Effect and mechanism of dihydroartemisinin on proliferation, metastasis and apoptosis of human osteosarcoma cells.

Osteosarcoma represents an aggressive type of bone malignancy that poses a significant health threat. The objective of the current study was to analyze the effect and mechanism of dihydroartemisinin (DHA) on the proliferation, metastasis and apoptosis of human osteosarcoma cells. A gradient concentration of DHA (15, 25 and 35 µmol.L-1) was used to stimulate the cells, along with control and Dimethyl sulfoxide (DMSO). The phenotypic outcomes were characterized using MTT assay, clone formation assay, Hoechst 33258 staining assay, luciferase reporter plasmid assay, Western blot and wound healing assay. In addition, IBM SPSS Statistics 18.0 software was applied for statistical analysis and all experimental data were expressed as mean ± s.d. Analysis of variance (ANOVA) was applied to compare the differences among multiple groups. Our results demonstrated that DHA inhibited the proliferation and metastasis of osteosarcoma cells and promoted the apoptosis in the cytomorphosis.

Colonisation of prosthetic grafts by immunocompetent cells in a sheep model.

The present study examined the distribution of immunocompetent cells in synthetic vascular grafts in an experimental sheep model. Sixty-two adult Merino sheep underwent synthetic patch closure of a longitudinal arteriotomy in the left common carotid artery. The synthetic patch materials used were gelatin sealed Dacron (n=10), fluoropassivated Dacron (n=10), Fluoropassiv (n=12), polyurethane (n=10), expanded polytetrafluoroethylene (n=10) and carbon-lined expanded polytetrafluoroethylene (n=10). The sheep were sacrificed after four weeks when the prosthetic patches were harvested and fixed in 10% neutral buffered formalin. Transverse sections were taken along the graft and paraffin embedded. Serial sections were stained with cell type specific antibodies to identify T-lymphocytes (CD3(+)), dendritic cells (S-100(+)), endothelial cells (von Willebrand factor(+)) and smooth muscle cells (smooth muscle alpha-actin(+)). All six graft types contained CD3(+) and S-100(+) cells in the neointima, within the synthetic matrix and in the perigraft layer. Three different tissue responses to synthetic materials were observed and the grafts were classified accordingly into three groups: (1) gelatin sealed Dacron, fluoropassivated Dacron and Fluoropassiv; (2) expanded polytetrafluoroethylene and carbon-lined expanded polytetrafluoroethylene; (3) polyurethane. The three synthetic materials in Group 1 showed almost identical reactions with least accumulation of immunocompetent cells within the synthetic material but greater accumulation of immuno-inflammatory infiltrates in the perigraft vascular tissue. In this group, new vessels penetrated into the synthetic material and there was prominent formation of foreign body (giant) cells. Group 2 showed greater accumulation of immunocompetent cells within the synthetic material itself but only sparse immuno-inflammatory infiltrates in the perigraft tissue. Group 3 showed a high degree of inflammatory response within both the synthetic material and the perigraft vascular tissue. These observations demonstrate that immunocompetent cells colonise the synthetic matrix of grafts and accumulate in the perigraft tissue, but inflammatory responses vary in different graft types.

Development of intimal hyperplasia in six different vascular prostheses.

OBJECTIVES: to compare six vascular prostheses for the development of intimal hyperplasia (IH) in a sheep model. MATERIAL AND METHODS: prostheses tested were gelatin sealed Dacron (GSD), fluoropassivated Dacron (FPD), FluroIpassiv TM (FD), expanded polytetrafluoroethylene (ePTFE), carbon-lined expanded polytetrafluoroethylene (CL-ePTFE) and vascular access graft (VAG). Sixty-two adult female Merino sheep (35-45 kg) were used. Elliptical graft patches were implanted into the left common carotid artery using one of the six graft types: GSD (n=10), FPD (n=10), FD (n=12) VAG (n=10), ePTFE (n=10), or CL-ePTFE (n=10). Four weeks later grafts were removed for histopathological assessment and measurement of the degree of IH obtained on a computerised image analysis system. RESULTS: IH indices were significantly less for FPD (0.191+/-0.095, p<0.05), FD (0.199+/-0. 081, p<0.05), ePTFE (0.213+/-0.078, p<0.05) and CL-ePTFE (0.161+/-0. 066,p<0.01), compared to the GSD group (0.287+/-0.077). The VAG group (0.257+/-0.091) showed no difference compared to GSD. There was no significant difference between the FPD, FD, ePTFE and CL-ePTFE grafts. CONCLUSION: this study indicates that less IH occurred in the two-ePTFE grafts and two fluoropolymer coated Dacron grafts than in gelatin sealed Dacron or polyurethane grafts.

Does in situ replacement of a staphylococcal infected vascular graft with a rifampicin impregnated gelatin sealed Dacron graft reduce the incidence of subsequent infection?

BACKGROUND: The aim of this study was to treat methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis prosthetic vascular graft infections by in situ replacement with a rifampicin bonded Gelsoft graft. METHODS: Interposition grafts were placed in the carotid artery of 56 sheep and the graft surface directly inoculated with 10(8) colony forming units of MRSA or S. epidermidis. At three weeks, grafts were harvested and sheep allocated to three groups. In the MRSA group, sheep received grafts soaked in 1.2 mg/ml (12), 10 mg/ml (10) and no rifampicin (7). For S. epidermidis, sheep received grafts soaked in 1.2 mg/ml (10), 10 mg/ml (9) and no rifampicin (8). There were two deaths, in the MRSA study group. Remaining sheep were euthanased and grafts harvested three weeks following regrafting. Swabs were taken to assess bacterial growth in the perigraft tissues, and external and internal graft surfaces. A 3-5 mm segment of graft was incubated in broth medium. RESULTS: For MRSA, no statistical difference between the groups was reached for any of the measured parameters. For S. epidermidis, a significant reduction was reached for total infected specimens in the 10 mg/ml group compared to both control (p<0.001) and 1.2 mg/ml (p<0.005) groups. Graft re-infection was also less likely to occur with S. epidermidis than MRSA. CONCLUSIONS: Replacement of S. epidermidis infected vascular grafts with 10 mg/ml rifampicin soaked Gelsoft graft is effective in reducing subsequent S. epidermidis infection. This conclusion cannot be extended to MRSA infected vascular grafts.

Verapamil reduces intimal hyperplasia in a sheep carotid artery patch graft model.

BACKGROUND: The current study investigated the effect of verapamil on the development of intimal hyperplasia (IH) using a sheep model. METHODS: A gelatin-sealed Dacron patch graft was implanted into the left common carotid artery in 40 adult Merino sheep. Sheep were then randomly allocated to four groups. Group 1 were controls given no treatment (n = 10). Groups 2-4 were treated with intravenous verapamil 0.5 mg/kg per day in two divided doses for different lengths of time. Group 2 (n = 10) received treatment for 1 week; group 3 (n = 10) received treatment for 2 weeks and group 4 (n = 10) received treatment for 4 weeks. All sheep were killed at 4 weeks and the grafted segments of artery were harvested for IH assessment by an image analysis system. RESULTS: The IH index from the three groups treated with verapamil was significantly less than that of the control (group 1,0.287+/-0.077: group 2,0.205+/-0.064, P<0.05; group 3, 0.193+/-0.059, P<0.01; group 4,0.171+/-0.046, P<0.01). CONCLUSION: The present study suggests that verapamil inhibits the development of IH even when treatment is given for only 1 week.

Suppression of intimal hyperplasia with low molecular weight heparin in a sheep model.

BACKGROUND: Both unfractionated heparin (UH) and low molecular weight heparin (LMWH) in therapeutic anticoagulant doses have been shown to inhibit the development of intimal hyperplasia (IH) but with an increased risk of haemorrhage. In this study we investigated the effect of a "low dose" and "high dose" of UH and LMWH on the inhibition of IH together with their effect on plasma anti-Xa activity (AXa) and activated partial thromboplastin time (APTT) using a carotid artery sheep model. METHODS: A gelatin sealed Dacron patch graft was implanted into the common carotid artery of sheep which were randomly allocated to a control group (Group 1, n=10) or to one of four treatment groups receiving either low-dose LMWH enoxaparin 1 mg/kg/day (group 2, n=11), high-dose LMWH enoxaparin 2 mg/kg/day (Group 3, n=13), low-dose UH 125 u/kg/day (Group 4, n=10) or high-dose UH 250 u/kg/day (Group 5, n=10). The LMWH was administered subcutaneously once daily for four weeks and the UH in two divided doses per day for four weeks. During the treatment period, AXa and APTT were assayed from blood collected prior to and at 1 and 2 h after heparin administration on day 3, 7, 14, 21 and 28. On day 28, all animals were sacrificed and grafts were collected for analysis after taking blood samples prior to, then at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h following the last injection. Measurements of intimal thickness were obtained under light microscopy from eight transverse sections of each grafted artery using an eyepiece graticule. RESULTS: IH measurements (Mean+/-SD) were: Group 1 (controls) 288+/-86 microm, Group 2 (low-dose LMWH) 222+/-50 mm (p<0.05 compared to Group 1), Group 3 (high-dose LMWH) 203+/-78 microm (p<0.01), Group 4 (low-dose UH) 275+/-61 microm, and Group 5 (high-dose UH) 206+/-71 microm (p<0.01). There was no significant difference between Groups 2, 3 and 5. Groups 2, 3 and 5 demonstrated significant AXa during the 28 days period of which Groups 2 and 5 showed a significant increase in AXa levels with time. In the 24 h study after the last dose of treatment both Groups 2 and 3 showed longer AXa than group 5 (12-24 h vs 8 h). When compared to the control group, significant elevation of APTT was demonstrated in Groups 3 and 5. Group 5 had significantly longer APTT than Group 3. In the 24 h study, APTT reflected the changes of AXa in all groups. CONCLUSIONS: In this study the LMWH enoxaparin was effective in reducing the formation of IH both at a standard anticoagulant therapeutic dose of 2 mg/kg/day and also at a lower dose of 1 mg/kg/day.

Does in situ replacement of a staphylococcal infected vascular graft with a rifampicin impregnated gelatin sealed Dacron graft reduce the incidence of subsequent infection?

BACKGROUND: The aim of this study was to treat methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis prosthetic vascular graft infections by in situ replacement with a rifampicin bonded Gelsoft graft. METHODS: Interposition grafts were placed in the internal carotid artery of 56 merino sheep and the graft surface directly inoculated with 10(8) colony forming units (CFU) of MRSA (29) or S. epidermidis (27). At three weeks, grafts were harvested and sheep allocated to three groups. In the MRSA infected group, sheep received grafts soaked in 1.2 mg/ml (12), 10 mg/ml (10) and no (7) rifampicin. For S. epidermidis, sheep received grafts soaked in 1.2 mg/ml (10), 10 mg/ml (9) and no (8) rifampicin. There were two deaths, in the MRSA study group, one each from the rifampicin treated groups. The remaining sheep were euthanased and grafts harvested three weeks following regrafting. Grafts at harvests were assessed for perigraft abscess formation, anastomotic disruption and graft thrombosis. Swabs were taken to assess bacterial growth in the perigraft tissues, and external and internal graft surfaces. A 3-5 mm segment of graft was incubated in a broth medium. For S. epidermidis the remainder of the graft was ground and then incubated in a broth medium. RESULTS: For MRSA, no statistical difference between the groups was reached for any of the measured parameters. For S. epidermidis, a significant reduction was reached for total infected specimens in the 10 mg/ml group compared to both control (p<0.001) and 1.2 mg/ml (p<0.005) groups. Graft reinfection was also less likely to occur with S. epidermidis than MRSA. CONCLUSIONS: In conclusion, replacement of S. epidermidis infected vascular grafts with 10 mg/ml rifampicin soaked Gelsoft graft is effective in reducing subsequent S. epidermidis infection. This conclusion cannot be extended to MRSA infected vascular grafts.

T-cell receptor repertoires utilized in response to linear peptides representing an immunodominant MHC class II restricted T-cell epitope are far more diverse than that utilized in response to the same epitope in the nominal antigen.

Previous analyses of the T-cell receptor (TCR) repertoire utilized in response to the 1-102 fragment of the lambda cI repressor protein and specific for the immunodominant amino acid 12-26 region in the context of I-Ek, have shown this repertoire to be extremely restricted. In contrast, here we show that the TCR repertoires utilized in two strains of I-Ek expressing mice in response to two linear peptides representing this immunodominant region are diverse. Despite their extensive diversity, these repertoires are somewhat overlapping. In addition, structural similarities were observed between the full lambda cI fragment (1-102) and peptide elicited TCR repertoires, including frequent use of the Valpha2 family of gene segments, particularly among peptide (12-26) elicited TCRs cross-reactive with 1-102/I-Ek. Nevertheless, these data indicate that it may be difficult to mimic the immune response to an immunodominant epitope of a protein antigen via immunization with linear peptides containing the amino acid sequence of that epitope. Possible explanations for differences in the levels of TCR diversity among T cells responding to an epitope present in a nominal antigen as compared to T cells responding to linear peptide antigens containing this same epitope are discussed.

An increased concentration of rifampicin bonded to gelatin-sealed Dacron reduces the incidence of subsequent graft infections following a staphylococcal challenge.

The purpose of this study was to determine if 10 mg/ml rifampicin bonded to gelatin-sealed Dacron (Gelsoft) reduced staphylococcal infection. Grafts soaked in rifampicin were interposed in the left carotid artery of 20 merino sheep and then inoculated with 10(8) colony-forming units of MRSA (10 sheep) or a slime producing Staphylococcus epidermidis (10 sheep). Grafts were harvested at 3 weeks, and perigraft abscess, anastomotic disruption and graft occlusion recorded. Swabs were taken to assess bacterial growth of the perigraft tissues, and external and internal graft surface. Grafts segments were incubated in broth medium. Results were compared with previously published results that used graft that were not soaked in rifampicin (control) and grafts soaked in 1.2 mg/ml rifampicin. A total of 4/50 cultures were positive and significantly reduced for S. epidermidis compared with the control group of 30/50 (P < 0.05) and the 1.2 mg/ml group of 13/45 (P < 0.05). For the methicillin resistant staphylococcus aureus (MRSA) group, 6/40 cultures were positive, which was significantly reduced compared with the control group (38/40, P < 0.05) and the 1.2-mg/ml group (19/32, P < 0.05). In conclusion an increased concentration of rifampicin significantly reduced the incidence of prosthetic vascular graft infection following a challenge of MRSA or S. epidermidis.

Optimal dose and duration of heparin for inhibition of intimal hyperplasia.

BACKGROUND: A Dacron patch graft was placed in the common carotid artery of sheep to assess the effect of three different doses of unfractionated heparin administered for periods of one to four weeks. MATERIALS AND METHODS: Animals were divided into a control group (n=6) in which animals received a patch graft but no heparin and three main treatment groups on the basis of the dose of heparin received. Group 1 animals (n=24) received 140 units per kg daily in two divided doses. Group 2 (n=24) received 280 units per kg daily and Group 3 (n=24) received 420 units per kg daily. Each treatment group was further divided into four subgroups on the basis of the duration of treatment. Groups 1(i), 2(i), 3(i) had treatment for one week. Groups 1(ii), 2(ii), 3(ii) had treatment for two weeks. Groups 1(iii), 2(iii), 3(iii) had treatment for three weeks. Groups 1(iv), 2(iv), 3(iv) had treatment for four weeks. There were six animals in each subgroup giving a total of 72 treated sheep. Animals were sacrificed at four weeks and an intimal measurement in pm was obtained under light microscopy from a transverse sectioned segment of artery containing the patch graft using an eyepiece graticule. RESULTS: There were no haemorrhagic complications. Control animals had a mean intimal thickness of 176 pm. All treatment groups had significantly less IH--Group 1 131.9 (p<0.01), Group 2 138.5 (p<0.05), Group 3 117.8 (p<0.01). There were no significant differences between the treatment groups. There was significantly less IH in animals treated for four weeks (106.3) compared to one week (140.2, p<0.05). CONCLUSIONS: This study indicates that inhibition of IH can be achieved by heparin with optimal effect from a four week treatment period.

Inhibition of fibro-intimal hyperplasia in a polytetrafluoroethylene vascular graft with standard heparin and low molecular weight heparin.

BACKGROUND: Fibro-intimal hyperplasia is a significant cause of late prosthetic vascular graft failure. The influence of standard unfractionated sodium heparin (SH) and low molecular weight heparin (LMWH) on intimal hyperplasia was studied in a sheep polytetrafluoroethylene (PTFE) end-to-side carotid artery vascular graft model. METHODS: Sodium heparin was administered subcutaneously in a dose of 7250 units twice daily (n = 6). Enoxaparin, an LMWH, was administered subcutaneously in two different dose regimens of 2 mg/kg (n = 6) and 5 mg/kg (n = 6) daily. At 1 month the animals were killed and the grafts sectioned. An intima to media ratio was obtained under light microscopy, using an eyepiece graticule, from measurements taken from the floor of the native artery. Results were analysed with a rank-sum correlation test. RESULTS: Control sheep had a median ratio of 0.104, an SH level of 0.021, a low-dose LMWH level of 0.035 and a high-dose LMWH level of 0.017. There was a significant difference between control and SH (P < 0.001), control and high-dose LMWH (P < 0.05), and SH and low-dose LMWH (P < 0.05), but there were no differences between SH and high-dose LMWH or between the two LMWH groups. Both LMWH groups recorded high anti-Xa levels but only slight prolongation of thrombin time. CONCLUSIONS: Low molecular weight heparin has a similar effect to SH on the inhibition of fibro-intimal hyperplasia in this animal model but is safer, with less bleeding potential, and is more convenient to administer as a once-daily dose.

Prophylaxis against Staphylococcus epidermidis vascular graft infection with rifampicin-soaked, gelatin-sealed Dacron.

An animal model was used to assess the efficacy of rifampicin-impregnated, gelatin-sealed Dacron in the prevention of vascular graft infections caused by Staphylococcus epidermidis. Under a general anaesthetic an interposition graft was placed into sheep carotid artery. On completion of the operation 1 ml of normal saline containing 10(8) colony forming units (cfu) of a slime-producing S. epidermidis was inoculated directly onto the graft. After 3 weeks the graft was harvested. Swabs were taken of perigraft tissues, and of external and internal aspects of the graft. A 3-5-mm segment of the graft was incubated in broth medium and a second segment was ground for 5 min and incubated in broth medium. The presence of abscess formation and anastomotic disruption was assessed. Ten sheep received a gelatin-sealed Dacron graft (control), while nine received the same graft impregnated with rifampicin at a concentration of 1.2 mg/ml (treated). Eight of 10 control grafts were infected, with 30 of 50 possible cultures positive, compared with four of nine treatment grafts infected (P = 0.13) and 13 of 45 cultures positive (P = 0.004). The control group had four abscesses and two anastomotic disruptions; the treatment group had no abscesses (P = 0.05) or anastomotic disruptions (P = 0.26). Other organisms were isolated from nine of the 12 infected grafts, most commonly Staphylococcus aureus. There was no development of resistance to rifampicin. Rifampicin-impregnated, gelatin-sealed Dacron is successful at reducing the incidence of S. epidermidis vascular graft infection.

Rifampicin impregnated Dacron grafts: no development of rifampicin resistance in an animal model.

AIM: Rifampicin impregnated Dacron grafts have been shown to be effective at preventing vascular graft infection in different animal models. The development of resistance to rifampicin would be a major drawback to the widespread use of such a graft. We aimed to determine how readily this would occur by using a sheep animal model. METHODS: Under general anaesthetic a 2cm long, 5mm diameter Dacron interposition graft inpregnated with 1.2 mg/ml rifampicin was placed in the left carotid artery. An extreme challenge of methicillin resistant Staphylococcus aureus (MRSA) using an inoculum of 10(9) colony forming units was placed directly onto the graft. The grafts were harvested at 3 weeks and cultures of the graft and tissues were taken. The presence or absence of any abscess formation, anastomotic disruption and graft thrombosis was noted. Any positive growths were identified and if found to be the same as the inoculum, the bacteria were used as the inoculum for another sheep. This was repeated once more. Thus we started with three sheep initially and used a total of nine sheep. RESULTS: There were no deaths. All grafts were infected with the same MRSA strain, confirmed on phage typing. There were three abscess and one anastomotic disruption. Seven of the grafts were occluded. The minimal inhibitory concentration (MIC) of the infecting inoculum and the bacteria retrieved were determined using the agar dilutional method. The MIC for the three initial inocula was < 0.007 mg/l. All subsequent strains isolated had an MIC of < 0.015 mg/l. This was a difference of one dilution and not significant. CONCLUSION: There was no development of rifampicin resistance using this animal model.

Comparison of fluoropolymer passivated Dacron and polytetrafluoroethylene grafts in a sheep model.

The search for an optimal prosthetic vascular graft suitable for small vessels continues. The aim of this study was to compare a fluoropolymer passivated Dacron graft with a standard polytetrafluoroethylene graft. A sheep animal model was used. Under general anaesthesia a 5-cm length of 5-mm diameter graft was interposed into the left carotid artery. After an interval of 1, 2 or 3 months the sheep were reanaesthetized and a carotid angiogram performed before harvesting the graft. Graft patency was assessed by macroscopic inspection, angiography and histological examination. Overall, four of eight polytetrafluoroethylene grafts and three of ten fluoropolymer passivated Dacron grafts remained patent with similar patency rates at 1, 2 and 3 months. Histological examination of all available grafts showed evidence of fibrointimal hyperplasia. All but one of the occluded grafts had fibrointimal hyperplasia.