Synergy is achieved by complementation with Apo2L/TRAIL and actinomycin D in Apo2L/TRAIL-mediated apoptosis of prostate cancer cells: role of XIAP in resistance

BACKGROUND: Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis. METHODS: We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot. RESULTS: Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D. CONCLUSION: This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.     

Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high Bcl-2 expression

An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7beta-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation.     

Butyrate sensitizes human colon cancer cells to TRAIL-mediated apoptosis

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor family, induces apoptosis in TRAIL-sensitive tumors through the activation of the caspase pathway. Sodium butyrate (NaBT) induces differentiation and apoptosis in certain colorectal cancers; the molecular mechanisms for these effects have not been clearly defined. The purpose of our study was to determine whether NaBT sensitizes TRAIL-resistant human colon cancer cells to the effects of TRAIL. METHODS: Human colon cancer cells (KM12C, KML4A, and KM20) that are resistant to TRAIL treatment alone were treated with TRAIL (100 ng/mL), NaBT (5 mmol/L), or a combination of these agents and harvested for total RNA and protein. Western blots were performed to assess intracellular expression of Flice-like inhibitory protein (FLIP), a caspase inhibitor. Percent-specific apoptosis, relative caspase-3 activity, and Annexin-V immunofluorescence were determined at 24 and 48 hours. Cell cycle--related gene expression was assessed by RNase protection. RESULTS: Treatment with NaBT for 24 and 48 hours decreased FLIP protein expression in all cell lines. Furthermore, NaBT sensitized these resistant cancer cells to the effects of TRAIL with significant increases noted in cell death, caspase-3 activity, and Annexin-V staining compared with NaBT alone. CONCLUSIONS: Our findings suggest that the reduction of FLIP protein levels by NaBT renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL with agents (such as NaBT, which target proteins that prevent cell death) may provide a more effective and less toxic regimen for the treatment of resistant colon cancers.     

Human mesangial cells are resistant to productive infection by multiple strains of human immunodeficiency virus types 1 and 2.

Human immunodeficiency virus-associated nephropathy (HIVAN) is a recognized clinical entity of unknown pathogenesis. A role for viral infection of renal cells in the initiation of this process at present is an intriguing but untested hypothesis. Studies in primate models of acquired immunodeficiency syndrome (AIDS) suggest that injury to the mesangial cell may be central to the sclerosing glomerular lesion characteristic of HIVAN. We therefore tested the infectibility of human mesangial cells (HMC) in vitro by a variety of strains of HIV chosen to include a spectrum of tropisms for different cell types. Productive infection of mesangial cells could not be demonstrated using any of the virus strains. Nonetheless, HIV infection of intrinsic renal cells remains an attractive area of inquiry for understanding the natural history of HIVAN.     

Ceramide induces apoptosis in neuroblastoma cell cultures resistant to CD95 (Fas/APO-1)-mediated apoptosis

BACKGROUND/PURPOSE: Spontaneous tumor regression is a well-known characteristic in neuroblastomas. Because preliminary reports have shown that regression may be caused by apoptosis (a lethal cascade mediated by the CD95 (APO-1/Fas)-receptor), we analyzed the expression of CD95-receptors in 5 human neuroblastoma cell lines. Ceramides (known stimuli of apoptosis downstream from the CD95-receptor complex) also were used to test whether apoptosis would be induced in neuroblastoma cell cultures resistant to CD95-mediated programmed cell death. METHODS: The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was detected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2-yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after propidium iodide staining of the DNA. RESULTS: CD95-receptor expression was found on all neuroblastoma cell lines. Stimulation of the CD95-receptor of the malignant glioblastoma cell line LN229 (positive control) with either anti-CD95-antibody or CD95-ligand induced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95-antibody or the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 micromol/L C2- and C6-ceramide. CONCLUSIONS: CD95-receptors are present on neuroblastoma cell lines, and these cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD95-ligand. Apoptosis is induced, however, when these cells are treated with ceramide. A signal blockage downstream from the CD95-receptor complex and upstream of ceramide may account for this finding, and the "cellular FLICE inhibitory protein" (cFLIP) may be primarily responsible.     

不同强度张应力影响成骨细胞分化的体外实验研究

目的 探讨不同强度的张应力对人成骨细胞分化的影响.方法 根据骨假体界面应力有限元分析数据和细胞水平应力扩增理论,分别以0.8%、1.6%、2.4%和3.2%的强度在体外对人成骨细胞连续48 h施加1 Hz的牵张应力,观察碱性磷酸酶活性的变化和其他成骨相关基因的表达.结果 成骨细胞碱性磷酸酶活性的提高出现在0.8%和1.6%的应变水平;2.4%和3.2%的应变促进骨钙素的表达;3.2%的应变增强Cbfal/Runx2的表达;与静力对照相比,所有应变组Ⅰ型胶原基因的表达增强;与1.6%应变组相比,Ⅰ型胶原基因的表达在0.8%时减弱,在3.2%时增强(P<0.05).结论 较高强度的应变促进与基质成熟相关的基因的表达,而低强度的应变刺激碱性磷酸酶活性的提高.Ⅰ型胶原基因表达对张应力的应答呈强度依赖性.     

Monoclonal antibodies to ROS 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes

We have raised a panel of 15 monoclonal antibodies (MAbs) recognizing cell surface antigens of the rat osteoblast-like cell line ROS 17/2.8. The MAbs were selected on the basis of preferential binding to ROS 17/2.8 cells compared to ROS 25/1 cells. Immunohistochemical studies of antigen localization on cryostat sections of rat calvaria, long bone, and soft tissues demonstrated that five of these MAbs, UBIM 1, 2, 3, 12, and 17, recognize antigens that are restricted to normal rat osteoblasts and chondrocytes. The antigens appear to be localized to the cell surface of the osteoblast, with no apparent staining of bone matrix in either undecalcified or decalcified sections. In vitro, these MAbs recognize cell surface antigens present on two additional cell lines, ROS 24/1 and Rat 2 cells, and on the adherent cell population cultured from rat long bone marrow. Of these MAbs, three (UBIM 1, 2, and 3) recognize high-molecular-weight antigens of Mr 200,000-225,000. This study has also identified cell surface antigens of ROS 17/2.8 cells that are not expressed by osteoblasts in vivo. MAbs UBIM 9 and 21 bind to marrow cells in long bone sections, to the 7-day-old nonadherent cell population from cultured marrow, and to lymphoid tissue in sections of spleen. Another four MAbs (UBIM 10, 11, 14, and 22) bind to a variety of cells and tissues both in vitro and in vivo. Studies of the interactions of this panel of MAbs with osteogenic tissues and cell lines may have an important impact on the understanding of osteoblast physiology.     

Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes

Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.     

MCH-/- mice are resistant to aging-associated increases in body weight and insulin resistance

Ablation of the hypothalamic peptide, melanin-concentrating hormone (MCH), leads to a lean phenotype and resistance to diet-induced obesity. Observation of MCH(-/-) mice at older ages suggested that these effects persist in mice >1 year old. Leanness secondary to caloric restriction is known to be associated with improved glucose tolerance as well as an overall increase in life span. Because the MCH(-/-) model represents leanness secondary to increased energy expenditure rather than caloric restriction, we were interested in determining whether this model of leanness would be associated with beneficial metabolic effects at older ages. To assess the effects of MCH ablation over a more prolonged period, we monitored male and female MCH(-/-) mice up to 19 months. The lean phenotype of MCH(-/-) mice persisted over the duration of the study. At 19 months, MCH(-/-) male and female mice weighed 23.4 and 30.8% less than their wild-type counterparts, a result of reduced fat mass in MCH(-/-) mice. Aged MCH(-/-) mice exhibited better glucose tolerance and were more insulin sensitive compared with wild-type controls. Aging-associated decreases in locomotor activity were also attenuated in MCH(-/-) mice. We also evaluated two molecules implicated in the pathophysiology of aging, p53 and silent inflammatory regulator 2 (Sir2). We found that expression of the tumor suppressor protein p53 was higher in MCH(-/-) mice at 9 and 19 months of age. In contrast, expression of Sir2 was unchanged. In aggregate, these findings suggest that MCH ablation improves the long-term outcome for several indicators of the aging process.     

人成骨细胞与外周血单核细胞共同培养诱导破骨细胞样细胞

目的 探讨人外周血单核细胞与原代人成骨细胞共同培养体外转化为破骨细胞样细胞的条件,并观察其体外生长,功能情况。方法 分别分离培养人成骨细胞,外周血单核细胞,并在含1,25（OH）2D3（10^-7mol/L）,地塞米松（10^-8mol/L)及巨噬细胞集落刺激因子（M－CSF）（25ng/ml)的条件液中共同培养,用相差显微,抗酒石酸酸性磷酸酶染色（TRAP）观察破骨细胞样细胞生长情况,应用扫描电镜（SEM）观察骨片隐窝以反映其功能。结果 共同培养中的单核细胞逐渐融合;TRAP染色阳性多核细胞在第7天明显增多;骨片隐窝呈现多种形态。结论 人原代成骨细胞与人外周血单核细胞共同培养可以诱导出破骨细胞样细胞,但应该选择分化程度较低的细胞以及控制接种密度。     

Murine dendritic cells that are resistant to maturation are unable to induce tolerance to allogeneic stem cells

Induction of donor-specific hyporesponsiveness would minimize the need for intensive immunosuppression in the clinical setting of graft rejection and dendritic cells (DCs) might be useful tools for this purpose. Besides their ability to induce immunogenic T-cell responses, these antigen presenting cells can lead to T-cell anergy, when antigen presentation occurs in the absence of costimulation as is the case in immature DCs (iDCs). In continuance of publications reporting on the use of iDCs to induce tolerance to various organs, we set out to determine whether tolerance could be induced in a model of allogeneic stem cell transplantation. Immature DCs were obtained by culture with very low concentrations of GM-CSF and by treating DCs with Dexamethasone (Dex). We show that these DCs express low levels of MHCII and costimulatory molecules and that this immature phenotype is retained after application of maturation stimuli. We also prove that these alternatively activated DCs are unable to induce T-cell proliferation in vitro. When used in vivo however, these tolerogenic DCs do not provide tolerance to fully mismatched or haploidentical stem cells.     

酒精诱导体外培养成骨细胞凋亡及Bcl-2、Bax mRNA表达的实验研究

目的 观察酒精对体外培养成骨细胞凋亡的影响及凋亡相关基因Bcl-2、Bax mRNA表达变化.方法 体外分离培养成骨细胞,随机分组进行不同浓度酒精干预,利用AO/EB染色观察细胞凋亡形态学改变、DNA Ladder检测凋亡细胞DNA片段形成等定性方法证实凋亡发生.流式细胞技术定量测定细胞凋亡率.RT-PCR检测细胞凋亡相关基因Bcl-2、Bax mRNA表达.结果 不同浓度酒精干预后,成骨细胞发生皱缩,AO/EB染色出现凋亡细胞,随酒精浓度增加形成规则的DNA Ladder梯状条带,100mM浓度时最明显.流式细胞仪分析,与对照组(3.42%±1.07%)相比,100 mM酒精干预48h后细胞凋亡率增加,为11.94%±1.14%,差异有统计学意义(P<0.05).与对照组比较,成骨细胞经100 mM酒精干预培养24 h后,Bcl-2 mRNA表达强度降低66%,Bax mRNA表达强度增高11%,Bcl-2/Bax比值下降69%,差异均有统计学意义(P<0.05).结论 酒精引起体外培养成骨细胞凋亡,是酒精性骨损害的发生机制之一.凋亡相关基因Bcl-2 mRNA表达下调和Bax mRNA表达上调与酒精引起成骨细胞凋亡有关.     

Anticancer efficacy of Apo2L/TRAIL is retained in the presence of high and biologically active concentrations of osteoprotegerin in vivo

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that binds to the ligand for receptor activator of nuclear factor κB (RANKL) and inhibits bone resorption. OPG can also bind and inhibit the activity of the TNF‐related apoptosis‐inducing ligand (Apo2L/TRAIL), raising the possibility that the anticancer efficacy of soluble Apo2L/TRAIL may be abrogated in the bone microenvironment where OPG expression is high. In this study we used a murine model of breast cancer growth in bone to evaluate the efficacy of recombinant soluble Apo2L/TRAIL against intratibial tumors that were engineered to overexpress native full‐length human OPG. In vitro, OPG‐overexpressing breast cancer cells were protected from Apo2L/TRAIL‐induced apoptosis, an effect that was reversed with the addition of soluble RANKL or neutralizing antibodies to OPG. In vivo, mice injected intratibially with cells containing the empty vector developed large osteolytic lesions. In contrast, OPG overexpression preserved the integrity of bone and prevented breast cancer–induced bone destruction. This effect was due primarily to the complete absence of osteoclasts in the tibias of mice inoculated with OPG‐transfected cells, confirming the biologic activity of the transfected OPG in vivo. Despite the secretion of supraphysiologic levels of OPG, treatment with Apo2L/TRAIL resulted in strong growth inhibition of both empty vector and OPG‐overexpressing intratibial tumors. While Apo2L/TRAIL‐induced apoptosis may be abrogated in vitro by OPG overexpression, the in vivo anticancer efficacy of recombinant soluble Apo2L/TRAIL is retained in the bone microenvironment even in the presence of biologically active OPG at supraphysiologic concentrations. © 2011 American Society for Bone and Mineral Research.     

Thrombospondin-1 null mice are resistant to hypoxia-induced pulmonary hypertension

Background and objective

Chronic hypoxia induces pulmonary hypertension in mice. Smooth muscle cell hyperplasia and medial thickening characterize the vasculature of these animals. Thrombospondin-1 null (TSP-1^-/-) mice spontaneously develop pulmonary smooth muscle cell hyperplasia and medial thickening. In addition, TSP-1 produced by the pulmonary endothelium inhibits pulmonary artery smooth muscle cell growth. Based on these observations we sought to describe the pulmonary vascular changes in TSP-1^-/- mice exposed to chronic hypoxia.

Methods

We exposed TSP-1^-/- and wild type (WT) mice to a fraction of inspired oxygen (FiO2) of 0.1 for up to six weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Additionally, right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by right ventricle/left ventricle + septum ratios (RV/LV+S) were measured to evaluate pulmonary hypertensive changes. Finally, acute pulmonary vasoconstriction response in both TSP-1^-/- and WT mice was evaluated by acute hypoxia and U-46619 (a prostaglandin F2 analog) response.

Results

In hypoxia, TSP-1^-/- mice had significantly lower RVSP, RV/LV+S ratios and less pulmonary vascular remodeling when compared to WT mice. TSP-1^-/- mice also had significantly lower RVSP in response to acute pulmonary vasoconstriction challenges than their WT counterparts.

Conclusion

TSP-1^-/- mice had diminished pulmonary vasoconstriction response and were less responsive to hypoxia-induced pulmonary hypertension than their wild type counterparts. This observation suggests that TSP-1 could play an active role in the pathogenesis of pulmonary hypertension associated with hypoxia.     

Enhancement of Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in non-small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein.

OBJECTIVE: Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that promotes apoptosis specifically in tumor cells. Because not all cancer cells are susceptible to Apo2L/TRAIL, the aim of our study was to determine whether non-small cell lung cancer cells can be sensitized by chemotherapeutic agents for Apo2L/TRAIL-induced apoptosis. In addition, endogenous expression levels of the caspase-inhibiting cellular protease caspase-8 inhibitory protein (C-FLIP) were measured to investigate partial resistance to Apo2L/TRAIL. METHODS: Six human lung cancer cell lines (A549, NCI-H358, Calu1, Calu6, SkMes1, and SkLu1) were incubated with soluble Apo2L/TRAIL and two different concentrations each of cisplatin, paclitaxel, doxorubicin, 5-fluorouracil, and camptothecin. After 24 hours the rate of apoptosis was measured by annexin V/propidium iodide staining followed by FACScan analysis. Expression levels of C-FLIP in cell lines and lung cancer biopsy specimens were determined by Western blotting. RESULTS: Treatment of lung cancer cells with Apo2L/TRAIL alone resulted in apoptotic cell death in four cell lines (P <.001). Combining Apo2L/TRAIL and chemotherapeutic agents enhanced the rate of apoptosis significantly. Statistical analysis revealed a synergistic effect of Apo2L/TRAIL in combination with 1.8 mmol/L camptothecin and 100 micromol/L cisplatin, each in four of the six cell lines (P <.002). Western blot analysis showed that sensitization to Apo2L/TRAIL did not correlate with the expression of cellular protease caspase-8 inhibitory protein. Furthermore, no increased cellular protease caspase-8 inhibitory protein levels relative to those in normal lung tissue could be found in non-small cell lung cancer specimens from 12 patients. CONCLUSION: Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cell lines is significantly enhanced by chemotherapeutic agents. Resistance and sensitization to Apo2L/TRAIL are not correlated with the endogenous expression level of cellular protease caspase-8 inhibitory protein, implying that in non-small cell lung cancer other mechanisms are responsible for inhibition of the Apo2L/TRAIL pathway. Even though the molecular mechanism remains unclear, the combination of Apo2L/TRAIL with chemotherapy may be a promising treatment modality for non-small cell lung cancer.     

Human chondrocyte apoptosis in response to mechanical injury

OBJECTIVE: The effect of mechanical injury on chondrocyte viability and matrix degradation was studied. It was proposed that mechanical injury to human cartilage explants results in chondrocyte apoptosis with associated loss of glycosaminoglycans. DESIGN: Full thickness human cartilage explants, 5 mm in diameter were subjected to a single static mechanical stress of 14 MPa for 500 ms under radially unconfined compression. Glycosaminoglycan (GAG) release and percentage of cells undergoing apoptosis were measured at 96 h after injury. To establish the time course of apoptosis, explants were subjected to 30% strain and cultured for varying intervals up to 7 days after injury. A group of loaded explants were also treated with the broad spectrum caspase inhibitor z-Vad.fmk after injury. RESULTS: Internucleosomal DNA fragmentation as one indicator of apoptosis was observed in 34% (S.D.+/-11) of chondrocytes at 96 h in response to mechanical loading at 14 MPa, compared to 4% (S.D.+/-2) in the non-loaded explants. Evidence for cell death induction via apoptosis was also obtained by electron microscopy and caspase cleavage of cytokeratin. GAG release was also higher for the loaded explants, mean 1.9% (S.D.+/-0.14) of total GAG content, compared to control explants, mean 0.8% (S.D.+/-0.28). The percentage of apoptotic cells also correlated with the level of GAG release into the culture media. The percentage of apoptotic chondrocytes demonstrated a progressive increase from 6 h to 7 days post-injury. When loaded explants were cultured in z-Vad.fmk after injury, a 50% reduction in apoptosis rates was seen. CONCLUSIONS: These results demonstrate that mechanical injury induces chondrocyte apoptosis and release of GAG from the matrix. The time course suggests that a therapeutic window may exist where apoptosis could be inhibited. This potentially identifies a new approach to chondroprotection.     

Rabbits treated with chronic isepamicin are resistant to mivacurium and rocuronium

We compared the dose-response relationships and the neuromuscular blocking effects of mivacurium and rocuronium after chronic isepamicin therapy for 7 days in 56 anesthetized rabbits. Train-of-four stimuli were applied every 10 s to the common peroneal nerve, and the force of contraction of the tibialis anterior muscle was measured. Chronic isepamicin therapy is associated with a rightward shift of the mivacurium and rocuronium dose-response curves. The effective dose for 50% twitch depression of mivacurium and rocuronium increased significantly, from 16.9 +/- 4.8 and 56.5 +/- 5.3 microg/kg, respectively, with placebo to 30.6 +/- 5.3 and 75.6 +/- 4.7 microg/kg, respectively, during isepamicin therapy. The isepamicin rabbits receiving mivacurium 0.18 mg/kg or rocuronium 0.6 mg/kg had an accelerated recovery from neuromuscular blockade compared with those receiving placebo. The results of this study show that mivacurium and rocuronium have both a decreased effect and a shorter duration of action in rabbits when used during concurrent isepamicin therapy. IMPLICATIONS: We studied the dose-response relationships and the neuromuscular blocking effects of mivacurium and rocuronium during chronic isepamicin therapy in rabbits. Mivacurium and rocuronium have both a decreased effect and a shorter duration of action during chronic aminoglycoside antibiotic therapy in rabbits.