0.05). No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.

Conclusions  RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

     

Expression of CD123 and CD114 on the bone marrow cells of patients with myelodysplastic syndrome

Background Recent studies have shown that interleukin-3 receptor α （CD123） is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis.The expression of CD123 may also be high in patients with myelodysplastic syndrome （MDS）.In this study, the expression and clinical significance of CD123 and granulocyte colony stimulating factor （G-CSF） receptor （CD114） on the bone marrow cells of patients with MDS were investigated to explore the molecular marker of the malignant clone of MDS.Methods Forty-two patients with MDS, who were diagnosed in the Hematological Department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study.Fluorescence activiated cell sorter （FACS） was used to measure the expression of CD123 on CD34＋CD38- cells and CD114 on CD34＋cells of the bone marrow of these patients and controls and the clinical significance was analyzed.The expression of CD114 on CD123＋CD34＋CD38- cells was further measured to explore the molecular marker of the malignant clone in MDS.Results MDS patients displayed significantly higher proportion of CD34＋CD38-/CD34＋ （（14.03±5.27）%） than normal controls （（7.70±4.36）%, P 〈0.05）.The expression rate of CD123＋CD34＋CD38-/CD34＋CD38- was significantly higher in MDS patients （（48.39±28.15）%） than that in normal controls （（8.75±11.71）%, P 〈0.01）.The expression level of CD123 was significantly correlated with the proportion of bone marrow blasts （r=0.457, P 〈0.05）.The expression rate of CD114＋CD34＋/CD34＋ was lower in MDS patients （（33.05±21.71）%） than that in normal controls （（38.99±19.07）%） but was not statistically significant （P 〉0.05）.The expression of CD114 on CD123＋CD34＋CD38- cells （（34.82±29.58）%） was significantly lower than that on CD123-CD34＋CD38- cells （（53.48±27.41）%） of M DS patients （P 〈0.05）.Conclusions MDS patients displayed higher proportion of CD34＋CD38-/CD34＋ than normal controls.CD123 was highly expressed in the bone marrow of the patients with MDS, significantly correlated with the proportion of bone marrow blasts, and thus might be the marker of MDS malignant clone.CD123＋CD34＋CD38- cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responds worse to G-CSF in vitro and in vivo.     

Relationship between the Expression of RASSF1A Protein and Promoter Hypermethylation of RASSF1A Gene in Bladder Tumor

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma （BTCC）. The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR （MSP）. The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF 1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.     

Effect of kidney-reinforcing,blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells from chronic aplastic anemia patients

OBJECTIVE:To explore the effect of kidney-reinforcing,blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells(MSCs) from patients with chronic aplastic anemia(CAA).METHODS:We used threeTraditional Chinese Medicine recipes,namely a kidney-reinforcing recipe(KRR),blood-activating and stasis-removing recipe(BASRR),and kidney-reinforcing,blood-activating and stasis-removing recipe(KRBASRR),and a normal saline control to prepare herbal medicine serum in Sprague Dawley rats.Thirty CAA patients were enrolled in the experimental group,including 17 kidney-Yang deficient patients and 13 kidney-Yin deficient patients.Ten healthy individuals were included in the control group.MSCs were isolated from bone marrow samples,and the cell density was observed to measure their proliferation ability by microscopy on days 2,7,and 14 after isolation.In addition,the expression of adhesion molecules of bone marrow MSCs(CD106,CD49d,CD31 and CD44) were detected by flow cytometry after 48 h of treatment with the four different herbal medicine serums.RESULTS:The proliferation of MSCs from kidney-Yang deficient and kidney-Yin deficient patients was weaker than that of MSCs from the control group.The expression of all adhesion molecules of bone marrow MSCs from CAA patients was obviously lower than that in the control group(P< 0.01).The expression of CD49d and CD31 in MSCs from patients with a kidney-Yin deficiency was lower than in those with a kidney-yang deficiency(P< 0.05 and P<0.01,respectively).For kidney-Yang deficient patients,CD31 expression in the KRBASRR group was significantly higher than that in the BASRR group(P<0.01),while CD44 in the KRBASRR group was significantly higher than that in both KRR and BASRR groups(P<0.01).For kidney-Yin deficient patients,CD106 and CD49d expression in the KRBASRR group was obviously higher than that in the KRR group(P<0.05),while CD31 and CD44 expression in the KRBASRR group was significantly higher than that in both KRR and BASRR groups(P< 0.05 and P<0.01,respectively).CONCLUSION:The bone marrow microenvironment in CAA patients is abnormal.The effect of KRBASRR may be better than that of KRR and BASRR for kidney-Yang deficient and kidney-Yin deficient patients by improving the expression levels of MSC adhesion molecules.     

Prevalence of osteoporosis and vertebral fractures and related factors in patients with ankylosing spondylitis

Background Osteoporosis and vertebral factures are well recognized features in patients with ankylosing spondylitis （AS）.The aim of this study was to investigate the prevalence and risk factors of osteoporosis and vertebral fractures in patients with AS.Methods Fifty-nine AS patients and 40 healthy controls were enrolled.Bone mineral density （BMD） was measured using dual-energy X-ray absorptiometry （DEXA） at posterior-anterior （PA） lumbar,lateral lumbar and hip regions.Thoracic and lumbar X-rays were obtained for morphometric measurements.Clinical,biological and radiological statuses were evaluated with Bath Ankylosing Spondylitis Disease Activity Index （BASDAI）,Bath Ankylosing Spondylitis Metrology Index （BASMI）,Bath Ankylosing Spondylitis Functional Index （BASFI）,Bath Ankylosing Spondylitis Radiology Index-total （BASRI-t）,erythrocyte sedimentation rate （ESR） and the C-reactive protein levels.Results Osteoporosis was present in 32% of patients and 5% of controls according to lateral vertebral BMD measurements.Fracture was present in 31% of patients.The effect of some clinical and laboratory parameters on BMD status and vertebral fractures was analyzed in the patient group.Osteoporosis in lateral lumbar DEXA was associated with higher BASMI,BASFI,BASRI-t scores and ESR level.Low hip BMD was associated with low BMI and high BASFI and BASRI-t scores.Vertebral fractures were associated with advanced age,longer disease duration,longer duration since diagnosis,higher BASMI and BASRI-t scores,higher ESR level,reduced femoral and lateral lumbar BMD.Logistic regression analysis revealed that only BASRI-t score was significantly associated with low lateral spinal BMD and BMI and BASFI score were independently associated with low hip BMD.The presence of compression fractures was independently associated with BASRI-t score and low lateral lumbar BMD.Conclusions Osteoporosis and vertebral fractures in AS seem to be related to the extent of radiological involvement.A low lateral lumbar BMD     

Observations of fragile sites in patients with lymphoma and leukemia.

Chromosomal fragile sites analyses were performed in peripheral lymphocytes of 37 patients with lymphoma and 16 patients with leukemia, and also of 50 healthy individuals as controls. The results were: 1) The rates of chromosomal aberration and frequency of expression of fragile sites in patients with lymphoma and leukemia were significantly higher than those of normal controls. 2) There was a statistical association between 21 of 44 fragile sites and specific cancer breakpoints in patients with lymphoma and this was also the case with 19 of 30 fragile sites and specific cancer breakpoints in patients with leukemia. 3) Concordance between fragile sites and location of oncogenes in the diseases was established. The possible important role of fragile sites in the pathogenesis of lymphoma and leukemia is discussed.

     

E-cadherin expression and its clinical significance in 41 cases of spinal plasma cell myeloma

Objective： To investigate the expression level of E-cadherin in spinal plasma cell myeloma and its relationship with the clinicopathological indexes of spinal plasma cell myeloma. Methods： The expression of E-cadherin was detected in 41 cases of spinal plasma cell myeloma and 14 cases of simple bone cysts by using S-P （streptavidin-perosidase） immunohistochemical staining. The relationship between E-cadherin expression and the clinicopathological features was analyzed. Results： In spinal plasma cell myelomas, the positive expression rate of E-cadherin （46.34%） was higher than that in simple bone cyst tissues （all negative） （P〈0.01）. And the expression of E-cadherin had no obvious relationship with patients＇ age, gender, clinical classification or M-component （P〉0.05）, but had relationship with the classification of light chain. The expression rate in lambda chain subgroup was lower than that in the others （P〈0.05）. We also found the E-cadherin negative patients had a higher overall survival rate than those who had strong E-cadherin expression （P〈0.05）. Conclusion： E-cadherin expressed higher in spinal plasma cell myelomas than in simple bone cyst tissues and its expression had relationship with the light chain classification in spinal plasma cell myeloma. The patients with no stain of E-cadherin may have a poor prognosis. Detecting the type of light chain associated with expression of E-cadherin might be helpful in evaluating the outcome of spinal PCM patients.     

The effects of soybean isoflavaones on the mRNA expression of OPG, ODF and M-CSF in bone tissue of ovariectomized rat

Objective： To observe the effects of the soybean isoflavaones on the rnRNA expression of osteoprotegerin（OPG）, osteoclast differentiation factor（ODF） and macrophage colony stimulating factor（M-CSF） in bone tissue of ovariectomized rat, and to investigate the possible pathway to prevent and treat postrmenopausal osteoporosis using soybean isoflavaones. Methods： Thirty healthy adult SD rats were randomly divided into 3 groups： sham-operated group, ovariectomized group and soybean isoflavaones treated group. All rats were ovariectomized except those in sham-operated group. The bone density of the 3-6th lumbar vertebrae was detected after 12 weeks. Total RNA was extracted from femur bone and the mRNA expression of OPG, ODF and M-CSF was examined by real time PCR. Results： Soybean isoflavaones increased the bone density of the lumbar vertebrae in ovariectomized rat and up-regulated the expression of OPG, whereas down-regulated the expression of M-CSF and the ratio of ODF to OPG. Conclusion： The effects of soybean isoflavaones on postmenopausal osteoporosis are tightly correlated with OPG, M-CSF and the ratio of ODF to OPG.     

Dysregulation of E-cadherin in chronic rhinosinusitis with nasal polyps

E-cadherin is a key epithelial protein and adhesive molecule. This study detected the E-cadherin expression in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and controls, and analyzed its possible role in the pathogenesis of CRSwNP. The expression of E-cadherin was detected by using Western blotting and immunohistochemistry in controls and patients with CRSwNP. Computed tomography (CT) scan findings were scored. The results showed that the E-cadherin expression was up-regulated in patients...     

Comparison of Fresh Blood Cell Nature Immune Reaction of Children Patients with SLE or RA

To research the erythrocytes of condition of innate immunity in the children patients with RA and SLE, comparing with changes of activity of CD35 （CR1） on the erythrocyte between these two kinds of diseases, and further discuss the pathogenesis of these two kinds of diseases in children. The reaction of innate immunity of erythrocytes, lymphocytes and neutrophils which isolated from fresh blood of patients with RA and SLE to tumor cells was measured, and then the levels of expression of CD35 by using flow cytometry （FCM） were measured. The rosette rate of erythrocyte, lymphocytes and granulocytes adhering to cancer cell in the children patients with RA were higher than counterparts of normal controls （P〈0.001） respectively; the rosette rate of erythrocyte, lymphocytes and granulocytes adhering to tumor cells in the children patients with SLE were lower than counterparts of normal controls （P〈0.001） respectively. The rosette rate of these to tumor cells of children patients with RA, showed the highest figure change during different phases, including progression phase〉stationary phase 〉 extinction phase. And the rosette rates in these three groups were significantly higher than ones of that in normal controls. The expression of CD35 on the erythrocyte of children patients with RA was significantly higher than that of normal controls （P〈0.001）, while that of patients with SLE was significantly lower than that of normal controls （P〈0.001）. The CD35 on the red cells showed significantly positive correlation （γ=0.804, P〈0.01） with adhering erythrocytes and positively correlation with the rate of lymphocytes （P〈0.05）. The native immune function of children patients with SLE or RA was out of balance, and perhaps its changes have a relation with activity of diseases. It was suggested that the erythrocyte immunity function may be a new indicator to the condition of children patients with RA or SLE to judge the development and prognosis.