Necroptosis of nucleus pulposus cells involved in intervertebral disc degeneration through MyD88 signaling.

Background context: Low back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear. Purpose: To study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling. Study design: The expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro. Methods: Human disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells. Results: In vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM). Conclusion: Our results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Exosomes derived from olfactory ensheathing cells provided neuroprotection for spinal cord injury by switching the phenotype of macrophages/microglia.

Transplantation of olfactory ensheathing cells (OECs) has been demonstrated to be beneficial for spinal cord injury (SCI) by modulating neuroinflammation, supporting neuronal survival and promoting angiogenesis. Besides OECs, the conditioned medium (CM) from OECs has also been proved to have therapeutic effects for SCI, indicating that the bioactive substances secreted by OECs are essential for its protective effects. Nevertheless, there is still little information regarding the underlying mechanisms. Considering that exosomes are crucial for intercellular communication and could be secreted by different types of cells, we speculated that the therapeutic potential of OECs for SCI might be partially based on their exosomes. To examine whether OECs could secret exosomes, we isolated exosomes by polyethylene glycol-based method, and identified them by electron microscopy study, nanoparticle tracking analysis (NTA) and western blotting. In view of phagocytic ability of microglia and its distinct roles in microenvironment regulation after SCI, we then focused the effects of OECs-derived exosomes (OECs-Exo) on microglial phenotypic regulation. We found that the extracted OECs-Exo could be engulfed by microglia and partially reverse the LPS-induced pro-inflammatory polarization through inhibiting NF-κB and c-Jun signaling pathways in vitro. Furthermore, OECs-Exo were found to inhibit the polarization of pro-inflammatory macrophages/microglia while increased the numbers of anti-inflammatory cells after SCI. Considering that the neuronal injury is closely related to the activation state of macrophages/microglia, co-culture of microglia and neurons were performed. Neuronal death induced by LPS-treated microglia could be significantly alleviated when microglia treated by LPS plus OECs-Exo in vitro. After SCI, NeuN-immunostaining and axonal tract-tracing were performed to assess neuronal survival and axon preservation. Our data showed that the OECs-Exo promoted the neuronal survival and axon preservation, and facilitated functional recovery after SCI. Our findings provide a promising therapeutic strategy for SCI based on exosome-immunomodulation.

Surgical treatment of the lower cervical brucellosis with osteoporosis in the northwest region of China: review of 22 cases.

OBJECTIVE: To analyze the surgical treatment of patients with cervical brucellosis with osteoporosis over a 4-year period in Northwest China. METHODS: From 2013 to 2018, 22 patients (12 males and 10 females) with lower cervical spine brucellosis (C3-C7) underwent anterior lesion debridement, decompression, bone grafting and internal fixation combined with posterior bone graft fusion and internal fixation (ADDF+PIF). The follow-up period averaged 37.4 months (ranging from 24 to 57 months). RESULTS: Involvement of 1 vertebra was observed in 3 patients, involvement of 3 vertebrae was observed in 9 patients, and involvement of 3 vertebrae was observed in 10 patients. Before surgery, 1 patient had Frankel grade B, 2 had grade C, 9 had grade D, and 10 had grade E. In the final follow-up, 12 patients had neurological deficits, 10 patients improved by one grade, 6 patients improved by two grades, and the neurological status of 6 patients remained unchanged. In all cases, it was observed that bone fusion required 6.8 months on average. The kyphosis Cobb angle was enhanced from an average of 11.5° preoperatively (range 0°-24°) to 0.13° postoperatively (range 1°-5°), and there was no vital loss of correction in the follow-up. CONCLUSIONS: ADDF+PIF is an effective and safe treatment for patients with lower cervical brucellosis with osteoporosis.

Safety and Efficacy of Minimally Invasive Transforaminal Lumbar Interbody Fusion Combined with Gelatin Sponge Impregnated with Dexamethasone and No Drainage Tube after Surgery in the Treatment of Lumbar Degenerative Disease.

OBJECTIVE: The aim of the present study was to use a gelatin sponge impregnated with dexamethasone, combined with minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) and no drainage tube after the operation for early postoperative recurrence of root pain caused by edema. METHODS: A prospective case series study was designed. From September 2015 to January 2018, eligible patients diagnosed with lumbar degenerative disease underwent MIS-TLIF combined with a gelatin sponge impregnated with dexamethasone and no drainage tube after surgery. The short-term clinical data were collected, such as visual analog scale (VAS) scores for low back pain and leg pain preoperatively and on postoperative days (POD) 1-10, time bedridden postoperatively, and length of hospital stay postoperatively. Long-term indicators include the Japanese Orthopaedic Association (JOA) score, the Oswestry Disability Index (ODI) score, and the 36-Item Short-Form Health Survey (SF-36) score, evaluated preoperatively and 1 week, 3 months, and more than 1 year postoperatively. RESULTS: Complete clinical data was obtained for 139 patients. All patients were followed up for more than 12 months (13.7 ± 3.3 months). The average bedridden period was 1.5 ± 0.4 days and hospital stays were 2.7 ± 0.9 days. The VAS score of leg and back pain on POD 1-10 were all decreased compared with preoperation (all P < 0.0001). At the last follow up, the VAS scores for back pain and leg pain (0.69 ± 0.47; 1.02 ± 0.55) and the ODI score (11.1 ± 3.5) decreased (all P < 0.0001), and the JOA score (27.1 ± 3.2) and the SF-36 (physical component summary, 50.5 ± 7.3; mental component summary, 49.4 ± 8.9) increased (all P < 0.0001) compared with preoperative values. Patients' early and long-term levels of satisfaction postoperatively were 92.8% and 97.8%, respectively. At POD 7 and the last follow-up, the improvement rate of the JOA score, respectively, was 41.8% ± 10.6% and 87.7% ± 8.2%, and clinical effects assessed as significantly effective according to the improvement rate of the JOA score was 16.5% and 66.9%, respectively. There were 2 (1.4%) cases with complications, including 1 (0.7%) case of wound infection and 1 (0.7%) case of deep vein thrombosis. There were no device-related complications or neurological injuries. CONCLUSION: Use of a gelatin sponge impregnated with dexamethasone combined with MIS-TLIF and no drainage tube after the operation, compared with previous studies, appears to be safe and feasible to reduce recurrent back pain and leg pain after decompression in the treatment of lumbar degenerative disease.

Spontaneous Fusion After Cervical Disc Arthroplasty: A Case Report and Literature Review.

OBJECTIVE: To report a rare case of spontaneous fusion (SF) following cervical disc arthroplasty (CDA), to review the related literature, and to propose a new measure to prevent it. METHODS: The course of a patient with SF is described here. The potential causes, risk factors, and preventive measure of SF after CDA published in previous studies have also been reviewed and discussed. RESULTS: A 63-year-old man presented with a 6-month history of progressive neck pain and developed left C-7 radiculopathy 4 years ago. Magnetic resonance imaging revealed disc herniation at the C6-C7 levels resulting in compression of the left C-7 nerve root. The patient underwent CDA at the C6-C7 levels, during which a PRESTIGE cervical disc device was implanted. He failed to follow-up regularly as recommended postoperatively because he was completely free from the pain in his neck and left upper limb. Four years later, he was readmitted with a 2-month history of occasional neck stiffness. Plain radiographs indicated complete radiographic fusion of the C6-C7 levels with trabecular bone bridging surrounding the cervical disc prosthesis, and dynamic imaging showed no motion. He was seen at regular follow-up visits for up to 60 months without special treatment, as his symptoms of neck stiffness were minor and his symptom has not worsened since then. CONCLUSION: SF after CDA is a rare condition that can be attributed to patient- or prosthesis-related causes, and its risk factors are diverse. SF after CDA did not affect the patient's clinical outcome, and no special treatment was required for it. Practitioners should be aware of this rare complication and advise patients of the risks before performing CDA.

Rare Bony Diastematomyelia Associated with Intraspinal Teratoma.

We report a rare case of bony diastematomyelia associated with intraspinal teratoma. The patient was surgically treated with bony diastematomyelia and intradural teratoma resection, followed by lumbar duroplasty, and posterior fusion from L2-L4 in order to maintain the spinal stability of the approached segments. Despite the risks, it was necessary to perform early surgical treatment because of rapid neurologic deterioration. The patient had a good postoperative outcome.

Quercetin prevents necroptosis of oligodendrocytes by inhibiting macrophages/microglia polarization to M1 phenotype after spinal cord injury in rats.

BACKGROUND: Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination, even leading to a permanent neurological deficit. Besides apoptosis, our previous study demonstrated that OLs underwent receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. Considering that necroptosis is always accompanied with pro-inflammatory response and quercetin has long been used as anti-inflammatory agent, in the present study we investigated whether quercetin could inhibit necroptosis of OLs and suppress the M1 macrophages/microglia-mediated immune response after SCI as well as the possible mechanism. METHODS: In this study, we applied quercetin, an important flavonoid component of various herbs, to treat rats with SCI and rats injected with saline were employed as the control group. Locomotor functional recovery was evaluated using Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay. In vivo, the necroptosis, apoptosis, and regeneration of OLs were detected by immunohistochemistry, 5'-bromo-2'-deoxyuridine (BrdU) incorporation. The loss of myelin and axons after SCI were evaluated by Luxol fast blue (LFB) staining, immunohistochemistry, and electron microscopic study. The polarization of macrophages/microglia after SCI and the underlying mechanisms were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. In vitro, the ATP and reactive oxygen species (ROS) level examination, propidium iodide (PI) labeling, and Western blotting were used to analyze the necroptosis of cultured OLs, while the signaling pathways-mediated polarization of cultured macrophages/microglia was detected by qRT-PCR and Western blotting. RESULTS: We demonstrated that quercetin treatment improved functional recovery in rats after SCI. We then found that quercetin significantly reduced necroptosis of OLs after SCI without influencing apoptosis and regeneration of OLs. Meanwhile, myelin loss and axon loss were also significantly reduced in quercetin-treated rats, as compared to SCI + saline control. Further, we revealed that quercetin could suppress macrophages/microglia polarized to M1 phenotype through inhibition of STAT1 and NF-κB pathway in vivo and in vitro, which contributes to the decreased necroptosis of OLs. CONCLUSIONS: Quercetin treatment alleviated necroptosis of OLs partially by inhibiting M1 macrophages/microglia polarization after SCI. Our findings suggest that necroptosis of OLs may be a potential therapeutic target for clinical SCI.

Intradural Lumbar Disc Herniation: A Case Report and Literature Review.

BACKGROUND: Lumbar disc herniation into the dural space is a very rare phenomenon of degenerative lumbar lesions in the elderly population, and its potential pathogenesis and natural course remain unclear. CASE DESCRIPTION: We describe a rare case of intradural lumbar disc herniation. A 68-year-old man presented with progressive lower back pain and radiating pain and numbness in both legs for 3 years. Magnetic resonance imaging revealed a large herniated disc at L4-L5. Posterior discectomy and fusion of the L4-L5 was performed after conservative treatment failed. Intraoperatively, only minimal disc fragments in the epidural space were found after meticulous probing following laminectomy of the L4-L5 vertebrae. The dorsal dura mater was saturated, tense, and bulged at the L4-L5 levels; additionally, an intradural mass was palpable and confirmed by intraoperative ultrasonography. Subsequently, dorsal middle durotomy was performed. Upon opening the dural sac, a large cauliflower-like mass similar to nucleus pulposus tissue was found near the arachnoid membrane. The mass was dissociative and could be completely resected. The dorsal dural incisions were closed after careful exploration, followed by fixation and fusion of the L4-L5 levels. Pathological examination revealed disc tissue with central balloon-type cystic degenerative changes. The patient's lower back pain and radiating pain and numbness of both legs improved remarkably postoperatively, and he became asymptomatic at 3 months postoperatively. CONCLUSION: Intradural lumbar disc herniation should be highly suspected when intraoperative findings are incompatible with findings from the preoperative imaging examination, and it could be further confirmed via intraoperative ultrasonography and pathological examination of the resected tissue from the dural space. Prompt surgery is recommended, and surgical results are usually favorable. We also reviewed the literature and discussed the potential pathogenesis, natural course, diagnosis, and treatment of intradural lumbar disc herniation.

Intensive care unit versus hospital floor: a comparative study of postoperative management of patients with adolescent idiopathic scoliosis.

BACKGROUND: Patients undergoing posterior spinal instrumentation and fusion surgery for adolescent idiopathic scoliosis were admitted to the intensive care unit until two years ago, at which time we changed our protocol to admit these patients to the general hospital floor following a brief stay in a postanesthesia care unit. This study compared postoperative management on a hospital floor with that in the intensive care unit for patients with adolescent idiopathic scoliosis undergoing posterior spinal fusion. METHODS: A retrospective review of 124 consecutive patients with adolescent idiopathic scoliosis treated with spinal fusion from August 2007 to August 2010 was performed. Inclusion criteria were a diagnosis of adolescent idiopathic scoliosis and posterior spinal instrumentation and fusion surgery. RESULTS: Of 124 patients, sixty-six were managed postoperatively in the intensive care unit and fifty-eight, on the hospital floor. The mean age at the time of surgery was fourteen years. A mean of eleven vertebral levels (range, six to fifteen levels) were fused. No significant difference between the groups was found with respect to the mean age at the time of surgery, mean weight, mean preoperative and postoperative Cobb angles, and mean number of levels fused (p ≥ 0.12). However, the use of analgesic and antianxiety medication, number of postoperative blood tests, days of hospital stay, and number of physical therapy sessions were significantly decreased in the floor group compared with the intensive care unit group (p ≤ 0.05). No patient from the floor group had to be admitted to the intensive care unit. The mean charge was $33,121 for the floor group and $39,252 for the intensive care unit group (p < 0.001). CONCLUSIONS: Initial postoperative management of patients with adolescent idiopathic scoliosis following a posterior spinal instrumentation and fusion surgery on a general hospital floor, rather than in an intensive care unit, was associated with a shorter hospital stay, fewer blood tests, less analgesic and antianxiety medication usage, and fewer physical therapy sessions at this high-volume, academic, tertiary-care children's hospital. In addition to improved patient outcomes, there was a significant decrease of 16% in hospital charges for the group that did not go to the intensive care unit.

The effect of bradykinin B2 receptor polymorphisms on the susceptibility and severity of osteoarthritis in a Chinese cohort.

BACKGROUND: The B2-bradykinin receptor (BDKRB2) has been reported to associate with onset and development of Osteoarthritis (OA); however, the role of BDKRB2 genetic polymorphisms in OA remains unknown. METHOD: A total of 245 patients with primary knee OA and 264 healthy volunteer were recruited. BDKRB2 gene polymorphisms, -58T/C and +9/-9 bp polymorphisms, were genotyped. RESULTS: The genotype distributions and allele frequencies of +9/-9 bp polymorphisms significantly differed between OA and control subjects. Logistic regression analysis showed carriers with -9/-9 genotype had a significantly increased risk for knee OA compared with the +9/+9 genotype (adjusted OR = 2.356, P < 0.001). The OR for -9 allele carriage was significantly higher than +9 allele carriage (adjusted OR = 1.52, P < 0.001). The +9/-9 bp polymorphisms also determined the OA radiographic severity. The presence of -9 bp was associated with severer OA. The -58T/C polymorphisms did not affect OA risk and severity. CONCLUSION: The +9/-9 bp polymorphisms of BDKRB2 gene may be used as a genetic marker for the susceptibility and severity of OA.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway.

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.

[Establishment and primary application of sandwich ELISA method to detect tenascin-C].

AIM: To establish a sandwich method to detect tenascin-c on the basis of preparation of monoclonal antibodies (mAbs) against tenascin-C (TN-C). METHODS: The ascites of three stains of mAbs (No. 1A8, 3H7 and 4D6) were prepared and purified. The mAbs were conjugated with HRP and paired, respectively. The recombinant TN-C was taken as standard to analyze the optimal combination between mAbs. The sera TN-C concentrations of patients with osteosarocoma and the normal persons were evaluated with the sandwich ELISA method. RESULTS: Among these mAbs, the sensitivity was obtained when combined the coated 1A8 with HRP-4D6. The sera TN-C significantly higher than the normal controls. CONCLUSION: The sandwich ELISA method to detect TN-C was established successfully. The sera TN-C concentrations of patients with osteosarcoma and the normal persons were found distinct with the sandwich method.

A novel recombinant immuno-tBid with a furin site effectively suppresses the growth of HER2-positive osteosarcoma cells in vitro.

Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.

Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits.

BACKGROUND: Hydroxysafflor Yellow A (HSYA), which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R) injury is still unknown. METHODS: Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6) were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA) level and increased superoxide dismutase (SOD) activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. CONCLUSIONS: These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

[ShRNA of Cyclin D1 decreased the proliferation of human osteosarcoma cell line SOSP-9607.].

AIM: To investigate the effect of Cyclin D1 shRNA on the apoptosis and proliferation of human osteosarcoma cell line SOSP-9607. METHODS: Human Cyclin D1 shRNA vector was stably transfected into SOSP-9607 osteosarcoma cells. The mRNA and protein cxpression levels of Cyclin D1 were detected by semiquantitative RT-PCR and Western blot respectively. The cell cycle and pretiferation of osteosarcoma cells were examined by FCM analysis and CCK-8 method, respectively. RESULTS: After stable transfection of Cyclin D1 shRNA, the expression of Cyclin D1 were inhibited at mRNA and protein levels. The proliferation of SOSP-9607 osteosarcoma cells was inhibited. The difference was significant compared with control groups (P<0.05). At the same time, Cyclin D1 shRNA transfection increased G0/1 phage content and decreased S phage content. CONCLUSION: Cyclin D1 shRNA could down-regulate the expression of Cyclin D1, effectively inhibit the proliferation of osteosarcoma cells, and have significant effect on the cell cycle.

scFv-mediated delivery of truncated BID suppresses HER2-positive osteosarcoma growth and metastasis.

Osteosarcoma is the most common primary malignant bone tumor, with high rates of metastasis. Here, we examined the expression of human epidermal growth factor receptor-2 (HER-2) in osteosarcoma cell lines with different metastatic potential, finding that the expression was correlated with metastasis of implanted tumors. We then introduced an expression vector encoding the e23sFv-PEA II-Bid Delta1-60 gene, composed of a HER2-specific single-chain antibody fused with domain II of Pseudomonas exotoxin A (PEA) and the carboxy end of truncated active Bid. We demonstrated that the e23sFv-PEA II-Bid Delta1-60 molecule selectively recognized and killed HER2-overexpressing osteosarcoma cells in vitro. Subsequently, we introduced the e23sFv-PEA II-bid Delta1-60 gene into BALB/c athymic mice bearing HER2-positive osteosarcomas using i.m. injections of liposome-encapsulated vector. Expression of the e23sFv-PEA II-Bid Delta1-60 gene suppressed tumor growth, significantly prolonged animal survival and inhibited metastasis, thereby suggesting it may represent a competitive approach to treating HER2/neu-positive osteosarcoma.