STAT3 expression in patients with fragility fractures and its effect on the biological function of osteoblasts.

To determine the expression of signal transducer and activator of transcription 3 (STAT3) in patients with fragility fractures (FFs) and its effect on the biological function of osteoblasts. The study included 32 patients with FFs who were diagnosed and treated in the research group and 30 concurrent healthy individuals in the control group. We observed STAT3 mRNA expression in the patients with FFs and controls and altered STAT3 mRNA to detect changes in the proliferation, invasion, and apoptosis of osteoblasts. The patients with FFs presented higher serum STAT3 mRNA expression than the controls (P < 0.05). We plotted receiver operating characteristic curves based on the STAT3 mRNA expression and found that the area under the curve for STAT3 mRNA was 0.856 (P < 0.05). Transfection of STAT3 mRNA mimics resulted in increased STAT3 mRNA expression, inhibited cell proliferation as detected by an MTT assay, and increased apoptosis rate, which was determined using flow cytometry with human fetal osteoblastic cell line 1.19 cells. STAT3 mRNA expression was elevated in the serum of patients with FFs and can be used as a biomarker for the diagnosis of the disease. Regulating STAT3 mRNA can inhibit the proliferation and induce the osteoblasts apoptosis.

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture.

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4+ T cells in patients with long bone fracture. We found that LAG-3+ cells represented approximately 13% of peripheral blood CD4+ T cells on average. Compared to LAG-3- CD4+ T cells, LAG-3+ CD4+ T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3+ CD4+ T cells expressed significantly higher levels of IL-10 and TGF-ß than LAG-3- CD4+ T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3+ CD4+ T cells. The frequency of LAG-3+ CD4+ T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4+ T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.

Downregulation of regulatory T cell function in patients with delayed fracture healing.

Bone fracture healing is a multistage regenerative process that requires the collaboration of various cell types, with approximately 5%-10% of fractures not healing properly. Accumulating evidence suggests that dysregulations in the immune system are associated with defective healing. In a cohort of 30 bone fracture patients between 50 and 62 years of age, 8 patients displayed delayed healing. Compared to the 22 normal healing patients, these 8 delayed healing patients presented significantly lower frequencies of CD4+ CD25hi Foxp3+ canonical regulatory T cells immediately following bone fracture and early on during the healing process. The CD4+ CD25+/hi T cells from delayed healing patients also presented reduced capacity to express transforming growth factor beta (TGF-ß), and presented reduced surface expression levels of inhibitory molecules, including CTLA-4 and Lag-3, compared to CD4+ CD25+/hi T cells from normal healing patients. Moreover, CD4+ CD25+/hi T cells from delayed healing patients were less potent in the suppression of CD4+ CD25- autologous conventional T cell proliferation, and presented reduced expansion capacity in response to interleukin (IL)-2 stimulation. Overall, our results demonstrated multiple reductions in regulatory T cell function in delayed healing patients that could produce long-lasting consequences in the bone fracture healing process.

Regulatory B cell is critical in bone union process through suppressing proinflammatory cytokines and stimulating Foxp3 in Treg cells.

Bone fractures may result in delayed union (DU) or non-union (NU) in some patients. Evidence suggests that the skewing of the immune system toward the proinflammatory type is a contributing factor. Because B cells were previously found to infiltrate the fracture healing site at abundant levels, we examined the regulatory B cells (Bregs) in DU/NU patients. In bone fracture patients with normal healing, the frequency of interleukin (IL)-10-expressing B cells was significantly upregulated in the early healing process (6 weeks post-surgery) and was downregulated later on (18 weeks post-surgery), whereas in DU/NU patients, the early upregulation of IL-10-expressing B cells was missing. The majority of IL-10-expressing B cells were concentrated in the IgM+ CD27+ fraction in both controls and patients. IgM+ CD27+ B cells effectively suppressed interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2 expression from CD4+ T cells, as well as IFN-γ and TNF-α expression from CD8+ T cells. The IgM+ CD27+ B cell-mediated suppression was restricted to the sample from the early healing time point in controls, as the IgM+ CD27+ B cells from normal healing patients later on or from DU/NU patients did not present significant regulatory function. In addition, culturing of CD4+ CD25+ Tregs with IgM+ CD27+ B cells from controls at early healing time point resulted in higher Foxp3 expression, a function absent in controls at later time point, or in DU/NU patients. In conclusion, our results support a role of B cell-mediated regulation early during the bone healing process.

A novel translocator protein 18 kDa ligand, ZBD-2, exerts neuroprotective effects against acute spinal cord injury.

Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N-benzyl-N-ethyl-2-(7,8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA-induced neurotoxicity in vitro. The present study aims to examine the role of ZBD-2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD-2 (10 mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of the spinal cord. High-dose ZBD-2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B-containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD-2 (10 mg/kg) increased phosphorylated Akt (p-Akt) and decreased the ratio of Bax/Bcl-2. These results demonstrate that ZBD-2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway.

Synthesis, antimalarial activity and cytotoxic potential of new monocarbonyl analogues of curcumin.

A series of novel monocarbonyl analogues of curcumin have been designed, synthesized and tested for their activity against Molt4, HeLa, PC3, DU145 and KB cancer cell lines. Six of the analogues showed potent cytotoxicity towards these cell lines with IC(50) values below 1 µM, which is better than doxorubicin, a US FDA approved drug. Several analogues were also found to be active against both CQ-resistant (W2 clone) and CQ-sensitive (D6) strains of Plasmodium falciparum in an in-vitro antimalarial screening. This level of activity warrants further investigation of the compounds for development as anticancer and antimalarial agents.

Objective Evaluation of Neurogenic Intermittent Claudication for Patients With Lumbar Spinal Stenosis Based on Plantar Pressure Analysis.

STUDY DESIGN: A cross-sectional study. OBJECTIVE: To quantify the severity of neurogenic intermittent claudication (NIC) for patients with lumbar spinal stenosis (LSS) based on the center of pressure trajectory. SUMMARY OF BACKGROUND DATA: NIC is one of the typical symptoms of LSS. So far, the severity level of NIC is mainly evaluated by the subjective description of patients, which might be biased by patients' background differences and thus lead to an ineffective diagnosis or inappropriate treatment for LSS. Therefore, it remains necessary to develop a reliable clinical technique for quantitative evaluation of NIC to achieve more effective therapy for LSS. MATERIALS AND METHODS: In the present study, the Footscan pressure system was used to detect the center of pressure trajectory. The real-time walking distance (rtWD) and the corresponding displacement of the medial-lateral center of pressure (ML-COP) were calculated based on the trajectory. The differences of ML-COP between LSS and control groups were analyzed using a one-way repeated measures analysis of variance. Regression and Pearson correlation analysis were used to investigate the correlation between rtWD and ML-COP, as well as the relation between the Oxford Claudication Score (OCS) and clinical evaluation indicators. RESULTS: The present study included 31 LSS patients and 31 healthy controls. There were no significant differences in demographic data between the two groups ( P >0.05). The results indicated that ML-COP would increase with the number of laps in the LSS group while not in the control group. Also, a linear relationship was identified between the ML-COP and rtWD for LSS patients ( R2 >0.80, P <0.05). Since the incremental rate of ML-COP for LSS patients was reflected by the regression coefficients of the linear regression analysis, thus the regression coefficients were defined as the claudication correlation coefficients (CCCs). In addition, it was indicated by the statistical analysis that there was a strong positive correlation between OCS and CCC ( r =0.96; P <0.001) and a medium negative correlation with final walking distance ( r =-0.67; P <0.001). It was also noticed that there was no significant correlation between the average ML-COP and OCS ( r =-0.03; P =0.864). CONCLUSIONS: The ML-COP of LSS patients would increase with the patients' walking distance. This incremental rate, characterized by the CCC, would be used as an effective indicator to quantify the severity level of the NIC for potentially more accurate and reliable diagnosis, evaluation, and treatment of LSS. LEVEL OF EVIDENCE: 3.

Covalent immobilization of VEGF on allogeneic bone through polydopamine coating to improve bone regeneration.

Objective: Promoting bone regeneration and repairing in bone defects is of great significance in clinical work. Using a simple and effective surface treatment method to enhance the osteogenic ability of existing bone scaffold is a promising method. In this article, we study the application of catecholic amino acid 3,4-dihydroxyphenylalanine (DOPA) surface coating chelated with vascular endothelial growth factor (VEGF) on allogeneic bone. Method: Allogeneic bone is immersed in DOPA solution and DOPA form polydopamine (PDA) with good adhesion. Electron microscopy is used to characterize the surface characteristics of allogeneic bone. MC3T3-E1 cells were tested for biocompatibility and osteogenic signal expression. Finally, a 12-week rabbit bone defect model was established to evaluate bone regeneration capability. Results: We found that the surface microenvironment of DOPA bonded allogeneic bone was similar to the natural allogeneic bone. VEGF loaded allografts exhibited satisfying biocompatibility and promoted the expression of osteogenic related signals in vitro. The VEGF loaded allografts healed the bone defect after 12 weeks of implantation that continuous and intact bone cortex was observed. Conclusion: The PDA coating is a simple surface modification method and has mild properties and high adhesion. Meanwhile, the PDA coating can act on the surface modification of different materials. This study provides an efficient surface modification method for enhancing bone regeneration by PDA coating, which has a high potential for translational clinical applications.

Effector memory regulatory T cells were most effective at suppressing RANKL but their frequency was downregulated in tibial fracture patients with delayed union.

Delayed union and nonunion occur in a minor subject of bone fractures, presenting ongoing challenges to treatment. RANKL, which promotes the differentiation of bone-resorbing osteoclasts, is thought to negatively impact bone healing. In this study, we recruited patients with isolated closed tibial fracture, who were later categorized into normal healing and delayed healing groups based on their healing progression. The regulatory T cell (Treg) compartment was then investigated in each patient. Based on CD45RA and CD62L expression, we distinguished circulating Treg cells into CD45RA+CD62L+ naive (N), CD45RA-CD62L+ central memory (CM), and CD45RA-CD62L- effector memory (EM) subsets. Compared to normal patients, delayed patients presented significantly lower EM Treg proportion and significantly higher N Treg proportion. Among the N, EM, and CM Treg cells, the EM Treg cells were the most potent at suppressing RANKL expression in T conventional (Tconv) cells. This functionality of EM Treg cells was present in both normal healing patients and delayed healing patients, and was dependent on IL-10, as neutralization of IL-10 resulted in significantly elevated RANKL expression. EM Treg cells presented the highest IL-10 and TGF-ß expression directly ex vivo, as well as after anti-CD3/anti-CD28/IL-2 stimulation. CM Treg cells did not present high expression of inhibitory cytokines ex vivo, but was capable of upregulating cytokine expression upon stimulation. N Treg cells, on the other hand, presented limited capacity to upregulate inhibitory cytokines. In summary, our study identified that, while the EM Treg cells were the most effective at suppressing RANKL, they in delayed union patients were present at lower frequencies with functional impairment, resulting in decreased RANKL suppression. Hence, bone-resorbing osteoclast formation may be favored in these patients thus suggesting a possible mechanism for delayed bone healing.

Partial Vertebrae Resection Laterally to Harvest Supplemental Autograft Bone for Anterior Cervical Discectomy and Fusion: A Technical Note and Outcomes.

PURPOSE: To evaluate the safety and effectiveness of partial vertebrae resection laterally through intervertebral space to harvest supplemental autograft bone for anterior cervical discectomy and fusion (ACDF). METHODS: Patients who accepted 1-segment (n = 19, 38.2 months follow-up) or 2-segment (n = 17, 40.4 months follow-up) ACDF with supplemental autograft bone were included. Cervical lordosis (CL), segmental lordosis (SL), anterior segment height (ASH), and posterior segment height (PSH) on neutrally lateral radiography, and intervertebral fusion rate on computed tomography were measured. The operation time, intraoperative blood loss, Japanese Orthopedic Association score, visual analog scale around the neck or arm, Neck Disability Index, and complications were also recorded. RESULTS: Mean operation time was 86.2 and 115.6 minutes, and the intraoperative blood loss was 41.7 and 79.4 mL in cases with 1-segment and 2-segment ACDF, respectively. At the final visit, the visual analog scale score and Neck Disability Index significantly decreased, and the Japanese Orthopedic Association score significantly increased. Significant increases were observed in the ASH, PSH, CL, and SL after 2-segment ACDF. Significant increases were observed in the CL and SL after 1-segment ACDF, but not in the ASH and PSH. All the ASH, PSH, CL, and SL kept unchanged at the final visit. All cases acquired definite intervertebral fusion, and the incidence of cage subsidence was 5.3% after 1-segment and 17.6% after 2-segment ACDF at the final visit. CONCLUSIONS: Partial vertebrae resection laterally through the intervertebral space was a safe and effective method to harvest supplemental autograft bone for the ACDF.

Stability evaluation of anterior external fixation in patient with unstable pelvic ring fracture: a finite element analysis.

BACKGROUND: The pelvic ring fractures (PRF) are commonly induced by the high-energy impact and will lead to unstable and sever injures. This study is aimed to explore the stability of anterior external fixation in treating pelvis fracture and evaluate the possibility for these kinds of patients to reduce bedridden time. METHODS: A patient with Tile B3 pelvis fracture was chosen in the research and the corresponding digital model was reconstructed according to the CT images and 3D scanning. Four angles of pelvis under vertical compression were employed in the finite element (FE) analyses. The stress distribution and micro-motion displacement were calculated to validate the instability of pelvis. RESULTS: The stress applied on the pelvis was ranged from 4.296 to 8.364 MPa in all postures. The stress applied on pins was less than 7.011 MPa during reclining, and reached 28.29 MPa when standing. The micro-motion displacement in reclining posture was ranged from 0.005 to 0.087 mm. The value increased to more than 1mm in standing posture. CONCLUSIONS: It was safety for patients with pelvis fracture to sit vertical or recline on the bed during nursing or having treatment, but standing or walking will generate inappropriate micro-motion. The existence of external fixation can reduce the possibility of complications caused by long-term bedridden.

Influence of Ceramic Debris on Osteoblast Behaviors: An In Vivo Study.

OBJECTIVE: Wear-induced aseptic loosening has been accepted as one of the main reasons for failure of total hip arthroplasty. Ceramic wear debris is generated following prosthesis implantation and plays an important part in the upregulation of inflammatory factors in total hip arthroplasty. The present study investigates the influence of ceramic debris on osteoblasts and inflammatory factors. METHODS: Ceramic debris was prepared by mechanical grinding of an aluminum femoral head and added to cultures of MC3T3-E subclone 14 cells at different concentrations (i.e. 0, 5, 10, and 15 µg/mL). Cell proliferation was evaluated using a Cell Counting Kit (CCK-8), and cell differentiation was assessed by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In addition, cell bio-mineralization was evaluated through alizarin red S staining, and release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) was measured through enzyme-linked immunosorbent assays (ELISA). Furthermore, mRNA expression of Smad1, Smad4, and Smad5 and protein expression of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. RESULTS: The ceramic debris had irregular shapes and sizes, and analysis of the size distribution using a particle size analyzer indicated that approximately 90% of the ceramic debris was smaller than 3.2 µm (2.0 ± 0.4 µm), which is considered clinically relevant. The results for mRNA expression of ALP, OCN, and OPN and alizarin red S staining indicated that cell differentiation and bio-mineralization were significantly inhibited by the presence of ceramic debris at all tested concentrations (P < 0.05, and the values decreased gradually with the increase of ceramic debris concentration), but the results of the CCK-8 assay showed that cell proliferation was not significantly affected (P > 0.05; there was no significant difference between the groups at 1, 3, and 5 days). In addition, the results of ELISA, RT-PCR, and western blotting demonstrated that ceramic debris significantly promoted the release of inflammatory factors, including TNF-α, IL-ß, and IL-6 (P < 0.05, and the values increased gradually with the increase of ceramic debris concentration), and also greatly reduced the mRNA expression of Smad1, Smad4, and Smad5 (the values decreased gradually with the increase of ceramic debris concentration) as well as protein expression of phosphorylated Smad1, Smad4, and Smad5. CONCLUSION: Ceramic debris may affect differentiation and bio-mineralization of MC3T3-E subclone 14 cells through the bone morphogenetic protein/Smad signaling pathway.

STAT3 promotes bone fracture healing by enhancing the FOXP3 expression and the suppressive function of regulatory T cells.

Signal transducer and activator of transcription 3 (STAT3) is a key signaling protein in the skeletal system as well as in the immune system. Accumulating evidence demonstrates that the inflammatory response is deeply involved in the healing process of bone fractures, but how the immune system is regulated during this process is unclear. In this study, we examined STAT3-mediated regulation of immunity in adult patients with closed tibia fracture. In all patients, the expression and activation of STAT3 peaked at around day 7 to day 14 after surgery, and gradually decreased during the rest of the healing period. At day 7 (peak STAT3 expression and phosphorylation), the CD4+ CD25+ T cells from bone fracture patients presented the highest level of STAT3 activation among lymphocyte subsets. Therefore, we investigated the role of STAT3 in CD4+ CD25+ T cells. The level of FOXP3 expression by CD4+ CD25+ T cells was directly correlated with the level of STAT3 phosphorylation in these cells. The level of STAT3 phosphorylation in CD4+ CD25+ T cells was also inversely correlated with the level of IFN-γ and TNF-α secretion in peripheral blood mononuclear cells. Inhibition of STAT3 significantly suppressed FOXP3 and IL-10 expression by CD4+ CD25+ T cells, as well as the ability of CD4+ CD25+ T cells to suppress T-cell IFN-γ and TNF-α secretion. Furthermore, early healers patients presented significantly higher STAT3 expression and phosphorylation than late healers, possibly due to the higher IL-6 and IL-10 levels in the serum of early healing patients. Together, these data demonstrated that STAT3 was beneficial to bone fracture healing, possibly by enhancing Treg-mediated suppression of counteracting inflammations, and suggested that STAT3 could be used as a prognostic marker to identify otherwise undistinguishable patients at risk of developing delayed union or nonunion.

Comparison of Monolateral External Fixation and Internal Fixation for Skeletal Stabilisation in the Management of Small Tibial Bone Defects following Successful Treatment of Chronic Osteomyelitis.

BACKGROUND: To compare the salvage rate and complication between internal fixation and external fixation in patients with small bone defects caused by chronic infectious osteomyelitis debridement. METHODS: 125 patients with chronic infectious osteomyelitis of tibia fracture who underwent multiple irrigation, debridement procedure, and local/systemic antibiotics were enrolled. Bone defects, which were less than 4 cm, were treated with bone grafting using either internal fixation or monolateral external fixation. 12-month follow-up was conducted with an interval of 3 months to evaluate union of bone defect. RESULTS: Patients who underwent monolateral external fixation had higher body mass index and fasting blood glucose, longer time since injury, and larger bone defect compared with internal fixation. No significant difference was observed in incidence of complications (23.5% versus 19.3%), surgery time (156 ± 23 minutes versus 162 ± 21 minutes), and time to union (11.1 ± 3.0 months versus 10.9 ± 3.1 months) between external fixation and internal fixation. Internal fixation had no significant influence on the occurrence of postoperation complications after multivariate adjustment when compared with external fixation. Furthermore, patients who underwent internal fixation experienced higher level of daily living scales and lower level of anxiety. CONCLUSIONS: It was relatively safe to use internal fixation for stabilization in osteomyelitis patients whose bone defects were less than 4 cm and infection was well controlled.

Chronic Osteomyelitis Increases the Incidence of Type 2 Diabetes in Humans and Mice.

Background: To compare the risk of type 2 diabetes (T2DM) between patients with and without chronic osteomyelitis (COM), both in humans and in mice, and to explore risk factors in COM patients who developed T2DM. Methods: One hundred seven patients with COM and 114 patients without COM were consecutively enrolled and retrospectively analysed. Clinical data concerning the time to develop diabetes, glucose metabolism, lipid metabolism, inflammatory factors, mental health and frequency of specialist visits were collected. A mouse model of osteomyelitis was used to verify the presence of impaired glucose metabolism and depression. All data were processed by SPSS. Results: The incidence of T2DM was 2.37-fold higher in patients with COM than in those without. In COM patients, subjects with T2DM (DDM) had higher BMI, less exercise and more frequent visits to specialists than those without (Con). Glucose and lipid metabolism were worse in patients with DDM. Patients with DDM had higher levels of white blood cells (12.9±2.1×109/L vs. 11.7±2.2×109/L, p=0.027), CRP (28.4±4.5 mg/L vs. 22.0±4.8 mg/L, p<0.001), TNF-α (13.5±5.0 pg/mL vs. 9.4±2.6 pg/mL, p= 0.003) and IL-6 (12.9±3.2 pg/mL vs. 9.2±2.7 pg/mL, p<0.001). Significantly increased fasting blood glucose concentrations and impairment of oral glucose tolerance tests were also observed in mice modelling osteomyelitis, which were accompanied by elevated TNF-α and IL-6 levels. Furthermore, the proportion of depression (63.2% vs. 35.2%, p=0.003) and severe anxiety (31.6% vs. 9.1%, p=0.002) were significantly higher in the DDM group. Osteomyelitis mice showed obvious depressive-like behaviours. The levels of TNF-α, IL-6, CRP, BMI, and LDL; lack of exercise; SAS; HAQ; and SF36 assessment were risk factors for the development of T2DM in COM patients. Conclusions: Chronic osteomyelitis increased the incidence of T2DM in both humans and mice. Inflammation, mental illness and lack of exercise were risk factors for the occurrence of T2DM in osteomyelitis. Comprehensive consideration of patient history, including metabolism and mental health, is needed in planning future treatment.

T-cell immunoglobulin domain and mucin domain 3 polymorphism affects cytokine expression in different cells and is associated with increased susceptibility to knee osteoarthritis.

Osteoarthritis (OA) is a group of mechanical abnormalities involving degradation of joints, including articular cartilage and subchondral bone. In China, knee OA accounts for the majority of the OA cases. Elevated immune responses and proinflammatory cytokine expressions have been identified in OA patients. Tim-3 plays critical roles in down-regulating T cell responses. Here, we first examined the correlation between Tim-3 polymorphisms and the risk of knee OA. Data showed that the percentage of rs200181745TG (-1622G/T) genotype was significantly higher in knee OA patients compared to the controls (odds ratio [OR]=2.23, 95% confidence interval [CI]: 1.19-4.89, P<0.001). We further investigated the effects of the two Tim-3 polymorphisms on cytokine expressions in different cells. Results showed that subjects with rs200181745TG genotype presented increased mRNA and protein levels of interferon gamma (IFN-γ) from CD4+ T cells (P<0.001), and elevated mRNA level of IFN-γ from CD8+ T cells (P<0.001). However, the Tim-3 polymorphisms did not affect the expression of tumor necrosis factor alpha (TNF-α) in CD4+ and CD8+ T cells. These findings indicate that Tim-3 polymorphism can affect cytokine expression in different cells and is associated with increased susceptibility to knee OA.

Therapeutic effects of OP-1 on metal wear particle induced osteoblasts injury in vitro.

Aseptic lossening is a main reason for the revision of total joint arthroplasty. Metal-wear particles induced deregulation of bone resorption or formation has been considered as the major process of aseptic lossening. Osteogenic protein-1 (OP-1) can be used to improve bone formation. However, such effect is not clearly understood after the metal-wear particles injury. Here, we investigated the molecular mechanisms by which OP-1 regulates the activity of bone formation and anti-inflammatory after injury. Results showed that OP-1 increased cell viability and bone formation ability of impaired osteoblast cells at 72 hours after being injured by cobalt particles. Pathway analyses revealed that both mRNA and protein levels of Smad1 and Smad5 were significantly increased upon the treatment of OP-1 in the cell injury model. Similarly, runt-related transcription factor 2 (Runx2) was also significantly upregulated in the OP-1 treated cells. Moreover, treatment with OP-1 inhibited the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-18 in cobalt impaired cells. Collectively, these results suggest that OP-1 could inhibit cobalt particles induced cell injury by activating Smad1, Smad5, and Runx2, and such procedure is accompanied by anti-inflammatory reaction.

Expression, Purification and in vitro Activity of Apoptin.

A novel protein named apoptin induces a p53-independent, Bcl-2 insensitive type apoptosis in various human tumor cells but not in normal cells. Apoptin is considered to be a promising candidate for safe and effective anti-tumortherapy. Moreover, apoptin may be important for fundamental studies on molecularbasis of apoptosis and cancer. Apoptin gene was subcloned into prokaryotic expression vector pPROEXHT and pBV220, respectively. The apoptin protein was obtained from pPROEXHT-apoptin expression system and not from pBV220-apoptin the former contains a 6xhistidine affinity tag for ease of purification. Expressed protein was purified by chromatography on a co-chelated affinity column and was renatured by dialysis. The renatured apoptin was able to induce purified Hela nuclei apoptosis in vitro even without the participation of the cytoplasm, showing that apoptin expressed in E.coli was active to induce apoptosis.

Transforming growth factor-beta polymorphisms and serum level in the development of osteosarcoma.

The transforming growth factor-beta (TGF-ß) signaling pathway plays critical roles in the development of various diseases. The current study investigated whether TGF-ß was involved in the pathogenesis of osteosarcoma from the genetic polymorphism perspective and serum level perspective. We first examined two TGF-ß1 polymorphisms, rs1800469C/T and rs1800470T/C, in 202 osteosarcoma patients and 216 healthy controls. Data revealed that the prevalence of rs1800470TT genotype and T allele was significantly elevated in patients than in controls (odds ratio [OR]=2.28, 95% confidence interval [CI]: 1.30-3.98, p<0.001, and OR=1.49, 95% CI: 1.14-1.96, p<0.001). Function analyses showed that healthy controls carrying rs1800470TT genotype had a significantly higher serum level of TGF-ß than those carrying the rs1800470CC genotype (191.1±15.7 pg/mL vs. 129.4±10.9 pg/mL, p=0.003). We then compared the serum level of TGF-ß between osteosarcoma patients and healthy controls. Results demonstrated a significantly increased serum level of TGF-ß in patients than in controls. Further analyses identified that patients with metastasis had augmented levels of serum TGF-ß than those without metastasis. These data indicate that TGF-ß may be closely involved in the pathogenesis of osteosarcoma.

Cytotoxic T-lymphocyte antigen-4 polymorphisms and susceptibility to Ewing's sarcoma.

The development of Ewing's sarcoma (ES) is a complex process, resulting from interplay between mutations in oncogenes and tumor suppressors, host susceptibility factors, and cellular context. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays important roles in downregulating the T-cell activation. Polymorphisms in the CTLA-4 gene have been shown to be associated with different autoimmune diseases and cancers. The current study evaluated the association of two CTLA-4 gene polymorphisms, -318C/T (rs5742909) and +49G/A (rs231775) with ES in the Chinese population. CTLA-4 polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 223 ES cases and 302 age-matched healthy controls. Data were analyzed using the chi-square test. Results showed that prevalence of the CTLA-4 gene +49AA genotype and +49A allele were significantly increased in ES patients compared to controls (odds ratio [OR]=2.03, 95% confidence interval [CI], 1.13-3.66, p=0.018; and OR=1.33, 95%CI, 1.03-1.72, p=0.027). Also, subjects with CA (-318, +49) haplotype had a 1.37-fold increased risk to develop ES (p=0.032). In addition, ES patients with metastasis had higher numbers of +49AA genotype than those with localized cases (OR=2.66, 95%CI, 1.14-6.22, p=0.022). These results indicate that the CTLA-4+49G/A polymorphism is a new risk factor for ES and may affect the prognosis of this cancer.