New insight into the virulence and inflammatory response of Staphylococcus aureus strains isolated from diabetic foot ulcers.

Staphylococcus aureus strains isolated from diabetic foot ulcers (DFUs) have less virulence, but still cause severe infections. Furthermore, hypovirulent S. aureus strains appear to be localized in the deep tissues of diabetic foot osteomyelitis, indicating that the unique environment within DFUs affects the pathogenicity of S. aureus. In this study, the cell-free culture medium (CFCM) of S. aureus strains isolated from DFUs exhibited higher cytotoxicity to human erythrocytes than those isolated from non-diabetic patients with sepsis or wounds. Among these S. aureus strains isolated from DFUs, ß-toxin negative strains have less virulence than ß-toxin positive strains, but induced a higher expression of inflammatory cytokines. Our study and previous studies have shown that the synergistic effect of phenol-soluble modulin α and ß-toxin contributes to the higher hemolytic activity of ß-toxin positive strains. However, lysis of human erythrocytes by the CFCM of ß-toxin negative strains was greatly inhibited by an autolysin inhibitor, sodium polyanethole sulfonate (SPS). A high level of glucose greatly reduced the hemolytic activity of S. aureus, but promoted the expression of interleukin-6 (IL-6) in human neutrophils. However, 5 mM glucose or glucose-6-phosphate (G6P) increased the hemolytic activity of SA118 (a ß-toxin negative strain) isolated from DFUs. Additionally, patients with DFUs with growth of S. aureus had lower level of serum IL-6 than those with other bacteria, and the CFCM of S. aureus strains significantly reduced lipopolysaccharide-induced IL-6 expression in human neutrophils. Therefore, the virulence and inflammatory response of S. aureus strains isolated from DFUs are determined by the levels of glucose and its metabolites, which may explain why it is the predominant bacteria isolated from DFUs.

The Skeletal Oncology Research Group Machine Learning Algorithm (SORG-MLA) for predicting prolonged postoperative opioid prescription after total knee arthroplasty: an international validation study using 3,495 patients from a Taiwanese cohort.

BACKGROUND: Preoperative prediction of prolonged postoperative opioid use (PPOU) after total knee arthroplasty (TKA) could identify high-risk patients for increased surveillance. The Skeletal Oncology Research Group machine learning algorithm (SORG-MLA) has been tested internally while lacking external support to assess its generalizability. The aims of this study were to externally validate this algorithm in an Asian cohort and to identify other potential independent factors for PPOU. METHODS: In a tertiary center in Taiwan, 3,495 patients receiving TKA from 2010-2018 were included. Baseline characteristics were compared between the external validation cohort and the original developmental cohorts. Discrimination (area under receiver operating characteristic curve [AUROC] and precision-recall curve [AUPRC]), calibration, overall performance (Brier score), and decision curve analysis (DCA) were applied to assess the model performance. A multivariable logistic regression was used to evaluate other potential prognostic factors. RESULTS: There were notable differences in baseline characteristics between the validation and the development cohort. Despite these variations, the SORG-MLA ( https://sorg-apps.shinyapps.io/tjaopioid/ ) remained its good discriminatory ability (AUROC, 0.75; AUPRC, 0.34) and good overall performance (Brier score, 0.029; null model Brier score, 0.032). The algorithm could bring clinical benefit in DCA while somewhat overestimating the probability of prolonged opioid use. Preoperative acetaminophen use was an independent factor to predict PPOU (odds ratio, 2.05). CONCLUSIONS: The SORG-MLA retained its discriminatory ability and good overall performance despite the different pharmaceutical regulations. The algorithm could be used to identify high-risk patients and tailor personalized prevention policy.

microRNA-23a promotes cell growth and metastasis in gastric cancer via targeting SPRY2-mediated ERK signaling.

[This retracts the article DOI: 10.3892/ol.2018.8374.].

Salvage surgery, chylothorax and pneumonia are the main drivers of unplanned readmissions after oesophagectomy for cancer.

OBJECTIVES: Unplanned readmissions after surgery can be cumbersome to patients and costly on healthcare resources. The aim of this single-centre study was to identify the independent risk factors for unplanned readmissions in patients who had undergone oesophagectomy for cancer. METHODS: We retrospectively reviewed the clinical records of 526 consecutive patients with oesophageal cancer who received transthoracic oesophagectomy and were discharged home between 2006 and 2017. Risk factors for unplanned readmission within the first 30 days from discharge were identified by multivariable competing risk analysis. RESULTS: The mean age of the study patients was 55.14 years and 93.7% were men. Squamous cell carcinoma was identified in 94.1% of the participants, and 68.0% received chemoradiotherapy. There were 299 (56.8%) patients who experienced at least 1 postoperative complication. Fifty-five patients (10.5%) experienced an unplanned readmission. The postoperative 90-day mortality rate among patients who experienced an unplanned readmission was significantly higher than that of cases who did not (9.1% vs 0.2%, respectively, P < 0.001). Multivariable analysis identified chylothorax [hazard ratio (HR): 3.86, 95% confidence interval (CI): 1.89-7.91, P < 0.001], pneumonia (HR: 1.98, 95% CI 1.03-3.82, P = 0.042) and salvage surgery (HR: 2.27, 95% CI: 1.10-4.69, P = 0.027) as independent risk factors for unplanned readmissions. CONCLUSIONS: Salvage surgery, postoperative chylothorax and pneumonia are the main drivers of 30-day unplanned readmissions in patients who had undergone oesophagectomy for cancer. Patients who required unplanned readmissions showed increased early mortality rates.

Antioxidant and Hepatoprotective Activities of Crude Polysaccharide Extracts from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), by Ultrasonic-Circulating Extraction.

We recently proposed, and successfully applied, a novel and efficient technique-ultrasonic-circulating extraction (UCE) integrating superfine pulverization-to extract and prepare antioxidant crude polysaccharides other natural active substances from Ganoderma lucidum. The aim of this study was to evaluate the antioxidant and hepatoprotective activities and active ingredients in the powder from UCE (UCEP) through comparison with powder from hot water extraction (HWEP). The DPPH radical, ABTS radical, superoxide anion, total antioxidant activity, and ferric-reducing antioxidant power assay results showed that the UCEP exhibited stronger (P < 0.01) in vitro antioxidant activity than the HWEP. The hepatoprotective activity of the extracts was evaluated against CCl4-induced oxidative damage in the liver. Measurements of reduced glutathione, superoxide dismutase, and malondialdehyde in rat liver; measurements of alanine transaminase, aspartate transaminase, and lactate dehydrogenase in rat blood; and Western blotting for antioxidant proteins of transforming growth factor-ß1, heme-oxygenase 1, and glutathione per-oxidase showed that the UCEP had antioxidant activity in vivo either similar to or slightly stronger than (P < 0.1) the HWEP. Further analysis of the active ingredients revealed that the UCEP and HWEP have similar mean yield and total triterpenoid content, but the former has significantly higher (P < 0.05) mean yield and total polysaccharide content than the latter. Our results suggest that the UCEP displays stronger antioxidant activities because of the larger amount of total polysaccharides; the UCEP may be able to be used as an antioxidant and liver protectant.

microRNA-23a promotes cell growth and metastasis in gastric cancer via targeting SPRY2-mediated ERK signaling.

microRNAs (miRs) serve important roles in various human cancer types. Recently, miR-23a has been indicated as an oncogene in gastric cancer, but the underlying mechanism remains unclear. In the present study, reverse transcription-quantitative polymerase chain reaction and western blot analysis was used to explore the effects of miR-23a in gastric cancer. Additionally, cell proliferation, migration and invasion were examined using an MTT assay, wound healing assay and Transwell assay, respectively. Furthermore, a luciferase reporter gene assay was used to confirm the target association. It was determined that miR-23a was significantly upregulated in gastric cancer tissues and cell lines compared with adjacent tissues, and a normal gastric epithelial cell line. Furthermore, its upregulation was significantly associated with cancer progression and poor prognosis of patients. Knockdown of miR-23a caused a notable reduction in the proliferation, migration and invasion of gastric cancer AGS cells. Sprouty homolog 2 (SPRY2) was then predicted to be target gene of miR-23a. A luciferase reporter gene assay data demonstrated that miR-23a has the ability to directly bind to the 3'-untranslational region of SPRY2 mRNA. Further investigation demonstrated that SPRY2 was significantly downregulated in gastric cancer tissues and cell lines, and the protein expression of SPRY2 was negatively regulated by miR-23a in AGS cells. Furthermore, knockdown of SPRY2 reduced the suppressive effects of miR-23a inhibition in AGS cell proliferation, migration and invasion. In addition, the activity of extracellular signal-regulated kinase (ERK) signaling was also inhibited by the miR-23a/SPRY2 knockdown in AGS cells. The present study indicated that miR-23a serves a promoting role in gastric cancer via targeting SPRY2 and downstream ERK signaling.

In vitro and in vivo antitumour activities of puerarin 6″-O-xyloside on human lung carcinoma A549 cell line via the induction of the mitochondria-mediated apoptosis pathway.

Context Pueraria lobata (Leguminoseae) shows cytotoxic effects against cancer cells; however, its active components remain unclear. Objective This study investigated the antitumour activity of puerarin 6″-O-xyloside (POS) on the human lung carcinoma A549 cell line. Materials and methods The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of POS (at 10, 20 and 40 µM) in vitro, and xenograft nude mice were established to evaluate the antitumour effect of POS (at 40 mg/kg/d) in vivo by 15 days intraperitoneal injection (ip). To explore its mechanism of action, flow cytometry was performed to determine the pro-apoptotic effect of POS (at 10, 20 and 40 µM). Subsequently, the expression of caspase-3, caspase-7, caspase-9, Bcl-2 and Bax in A549 cells were determined. Results POS showed significant cytotoxicity toward A549 cells (p < 0.05) by inducing apoptosis. Treatment with POS significantly upregulated the levels of caspase-3 (p < 0.01), caspase-7 (p < 0.01), caspase-9 (p < 0.01) and Bax (p < 0.01) in A549 cells, and Bcl-2 was downregulated (p < 0.01). Additionally, the in vivo animal study showed that POS significantly inhibited tumour growth in A549 cells (p < 0.01). Conclusion Our study demonstrated the POS has significant antitumour activities. The mechanisms are related to increased levels of caspase-3, caspase-7, caspase-9 and Bax, and reduced levels Bcl-2.

Hepatoprotective effect of ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells.

CONTEXT: Ganoderma triterpenoids (GTs) have been recognised as an important bioactive ingredient in Ganoderma Lucidum (Leyss. ex Fr.) Karst. (Polyporaceae), widely used for treating and preventing chronic hepatopathy of various etiologies. OBJECTIVE: The objective of this study is to better understand the hepatoprotective effect of GTs and to enhance their use in food supplement pharmaceutical and medical industries. MATERIALS AND METHODS: HepG2 cells were pretreated in the presence or absence of GTs (50, 100 and 200 µg/ml) for 4 h, then exposed with 60 µmol/L of t-BHP for an additional 4 h. The cell viability was evaluated by MTT method. ALT, AST and LDH production in culture medium and intracellular MDA, GSH and SOD levels were determined. Moreover, the total triterpenoid content and chemical constituents in GTs were detected by ultraviolet spectrophotometry and HPLC/Q-TOF-MS, respectively. RESULTS: GTs (50, 100 and 200 µg/ml) significantly increased the relative cell viability by 4.66, 7.78 and 13.46%, respectively, and reduced the level of ALT by 11.44%, 33.41% and 51.24%, AST by 10.05%, 15.63% and 33.64%, and LDH by 16.03%, 23.4% and 24.07% in culture medium, respectively. GTs could also remarkably decrease the level of MDA and increase the content of GSH and SOD in HepG2 cells. Furthermore, the total triterpenoid content in GTs was 438 mg GAAEs/g GTs. And 16 triterpenoids in GTs were identified or tentatively characterised. DISCUSSION AND CONCLUSION: Our results showed that GTs had potent cytoprotective effect against oxidative damage induced by t-BHP in HepG2 cells, thus suggesting their potential use as liver protectant.

Chemical composition, antimicrobial activity against Staphylococcus aureus and a pro-apoptotic effect in SGC-7901 of the essential oil from Toona sinensis (A. Juss.) Roem. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Toona sinensis (A. Juss.) Roem. (TSL), a popular vegetable in China, have anti-inflammatory, antidoting, and worm-killing effects and are used in folk medicine for the treatment of enteritis, dysentery, carbuncles, boils, and especially abdominal tumors. Our aim was to investigate the in vitro antimicrobial activity against Staphylococcus aureus and anticancer property of the essential oil from TSL (TSL-EO), especially the pro-apoptotic effect in SGC-7901. MATERIALS AND METHODS: TSL-EO obtained by hydrodistillation was analyzed by GC/MS and was tested in vitro against twenty clinically isolated strains of Staphylococcus aureus (SA 1-20), which were either methicillin-sensitive Staphylococcus aureus (MSSA) or methicillin-resistant Staphylococcus aureus (MRSA) and two standard strains viz. ATCC 25923 and ATCC 43300. The anticancer activity of TSL-EO was evaluated in vitro against HepG2, SGC7901, and HT29 through MTT assay. Moreover, the apoptosis-inducing activity of TSL-EO in SGC7901 cells was determined by Hoechst 33324 staining and flow cytometry methods. Also, the apoptosis-related proteins viz. Bax, Bcl-2 and caspase-3 were detected by western-blotting. RESULTS: GC-MS analysis showed that TSL-EO contained a high amount of sesquiterpenes (84.64%), including copaene (8.27%), ß-caryophyllene (10.16%), caryophyllene (13.18%) and ß-eudesmene (5.06%). TSL-EO inhibited the growth of both MSSA and MRSA, with the lowest MIC values of 0.125 and 1mg/ml, respectively. Treatment with TSL-EO for 24h could significantly suppress the viability of three different cancer cell lines (P<0.05). Furthermore, the apoptosis-inducing activity of TSL-EO in SGC7901 cells increased in a dose-dependent manner, potentially resulting from the up-regulated expression of Bax, caspase-3 and down-regulated expression of Bcl-2. CONCLUSIONS: TSL-EO possessed antibacterial activity against Staphylococcus aureus and significant cytotoxicity against cancer cells and particularly prominent pro-apoptotic activity in SGC7901 cells. These bioactivities were probably due to the high content of sesquiterpenes. Our results suggested that TSL-EO possessed potential health benefits and could serve as a promising natural food addictive.

Luminol as in situ light source in meso-tetraphenylporphyrin-mediated photodynamic therapy.

The light sources used in current photodynamic therapy are mainly lasers or light emitting diodes, which are not suitable to treat large-volume tumors and those located in the inner body. To overcome the limitation, we propose an in situ light source to activate the photosensitizer and kill the cancer cells directly. In the present work, we use luminol as light source and meso-tetraphenylporphyrin as the photosensitizer. According to the results, cells incubated with meso-tetraphenylporphyrin, subsequently triggered by luminol, decreased significantly in assays including cell viability and cytotoxicity, while the other groups showed only minor differences. The flow cytometric and fluorescent microscopy analysis showed similar results as well. In the analysis of cell death pathway, cell shrinkage was noticed after photodynamic therapy treatment, which might refer to apoptosis. Briefly, we suggest that luminol is a promising light source in meso-tetraphenylporphyrin-mediated photodynamic therapy for its greater penetration depth and well matched emission wavelength.

SC-1, a sorafenib derivative, shows anti-tumor effects in osteogenic sarcoma cells.

Despite significant advances in the treatment of osteosarcoma (OS), overall survival rate of OS patients has remained relatively constant for over two decades and novel approaches are needed to further improve prognosis. Here, we report the anti-tumor effect of SC-1, a novel sorafenib derivative that closely resembles sorafenib structurally but is devoid of kinase inhibitory activity, on OS cells through mediation of signal transducer and activator of transcription 3 (STAT3). SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis, and downregulated phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested OS cell lines (U2OS, HOS, and 143B). Expression of STAT3-driven genes, including cylcin D1 and c-myc, were also repressed by SC-1. Ectopic expression of STAT3 in 143B cells abolished apoptosis in SC-1-treated cells. Inhibition of SHP-1 decreased SC-1-induced apoptosis. SC-1 upregulated the activity of SHP-1 in tested OS cell lines in a dose-dependent manner. Finally, SC-1 reduced 143B tumor growth significantly in vivo, which was associated with downregulation of p-STAT3 and upregulation of SHP-1 activity. These data demonstrate that SC-1 has clinical potential for the treatment of OS patients.

Survival of patients aged over 80 years after Austin-Moore hemiarthroplasty and bipolar hemiarthroplasty for femoral neck fractures.

OBJECTIVES: Hemiarthroplasty is recommended for treatment of displaced femoral neck fractures in physically compromised elderly patients. The objective of this study was to analyze survival of patients aged >80 years after the implantation of either an Austin-Moore type prosthesis or a bipolar bearing prosthesis. METHODS: An Austin-Moore or bipolar hemiarthroplasty was implanted into 120 patients aged >80 years. Demographic data were collected. Survival rate at 5 years and factors related to mortality were analyzed. RESULTS: Sixty-two patients received Austin-Moore hemiarthroplasty, and 58 received bipolar hemiarthroplasty. No significant differences in gender, comorbid conditions, ASA scores, duration of hospitalization, intraoperative blood loss, duration from injury to operation, or postoperative morbidity between the two groups were found. However, patients who received the Austin-Moore hemiarthroplasty were older and had shorter operation time than those who received bipolar hemiarthroplasty. Kaplan-Meier estimates of 5 years survival were 40.0% for patients who received Austin-Moore hemiarthroplasty, and 62.9% for patients who received bipolar hemiarthroplasty. Cox proportional hazard regression analysis of risks factors of death revealed that patients who underwent Austin-Moore hemiarthroplasty were 2.0-fold more likely to die when compared to those who received bipolar hemiarthroplasty. CONCLUSIONS: Elderly patients who receive bipolar hemiarthroplasty may have a more favorable survival outcome when compared to those who receive unipolar hemiarthroplasty.

Treatment of unicameral bone cyst: surgical technique.

BACKGROUND: There is a variety of treatment modalities for unicameral bone cysts, with variable outcomes reported in the literature. Although good initial outcomes have been reported, the success rate has often changed with longer-term follow-up. We introduce a novel, minimally invasive treatment method and compare its clinical outcomes with those of other methods of treatment of this lesion. METHODS: From February 1994 to April 2008, forty patients with a unicameral bone cyst were treated with one of four techniques: serial percutaneous steroid and autogenous bone-marrow injection (Group 1, nine patients); open curettage and grafting with a calcium sulfate bone substitute either without instrumentation (Group 2, twelve patients) or with internal instrumentation (Group 3, seven patients); or minimally invasive curettage, ethanol cauterization, disruption of the cystic boundary, insertion of a synthetic calcium sulfate bone-graft substitute, and placement of a cannulated screw to provide drainage (Group 4, twelve patients). Success was defined as radiographic evidence of a healed cyst or of a healed cyst with some defect according to the modified Neer classification, and failure was defined as a persistent or recurrent cyst that needed additional treatment. Patients who sustained a fracture during treatment were also considered to have had a failure. The outcome parameters included the radiographically determined healing rate, the time to solid union, and the total number of procedures needed. RESULTS: The follow-up time ranged from eighteen to eighty-four months. Group-4 patients had the highest radiographically determined healing rate. Healing was seen in eleven of the twelve patients in that group compared with three of the nine in Group 1, eight of the twelve in Group 2, and six of the seven in Group 3. Group-4 patients also had the shortest mean time to union: 3.7 ± 2.3 months compared with 23.4 ± 14.9, 12.2 ± 8.5, and 6.6 ± 4.3 months in Groups 1, 2, and 3, respectively. CONCLUSIONS: This new minimally invasive method achieved a favorable outcome, with a higher radiographically determined healing rate and a shorter time to union. Thus, it can be considered an option for initial treatment of unicameral bone cysts.

Vascular compression syndrome of sciatic nerve caused by gluteal varicosities.

Sciatica is defined as pain or discomfort along the regions innervated by the sciatic nerve. Compression or irritation of lumbar spinal roots, most commonly because of lumbar disc herniation or spinal stenosis, causes sciatica in the vast majority of cases. Although it is rather uncommon, many pathologies have reported to cause nondiscogenic sciatica. A 70-year-old woman presented with intractable sciatic pain which was not elicited by posture change or cough. Sitting on the affected side provoked more pain than standing or walking. Magnetic resonance imaging revealed both spondylolisthesis with lumbar stenosis and compression of the gluteal portion of the sciatic nerve by varicotic gluteal veins. Given the atypical presentation of spinal root compression, gluteal vascular compressive neuropathy was suspected. Ligation and resection of varicotic vein resulted in relief of the patient's pain. To our knowledge, cases with varicosity-caused sciatica were limited in the literature review.

Treatment of unicameral bone cyst: a comparative study of selected techniques.

BACKGROUND: There is a variety of treatment modalities for unicameral bone cysts, with variable outcomes reported in the literature. Although good initial outcomes have been reported, the success rate has often changed with longer-term follow-up. We introduce a novel, minimally invasive treatment method and compare its clinical outcomes with those of other methods of treatment of this lesion. METHODS: From February 1994 to April 2008, forty patients with a unicameral bone cyst were treated with one of four techniques: serial percutaneous steroid and autogenous bone-marrow injection (Group 1, nine patients); open curettage and grafting with a calcium sulfate bone substitute either without instrumentation (Group 2, twelve patients) or with internal instrumentation (Group 3, seven patients); or minimally invasive curettage, ethanol cauterization, disruption of the cystic boundary, insertion of a synthetic calcium sulfate bone-graft substitute, and placement of a cannulated screw to provide drainage (Group 4, twelve patients). Success was defined as radiographic evidence of a healed cyst or of a healed cyst with some defect according to the modified Neer classification, and failure was defined as a persistent or recurrent cyst that needed additional treatment. Patients who sustained a fracture during treatment were also considered to have had a failure. The outcome parameters included the radiographically determined healing rate, the time to solid union, and the total number of procedures needed. RESULTS: The follow-up time ranged from eighteen to eighty-four months. Group-4 patients had the highest radiographically determined healing rate. Healing was seen in eleven of the twelve patients in that group compared with three of the nine in Group 1, eight of the twelve in Group 2, and six of the seven in Group 3. Group-4 patients also had the shortest mean time to union: 3.7 +/- 2.3 months compared with 23.4 +/- 14.9, 12.2 +/- 8.5, and 6.6 +/- 4.3 months in Groups 1, 2, and 3, respectively. CONCLUSIONS: This new minimally invasive method achieved a favorable outcome, with a higher radiographically determined healing rate and a shorter time to union. Thus, it can be considered an option for initial treatment of unicameral bone cysts.

A comparison of the effectiveness of dexmedetomidine versus propofol target-controlled infusion for sedation during fibreoptic nasotracheal intubation.

Fibreoptic intubation is a valuable modality for airway management. This study aimed to compare the effectiveness of dexmedetomidine vs target controlled propofol infusion in providing sedation during fibreoptic intubation. Forty patients with anticipated difficult airways and due to undergo tracheal intubation for elective surgery were enrolled and randomly allocated into the dexmedetomidine group (1.0 microg.kg(-1) over 10 min) (n = 20) or the propofol target controlled infusion group (n = 20). Intubating conditions and patient tolerance as graded by a scoring system were evaluated as primary outcomes. Intubation was successful in all patients. Satisfactory intubating conditions were found in both groups (19/20 in each group). The median (IOR [range]) comfort score was 2 (1-2 [1-4]) in the dexmedetomidine group and 3 (2-4 [2-5]) in the propofol group (p = 0.027), favouring the former. The dexmedetomidine group experienced fewer airway events and less heart rate response to intubation than the propofol group (p < 0.003 and p = 0.007, respectively). Both dexmedetomidine and propofol target-controlled infusion are effective for fibreoptic intubation. Dexmedetomidine allows better tolerance, more stable haemodynamic status and preserves a patent airway.

Establishment, characterization, virus susceptibility and transfection of cell lines from cobia, Rachycentron canadum (L.), brain and fin.

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate-buffered saline containing 0.25% trypsin at 25 degrees C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 degrees C in Leibovitz-15 medium containing 10% foetal bovine serum. The cells have been sub-cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.

Over-expression of receptor activator of nuclear factor-kappaB ligand (RANKL), inflammatory cytokines, and chemokines in periprosthetic osteolysis of loosened total hip arthroplasty.

Loosening of total hip arthroplasty (THA) caused by periprosthetic osteolysis induced by ultra-high molecular weight polyethylene (UHMWPE) particles is a major clinical problem. We investigated whether there are differences between loosened THA patients and primary THA patients in (1) receptor activator of nuclear factor-kappaB ligand (RANKL) expression on periprosthetic bone marrow cells; (2) RANKL levels, osteoprotegerin (OPG)/RANKL ratios, the levels of inflammatory cytokines and chemokines in synovial fluid. We used flow cytometric analysis to detect RANKL expression on periprosthetic bone marrow cells. We used enzyme-linked immunoassay and multiplex microsphere-based immunoassay to measure RANKL, OPG, cytokines, and chemokines in synovial fluid. We found loosened THA patients had higher RANKL expression on osteoblastic stromal cells, higher levels of RANKL, interleukin (IL)-6, IL-8, IL-10, interferon-gamma-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, monokine induced by interferon-gamma (MIG), and lower OPG/RANKL ratios in synovial fluid than primary THA patients. There was positive correlation between the levels of IL-6, IL-8, IL-10, IP-10, MCP-1, or MIG and RANKL levels in synovial fluid or RANKL expression on osteoblastic stromal cells. These suggest that UHMWPE particles induce over-expression of RANKL, IL-6, IL-8, IP-10, MCP-1, and MIG in human periprosthetic microenvironment. This results in periprosthetic osteolysis and loosening of THA.